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Abood
American Cancer Society
American Institute of Chemists
Center College
Council for Tobacco Research - USA (CTR) (Formerly Tobacco Industry Research Committee (TIRC))
Originally organized as the Tobacco Industry Research Committe(TIRC) in 1954, and renamed Council for Tobacco Research - USA, Inc. (CTR) in 1964.
Environmental Protection Agency (EPA)
Georgetown University
Kodak
Massachusetts Institute of Technology (MIT)
National Institutes of Health
National Institutes of Health (NIH)
National Science Foundation
Oklahoma City University
*Scientific Advisory Board (SAB) (Only use SAB with name of specific org.)
University of Oklahoma
University of Pittsburgh
Washington University in St. Louis
Named Person
Bruce, John B.
Conner, Mary
Crawford, Carol
Day, Billy
Day, Billy W.
Eisenberg, Arthur D., Ph.D. (CTR Assoc. Research Director 1991, Asst. Secretary 1997)
Defense
Karol, Meryl H.
Korsmeyer, Stan
Lippman, Marc
Liss, Alan R.
Mattison, Donald R.
Mccarty, Ken
Spitz, Douglas R.
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11 Jan 2006
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0157

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PROCESSING LNC0~fLNG .4/~PLICAYIONS BUDGETARY INFOP, MATION Appl. No.: ___~/ Total Requested Equipment Full Frog. Rept.: ~] Yes ~ No Abst. Frog. Rept: ~] Yes [~] No CATEGORY OF RESE.M~.CH VI Cardiovascular I-I Cell Biology I-'l Developmental Biology I~1 Epidemiology ~ Genetics COMMITTEE [q Abood I-I Amason I-I Bowden ~ Croce ~] Erikson Immunology I-I Feldman Application Stamped in: Copy of Fats Sh~t: _._~_~/ Appl. Acknowledged.,~// Numerical File Card [~ Miscellaneous [~ Neuroscicncc r-~ Pharmacology ~ Pulmonm'y [] Radicals [] Virology r-'l Gill I-1 Lynch I-I McAllister r-i Pierce [] Sabatini I-I Swain Cl Vogt GH F~ceS.l ~oc 50374179
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BUDGETARY INFORMATION Total Rec Year 1-'~ Year 3:~ uested Equipment Full Prog. Rept.: n Yes [~ No Abst. Prog. Rept: ~] Yes [~] No CATEGORY OF RESEARCH Cardiovascular Cell Biology Developmental Biology Epidemiology Genetics COMMITTEE [~ Abood ~I Am~on n Bowdcn [~ Croce ~ Erikson Immunology [~1 Fcldman Application Starnpcd in: ~ NumericaI File Card/.~ Miscellaneous Neurosciencc Pharmacology Pulmonary Radicals Vimlo~, {~ Gill I~ Lynch t"1 McAllister I~1 Pierce ~1 Sabatini l~ Swan [] Vogt 50374180
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THE COL~NCIL FOR TOB,%CC0 I~ESE~%RCH-U.~..{., INC. September 29, 1995 Billy Day, Ph.D. University of Pittsburgh Dept. of Environmental & Occupational Health RIDC Park 260 Kappa Drive Pittsburgh, PA 15238 Re: Application No. 4374 Dear Dr. Day: After careful review of your application entitled "Role and Mechanisms of Apoptosis in Breast Cancer Cells," we regret to advise you that we cannot fund your proposed study. We hope that you will be successful in f'mding support elsewhere for your investigation. JFG/mm
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He ~as £1:st au~ho~ on ~he syn~Izesls and biolo~iesl evaluation o£ the dichloro-twlarylcyclol~Openes in J. ~ed. Chem. in 1991 and he has been ~o:klnl: on ~hese c~ as pure antiest-z~L~:~ since r~en. Nost o~ his cited publlcatlons a~e in toxicology and aedlci~al cheais~'~ journals. 50374~82
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_ RB:)AClla)IP 14374 P~ge 2. The budget requeste 1l~ salary :~l~rt for ~i~(:; and 75~ for FIEDACTEO • A Blo-r~l lk~ntltltlvl Field Inversion Gel Sle~trophortsls apparatus at ~5,BBB Is I isted in the first year. The total request asks $7<J,~7 1'or the first oi' three Priority score 50374183
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T~ree genes are known %o be definitely involved in apophosis: p53 which promotes i%, hcl-2 ~/ch preven£s i%, and hax ~hich n~u£ralizes bcl-2. ~~ has recently found ~hat apophosis does occur in breas~ cancer cells. MCF-7 cells, which are ER(+)/~2(+) are resistant to apop£osis because ~.hey con£ain bcl-2; MDA-MB-23~ cells, wh/ch are ER(-)/EZ(-) are more susoep£ible because ~_hey con£ain more p53. He now proposes to probe breas~ cancer ~ lines with differing horc~one s~r~si%ivities wi~h agents %hat induce apop~osis, such as ZCC (which h~nds nucleer type II estrogen binding sites)t as w~ll as other agents w££h nuclear %argees like topo II and £~hulin; and measure 9.heir bcl-2, bax and pS3 levels. He has already done ~cmsiderable work on ~his project and ccme %0 ~he conclusion ~ha% breas£ cancer cells possess a normal apop£o%ic pathway %ha% is cell-cycle-dependent and protein synthesis-dependent and yields 50Kb DNA fragments ~ha% occur without ~he formation of nuclear apop~o%ic bodies. The proposed program is technically per£ec~ly feasible. R~-~)~ ~b.tained his ~h.D. in 1988 in Medicinal Chemis£ry a% Oklahc~a, wes 9.hen a postdo~coral fellow for ~hree years a% MIT in ~he Depaz'hmen% of Chemistry and Divi.sion of Toxicology (w~th where?; no publications?), and since 1991 has been Assis£an% Professor of Environmental and Occupational Heal~h and Pharmaceutical Sciences at Pi~cshurgh, It could well be ~hat %his somewha£ unusual training is exac~cly what is needed for ~his scme~ahat unusual hut potentially interesting projec~~~search program is indeed funde~ via a varle£y of mechanisms; and his budge% here is fine. RECOMMENDATION There are plusses and minuses here. On %he one hand~ studying %he effec~ of a variety o£ agents on %he levels of ~.hree pro£eins several cell lines is not v~ry exciting. ~~s very broadly %rained and I hay8 a feeling ~.hat ~he work will be done very ~Ii. Human hreas£ cancer is not just one disease; and 9.he agen£s %hat he will test do have dif£eren% relevant £arge%s. So I came cut wi£h a ~da£ion for approval with a priority of ~ .5.
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THE COUNCIL FOR TOBACCO ~R~SEARCH - U.S.A., INC. ~U~O~TIH~ ]~IO~(EDI~AL fNVK~TI~ATIOH (212) D~te: Jan~ 27, I995 Number: 4374 Discipline: Cancer NEW RESEARCH APPLICATION for the Fall 1995 SAB Meeting To: Prim~' Reviewer: Dr. Brennan Secondary Reviewer: Dr. 3oklik Applicant: Institution: Location: Project Titl¢: Billy W. Day, Ph.D. University of Pittsburgh Pittsburgh, Pennsylvania Role and Mechanisms of Apoptosis in Breast Caz~r Cells HISTORY: Preliminary Application number 5567 was considered by the reviewers and a full application was encouraged. REQUEST: Application Number 4374 requests $79.857 for the first year of a 3 y~ar project. Budget estimates for the second and third year are $~ and $80.555, respectively. Permanvnt equipment requested for year 1, $5000. DOCUMENTS SUBMITTED: 1. Application dated: May 31, 1995 2. Biographical sketches: Drs. Day (2 Pgs); Balachandran (2 Pgs) 3. Reprints: -4- 4. Manuscripts: -I- 5. L~tt~rs of collaboration and suppor~: None 6. Other documents: None GAH/mv! 50374185
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Research Grant Application TITLE OF STUDY (09t;OTE2~CEFD 70 Role and Mecha~i~=~ of Apopto~i~ in Breast Cancer Cell~ K~ WO~DS (L~M~T T~ F~V~) apoptosis, breast cancer, bax, hcl-2, p53 START DATE 1/1/96 F%ROPOBED DUPJ~T{ON 1, 2 o~3 I:~"~. EQUIPMENT (pago FUNDS ~EaUESTeD (~NOLUDI~S ~ID R--CT COSTS) YEaR 1 YF.~R 2 YEaR 3 79,857 77,733 80,555 5,000 -0- - 0 - PRINCIPAL INVESTIGATOR (do no': indicate any c>P.I, on this page) NAME (LAST, F[K~T M.L) AND DEGKEES: Day, Billy W. Ph.D. ACADEMIC o~ Assistant Professor PROFESSIOHAL TITLE: DEPARTMENT: Environmental & Occupational Health INSTITUTION: University of Pittsburgh STREET ADDRESS: RIDC Park, 260 Kappa Drive CITY, STATE (OR COUNTRY) ZiP CODE: Pittsburgh, PA 15238 MAIUNG ADDRESS IF DIFFERENT FROM ABOVE: TELEPHONE NUMBER: (412) 967-6500 INSTITUTIONAL OFFICIAL (acc~ptin~ for thB institution] ~'~: Mich~l M. Crouch POSITION TITLE:: Director, Office of Research University of Pittsburgh 350 Thackeray Hall Pittsburgh, PA 15260 T EL£P~O%E WJMBEFq (4!2) 624-7400 RNANCIAL ADMINISTRATOR (person to c~nta~t for budget infon~ati~n) Carol Crawford POS|TION TITL£: Grants & Contracts Officer M,A.IUNG ADDRESS: University of Pittsburgh 350 Thackeray Hail Pittsburgh, PA 15260 TELEPHONE NUMBER: (412) 624-7400 IMAKE CHECKS PAYABLE TO: University of Pittsburgh I MAIL CHECKS TO (NAME AND POSITION TITLE): William Lair4, Controller MAIUNGADD~SS: University of Pittsburgh Controller's Office/Research Accountln Lock Box 371220 Pittsburgh, PA 15251-7220 Dir-~clc, r~ Office of Research
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1. ~_P..T. : E~ili~,, W. Da)', Ph.D. c.~ c,f~oi~t : RoIe and ~,lech~nisms of Ap~ptos~s in Brea~ Cancer Ceils Do not ~e ~ou~ pa~e~ Apoptosis and the g~nes involved. The ro~e of apoptosis, programmed cell death, in cancer biology has recently gained both impo~ance and widespread aRention. Many toxicants reduce the proliferative potential of cnncer celIs and disrupt their passage theugh the ceil cycle. A xenob~otic can initiate the signals necessaw for indu~ion of apoptosis. The existence of a genetic program in the cell during a drag- induced death pathway suggests that modification in the expression of the responsible genes could have a profound effect on the development of cancer. A sequential or concomitant biochemical/genetic modulation that. augments the initial steps of the cascade should fuel the apoptotic event induced by antiproHferative xenobiotics. The genes bcl-2, bax, and p53, and their expressed protein products, have been linked to programmed cell death. The wild type (~) p53 gene product induces apoptosis, wh~le the bcl-2 gone product can inhibit apoptosis triggered by ~ p53. These a~ivities of both genes have been noted in breast, both clinically and in cell culture. The bct-2 gene encodes for a mitochondfial protein that is localized to the luminal cells of normal breast, considered to be the origin of malignant brehst disease. Several studies of breast tumors have shown that bcl-2 levels are positively correlated with estrogen receptor (ER) and progesterone receptor (PR) positivity. The bcl-2 produ~ is generally associated with small, ER positive (+), slowly proliferating, p53 negative tumors, and is not generally associated with large, ER negative (-), rapidly proliferating, p53 positive breast tumors. Hence, MCF-7 cells, which are ER(+)/estrogen sensitive (E2(+)), should be more resistant to apoptosis-inducing drqgs than MDA-MB-231 cells, which are ER(-)/E2(-). Breast cancer cells, however, been classified as 'not apoptosis proficient', because they consistently fail to produce the characteristic DNA fragmentation (- 180 bp) ladders on agarose gels in response to typical apoptotic stimuli. Over-expression of the bax gene has been shown to counter the death repressor activity of bcl-2 via the formation of a heterodimeric protein. The bax gene product is thus a dominant inhibitor of the bcl-2 protein. The ratio of bcl-2 to bax determines the su~ival or death of a cell to an apoptotic stimulus. Bax and its gene product are relatively recently discovered contributors to the apoptosis process and, to date, the~# h~ve b#en ~ repo~s on the involvement of b~ in breast cancer. Although these three genes have been associated with the apoptosis process, their relationship to xenobiotic-induced cell death is not clear, especially in the case of breast cancer. The screening of different human breast cancer cell lines with differing estrogen sensitivity for these genes during the xenobiotic-induced apoptosis process will provide useful information on cell death and cell proliferation of these cells, and may provide impo~ant insight into the development and progression of breast cancer. The working hypothesis of this proposal is that apoptosis can, based on our prelimina~ studies, be studied in human breast carcinoma cells in culture. Our broad goal is to elucidate the role and mechanisms of apoptosis in breast cancer ceils by use of a combination of: appl{cation of xenobiotics to the cells that cause induction of apoptosis; probing for large DNA fragments during the process; and molecular biological measurement of the responses of p53, bcl-2 and ba~ during the process.
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~ ~ot exceed ~e ~ac~ ~t:.ne~ The ob~e~ives of th~s research are to define the role and mechanism of apoptosis in cIonatly different human breast cancer cells. Specifically, we will first probe human breast c~ncer col; lines of d#ering hormonal sensitivity ~',,ith agents studied in our lab that we have preliminarily shown to induce apoptosis in the cells. To be examined are ER(+)tE2(+), ER(+)IE2(-), and (ER-)IE2(-) human breast cancer cell lines MCF-7, MCF-7/LY2, and MDA-MB-231. Concurrently we will study" the intera~ions of, and elucidate the roles of, three specific genes, b~, bcl-2 and p53, that have been shown to regulate apoptosis. The results from our studies will allow us to elucidate the role of gone regulation in the apoptosis of breast cancer cells. Therefore, the specific aims of this project are: 1. To investigate the nature of the apoptosis in MCF-7, MCF-7/LY2 and MDA-MB- 231 human breast cancer ceils. 2. To identify the role of the p53, bcl-2 and box gone products in these cell lines and correlate their relative expressions with the apoptosis induced by xenobiotics. We will first continue our studies, described in the prelimina~ studies section, with the novel agent Z-~-chlomchalcone (ZCC) to fully elucidate the mechanisms of apoptosis it induces. Since ZCC has a nuclear protein (nuclear type II estrogen binding site (EBS)) as its target, we will subsequently study the effects of ether nuclear-targeted agents that have known activity against human breast cancer cells: genistein, VM-26 and VP-16 (topoisomerase !1~); ~atoxin (topo II and tubulin); podophyllotoxin, taxol, and discodermolide (tubulin); Analog II, luteolin, quercetin and diethylslilbestrol (tubulin and nuclear type II EBS). 3. S~PORT~G DATA, E~E~~ DESIGN ~d PROCED~S. D~ not a~ch more ~ six ~fion~ pages (3a-3t). ~1 fi~es, c~, ~bles ~d references must fit ~ pages 3 - 3f. - Prolimlna~ stu~ies. We have been studying t~e investigational broad-spe~mm a~ti-breast cancer agent Z4,~-dichloro-2,3-diphenylcyclopropane (Analo~ II), a non- mutagenic "antiestrogen" (in animals) with low loxicity and anti4ubulin/nuclear type ESS activities, ~or the past several years. Its ma]o~ metabolite in these cel~s, ZGC, ~as ca. q0-fold higher activily in all of the endpoints we have studied (except ami-t~bolin), and we have pm~imlly cham~erized the biochemical nmtore of the apoptosis induced by this agent using a number of parameters Cytological examination o~ ZCC-troato8 breast cancer co~ls. T~ese studies were done primarily to assure that any n~clear-relate~ events that we cheese0 were ~ue 1o apoptotic, vers~s necrotic, processes. For cytological preparations, ce,s were washefl in PBS and immediately fixed in 4% buffered ~ormalin, rinsed in PBS, and slainefl with May-~runwald Giemsa for ~0 min, foIlowed by restainin~ for 20 rain using Wright Giemsa (~ :20). Sl~des were washe~ in water, mounted and photographed. From each slide, ~ 000 cell~ were scored for chromafin conaensation. During process o~ progamme~ cel~ death, t~e cytoskeleton reorgm~ize~ so t~a~ l~e cell ro~nOs up, the plasma membrane breaks down~ an~ t~e nucleus condenses. Although the ce~l lines used undergo c~o)ogical changes, the rates and types of nuclear changes differ. In the case of MCF-7 cells, the condensation of the nucleus is ve~ rema&able and the the cell morphology remains largely intact. Morphological changes in the nucleus are detecte~ 12 h a~er expesure to ZCC. By 20 h, 83% of the cells have ~ndensed nuclei. In contrast, c~ological changes occur as early as 4 h into ZCC treatment in MDA-MB-231 cells, with 60% Gf the coifs showing rounded morphology, while the nucleus becomesincistingu;shabre from thec~oplasm. The MCF-7/LY2 C C,_" 7FIDE NTL.'d_
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ceils also dispIay cytoIog[ca[ changes sim;]~r to MDA-MB-231 cells in response to ZCO, follow;rig a simiI3r time-course. Hours,, post-treatment .Control ZOO treated Ceil death pattern in MCF-7 cells with no detectable 180-200 base pair DNA fragmentation (percent of dead cells). 12 hr 2 21 16 hr 11 20 20 hr 9 39 Cell death pattern in MDA-MB-231 cells with no detectable 180-200 base pair DNA fragmentation (percent of dead cells). 4hr 1 11 8hr 1 11 12 hr 2 18 Percent of MCF -7 cells with condensed nuclei 12 hr 0.4 17 16 hr 1 32 20 hr 1 83 Percent of MDA-M8-231 cells with 'rounded' morphology and intense staining of the cytoplasm and nucleus 4 hr 3 60 8 hr 1 78 12hr 17 62 Percent of MCF-7/LY2 cells with 'rounded' morphology and intense staining of the cytoplasm and the nucleus 4 hr 3 58 8 hr 3 57 12 hr 6 45 Note: The condensation of the nuclei found in MCF-7 cells was much more marked than that seen in MDA-MB-231 and MCF-7/LY2 cells. ZCC treated MCF-7 control 16 hr 12 hr
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3~ ZCC treated MCF-7/LY2 ZCC treated ~DA-MB-231 control control 4 hr ir 4 hr Role of protein synthesis in the ZCC-induced programmed ceil death of breast cancer cells. Generally, programmed cell death requires de novo protein synthesis. Cycloheximide (CHX) can block or provoke cell death in response to appropriate stimuli. In the study of the role of protein synthesis in ZCC-induced cell death, cells were exposed to different concentrations of CHX. After 12 h of CHX treatment, cells were washed and treated with ZCC for 24 h. Cell viability was determined hemacytometrically using trypan blue exclusion. CHX alone did not reduce the viability of the cells. The concentrations of CHX used blocked protein synthesis in the cells. Thus, proteins essential for initiating a programmed cell death pathway were not present in these cells at the time of ZCC treatment. Our data indicate that considerable protection was provided by CHX in the case of MDA-MB-231 cells. The data also indicate that protein synthesis is a prerequisite for ZCC to initiate apoptosis in the MDA-MB-231 cells. 100 I I ! 0 5 10 20 Cycloheximide Concentration Ro/e of DNA synthesis in the programmed ceil death of the breast cancer cefls. To identify whether the DKIA synthesis phase (S-phase) af the ¢e115 cycle is the target far ZCC's action, cells were pre-treated with aifferent concentrations of a DNA synthesis inhibitor, hydroxyurea (HOU), for 24 h. Exposing the cells to HOU eliminates those cells that are already in S-phase Aff.er HOU treatment, ceils were washed and treated with ZCC for 24 h. Cell viabil:y ,,,,'as determined as above. Ce~ls used in these experiments nave an S-pn~se of i2 n. ~,','hen trea~_ed with ZCC, the taraet cells present
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are in the G1, G2, and M phases of the cell cycle. HOU-treated cells will enter the S- phase. HOU alone did not reduce viability of the MDA-MB-231 cells, nor modify the action of ZCC significantly. ZCC reduced the viability of the MDA-MB-231 ceils considerably, and a large number of these ceils were in the S-phase of the cell cycle. From the results obtained from the current experiments, it possible to state that the cells undergoing apoptosis in response to ZCC are in the S-phase of the cell cycle. Hydroxyurea Concentration (raM) DNA fragmentation in MCF-7 cells treated with ZCC. Apoptois is characterized by the appearance of nucleosomal oligomer DNA fragments, typically in multiples of 180-200 base pairs, displayed by subunit ladders appearing in agarose electrophoretic gels. This fragmentation is followed by chromatin condenstion, cell surface blebbing, and cell fragmentaion into a cluster of membrane-bound apoptotic bodies. Cells treated with ZCC at various time intervals up to 60 h were collected and the DNA was subjected to electrophoresis in an agarose gel (1.8%, 75 V, constant current for 6 h), using standard 100 base pair markers.as controls. Gels were examined under UV-transillumination after ethidium bromide staining. This conventional gel electrophoresis approach failed to reproducibly demonstrate any DNA cleavage in MCF-7 or MDA-MB-231 cells, although, on occasion, we saw evidence of laddering. Using a different approach to detect DNA fragmentation, MCF- 7 cells were treated with ZCC for 24 h, and at 6 h intervals aliquots of cells were removed, their DNA was extracted and subjected to Quantitative Field Inversion Gel Electrophoresis (QFIGE) analysis. Cells were suspended in a buffer containig 20 mM NaCI, 50 mM EDTA, and 10 mM Tris, pH 7.2, and equilibrated to 50 oC. An equal volume of 2% InCert agarose (FMC) was added and plugs were made (10s cells per plug) for QFIGE in disposble molds (BioRad). After solidification at 4 oC, plugs were incubated at 50 ~C overnight in 4.8 mM (0.2%) sodium deoxycholate, 1% SDS, 100 mM EDTA, pH 8.0, and 500ug/ml proteinase K, then washed four times with 50 mL of 50 mM EDTA and 20 mM Tris, pH 8.0. QFIGE was performed on 5 x 106 cells per lane using a FIGE Mapper Electrophoresis system in 1.5 % Pulsed Field Certified Agarose in 0.5% TBE. Electrophoresis parameters were : a 12 min continuous forward pulse at 150 V, followed by 30 h at 150 V using a 21% ramp from 0.9 secto 30 secin the forward (direction ar.d 0.3 sec to 10 sec in the reverse direclion at 10 cC. After electrophoresis, gelswere stained with 1.0 u.g/mLethid~umbromide for 30 rain. Gels were viewed by UV-:ra~sillum:nation and photographed Ti-e results show that the MCF-7 cells u,-,derc~o DNA fraomentation, ,uis:,laying 50 Kb fragments. Tn,s fragmentat :,q ',','as detected at the 6 b ,.ime :,~rmt cf ~.rea: .~.emt =_,:.d increasec in a time-
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dependent manner w;th the macdmL~m DNA fragmentation at 24 h time point of treatment. ~c~-7 hours .high zw 50 Kb region] ZCC-treated ~2~ ~:~i! Detection of Bax mRNA in ZCC-treated cells. The bax gene and its protein product are known to promote apoptosis in cell lines of various lineages, while the related bci-2 gene product inhibits apoptosis and promotes cell survival. Considering the clear protective role of bcl-2 in genotoxic stress-induced programmed cell death, up-regulation of its repressor, box, in a p53-dependent manner could be an important determinant of programmed cell death. The possibility of box gene up-regulation was determined in breast cancer cells following treatment with ZCC. Cells were treated with ZCC, collected at various time points, and RNA.was extraoted using the hot phenol method. In detail, cells were washed three times with PBS and suspended in 50 mM sodium acetate -1% SDS, mixed with an equal volume of phenol equilibtrated with 50 mM sodium acetate, then agitated for 15 min at 60 oC. The solution was cooled at 0 oC for 15 min, then centrifuged at 3000 rpm for 15 min. The aqueous layer was taken, made 0.3 M in sodium acetate, and the RNA was precipitated with ethanol. The pellet was rinsed in ethanol, dried, and resuspended in RNAse-free water. For dot blot hybridizations, RNA was denatured by heating at 70 oC for 3 min in 40 % formamide, 6 % formaldehyde, and MOPS, then chilled on ice for 2 min. Serial dilutions of RNA samples were applied to nitrocellulose membranes, pre-equilibrated with 2x SSC, using a dot blot apparatus. Filters were then baked and hybridized in 50% formamide, 5x SSC, 5x Denhardts, 50 mM sodium phosphate, 5% dextran sulfate, and 250 p.g/mL sonicated salmon sperm DNA for 6 h. After pre-hybridization, the membranes were hybridized for 20 h at 42 oC in 50 % formamide, 5x SSC, 50 mM sodium phosphate, 5x Denhardts, 10% dextran sutfate, 100 #g/mL sonicated salmon sperm DNA, and 5 x 106 cpm/mL nick-translated box probe (the box probe was a 229 base pair Pst 1-Barn HI cDNA fragment from the cloned full length cDNA of the human bax gene, a generous gift of Dr. Stan Korsmeyer, HHMI, Washington Univ., St. Louis). The filters were washed first at room temperature with 2x SSC and 0.1% SDS, and then in 0.I % SSC, 0.1% SDS at 56 oC with three buffer changes. Kodak Xomatic AR film was exposed to the filters using intensifying screens at -70 ~C. In summary, Ioa_~ was indeed detected in the cells, and treatment with ZCC caused an up- regulation in its expression. Tills is the first time that box has been characterized in breast cancer cells. Phosporima~ler analysis, shown below, reflects the time- dependent up-regulation of the box s~gnal (the 2 h time point was contaminated with protein). 5 0 ~.7,-: i 9£
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~000 ~000 ~000 I000 0 ~e (hour ~s) Summary ofprefiminary studies. When normal breast cancer cells become neoplastic, their cell cycle kinetics and proliferative pattern are drastically changed, especially with respect to the levels of available estrogen. Such cell populations either undergo programmed cell death or proliferation, the latter allowing some cells develop the neoplastic state. Previous studies by various investigators have shown that breast cancer cells, especially those that retain estrogen sensitivity, are not generally apoptosis proficient. The use of ZCC as an apoptosis inducing agent is important in this context. The present studies have shown that the breast cancer cells possess a novel apoptotic pathway that is cell cycle-dependent, protein synthesis- dependent, and is displayed by 50 Kb DNA fragmentation that occurs without the formation of nuclear apoptotic bodies. Further studies of breast cancer cells of differing levels of ER content/E2 sensitivity other apoptosis-inducing agents will assist our elucidation of the origin, development, and treatment of human breast cancers. Experimental design and procedures. The induction of apoptosis, determined from both the ability to cause morphological alterations and early cleavage of DNA into large (50-700 kb) fragments will be determined in MCF-7, MDA-MB-231, and MCF- 7/LY2 cells in vitro, the latter by quantitative field inversion gel electrophoresis, hybridization with the 32P-labelled alu I probe, and phosphoimaging techniques. Beginning with Analog II and ZCC, selected compounds will be studied to correlate their induction of apoptosis with their inhibition of proliferation of the cells, as well as with their cytotoxicities by clonogenic assays. The kinetics of these processes will in turn be correlated with levels and expression of p53, bax and bcl-2, measured by PCR and immunological techniques. In later experiments, after the endpoints have been correlated, we will insert one or more of the genes into the cells to determine if molecular biological treatments will be useful in the manipulation of the cells' apoptotic machinery. MCF-7 (28th passage), MCF-7/LY2 (both and obtained from Dr. Marc Lippman of the Lombardi Cancer Center at Georgetown University), and MDA-MB-231(obtained from ATCC) cells are routinely grown as monolayers in IMEM (no phenol red) with 5% heat inactivated fetal calf serum and 4 mlU/mL human insulin in 175 cm2 flasks at 37 oC in a 5% CO2-enriched humidified atmosphere. The complete medium is sterilized by filtration through a 0.20 pm filter. The cells receive an additional media change priortothe next treatment. Cells arerout,nely screenec~formycoplasma contamination. Cytostatic activity of the test corn,~ounds is measured by determination of growth curves for cell lines Cells (105 per well) are plated in triplicate in 6-well plates in IMEM with 5% felal calf serum (dex!ran-coated charcea!-stripped in the case of E2- responsive cells). Aftera24 h ar~acbmeni pencd,~he cels are grown in the pre~er~ce 502,741
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3~ of the test compound (in > 999:1 meo:ium-DMSO) for 0 to 144 h. The concentrations of drugs used is 0 and 10-9 to 10-4 M. At the time i~oirJts 0, 6, 12, 24, 48, 72, 98, 120 and 144 h, medium with nonadherent cells is removed and adherent cells are detached by a stream of medium (expeiIed from a syringe) or with trypsin. It shouId be noted that in the cases of MCF-7 and MCF-7/LY2 cell Ifnes, detached cells are a common occurrence even in healthy/untreated cultures, and are not a reliable indicator of on- going apoptotic processes. Remaining adherent cells are gently scraped from the plate with a teflon spatula. The medium and dWodged/scraped cells are combined, centrifuged, resuspended in HBSS, and an aliquot is mixed with an equal volume of 0.08 % trypan blue in HBSS. After a 10 rain period to allow for dye uptake in nonviabIe ceils, dye exclusion by viable ceils is determined microscopically using a hemacytometer. Cytotoxic activity is determined by a clonogenic assay. Cells in HBSS that were not used for counting from the above experiments, are plated in IMEM in with 10 % fetal calf serum in triplicate in 6-well plates at Iimiting dilution (500-10000 viable cells per wetl). The concentration of test drug for cytotoxicity evaluation is typically chosen from a concentration that shows a weak (10-40%) antiproliferative at time points o! 96 h and beyond. It is sometimes necessary to add 2000-10000 gamma-irradiated and killed 'seeding' cells at this point to promote attachment. After4 to 14 days of growth, depending on the growth rate of the specific cell line, cells are fixed with 25 % methanol, then stained with crystal violet. Colonies of > 50 cells are counted by microscopy. The clonogenic ability of the untreated cells is determined. Results of the drug treatments on clonogenic capacity are expressed as the fraction of the clonogenic percentage of treated cells vs. untreated cells. DNA fragmentation will be studied at selected (from morphological analyses as outlined above) timepoints and xenobiotic concentrations by QFIGE. After visualization by UV trans-illumination and photography, the DNA is nicked with UV light and transferred to a nylon membrane with alkali (0.4 N NaOH, 1.5 mM NaCI, 2-2.5 days) by capillary action. The membrane is neutralized in 0.5 M Tris, pH 7.0 and washed 2x with 0.15 NaCI, 15 mM Na citrate. The transferred DNA is hybridized for 16-24 h at 42 oC with 32P-o~-dCTP-labelled alu I sequence probe, the membranes are washed with buffered detergent, and are analyzed on a Phosphorlmager. Resulting luminescence is collected, transformed to 16-bit data and analyzed. Translational products of p53, bax and bcl-2 will be detected using commer- cially and collaboratively available antibodies to the proteins by immunohistochemical methods. The transcripts of these genes will be detected and quantilied by hybridi- zation and dot or northern blot analysis. RNA will be isolated and probed as described above. 8-Actin will serve as the internal control. Low copy numbers will be detected by adaptation of RT-PCR methods presently runnin9 in our labs. Appropriate primers are either available (Clonetech) or will be synthesized at the Univ. Pittsburgh DNA Synthesis Facility after analysis and Ioc.ation of appropriate sequences using the OLIGO primer design software from National Biosciences. Isolated RNA will be reverse transcribed with AMV r~ at 42 oC for 30 rain in the presence of deoxynucleo- tides and 3'-primer oligonucleotide, cDNA (0.3 pg) is mixed in 50 ~L PCR l:uffer with 5' and 3' primers and dNTPs, overlaid with mineral 0il, an{j subjected to 30-35 cycles of AT (£4 cC for 1 min, 55 oC for 1 rain, then 72 cC for 1 mirl). £roducts will be a~.alyzed by 10% PAGE after hybridzation with ~2P-labe ~ed probes, cDNA coqtroI amplifications w~tl be performed on G-actin w~th at RNApreparat~ons. 502,72.1 94
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~of. Day has a 740 ~q. f~ IaboratoD" at ff~e Center for Enxdronm~nml ~d Occupafion~ lab ~ a biolo~¢~ ~b~¢t rand tbr¢~ COz ~.¢ub~:or for ¢¢H ¢M~¢ e~ent~, ~d ~'o ~djoin~g 220 sq. ft. labs f~ MS ~d N~,~ cquipm~nh Sh~¢d t~dIifics ~¢lud¢ a 5~ sq. f¢ cen~ ins~ment la~rato~. PcrsonM ¢ompmers wi~ high quMib' pfintcm ~¢ av~abl¢, ~¢ Univ. of ~. hm~ V,~S ~d ~ clusmm avMlabI¢ by ¢~¢mct, as w¢~ as access to ~¢ ~tmb~gh Supercompufing Cm ~d its ¢~¢~s¢. T~¢ Dept. End*on. O¢¢up. HcM~ has scvcrM DEC, San SGI wor~mfio~s for ~ap~¢s m~ipulafio~ ~8 mol¢¢ul~ m~¢H~g. ~of. Day h~s a 120 o~e¢ ~ dcs~ t¢Icphon¢, m~em, sh~¢d facs~l¢ ma¢~¢ ~d cthcmct l~¢s, ~ksh¢lvcs ~d ~ng cabinet< Dr. Balachan~ h~ access to a 80 sq. ft. office s~l~Iy eq~p~. Ci¢fi¢~ ~d office sup~n s~f ~ av~Iab~e. ~quipment: A~osphcfi¢ prussic io~afion mass qua~l¢ mass r~ge 10 to 2400 ~z-mMfiply ch~gcd mass rage >1~,0~ mu; Paced 5~IA GC-MSD-EI ~d PC[ capab~fies; mass r~g¢ 10 to 650 ~u; V~ ~-2~ wi~ V~ opcm~g system, mul~u¢le~, 2D capabilities; HcwleR-Pack~d 1~ Series ~ ~LC ~ Diode A~y Dct¢ctor-fulI s¢~ ~ 2~-6~ ~m detection wi~ ~~ Che~tafion;. Scintillation co,tern, rcMgcrated ccnMfuges ~d ul~cenMthg¢, ineubato~, l~min~ flow hc~s, baJan¢¢s, mi~ofitcr plate reader, Coul~¢r counter, flow ¢~om~ter, ~IS, fluorescence and other ~os¢opcs, ¢l¢¢~ophorcsis cq~pmcnt, d~oom, 5. BUDGET JUSTIFICATION- Use this space to explain specific needs for items described on budget pages. All costs are incremented at a 6% increase per year. Billy W. Day will be the principal investigator for this project, and will be responsible tbr the overall research direction, guidance and cohesion, as well as all progress reports to the CTR. Dr. Day will directly participate in all aspects of each of the specific aims in this proposal. Dr. Day will dedicate at least 20% of his professional effort toward this project. A request is made to cover 10% of Dr. Day's salary. Raghavan Balachandran will be a Research Associate under Dr. Day's supervision on this grant. Dr. Balachandran is a seasoned cell biologist with outstanding experience and technical knowledge in cell culture. He has been working in our department since last year, performing studies on apoptosis in human breast cancer and ovarian cells, measuring/quantifying DNA fragmendon and apoptosis-reated events. A request is made for 75% of Dr. Balachandran's salary as he will carry out the studies described essentially full-time throughout the entire study period. He will be responsible for all aspects of the cell culture, treatment, biochemical and molecular biological studies described in the proposal. Fringe Benefits: Faculty (Err. Day) -- calculated at 33.7% until 07/01/96, at which point the rate increases to 35%. Staff (Dr. Balachandran) -- calculated at 37.7% until 07/01/96, at which point the rate increases to 38.34% as per University guidelines. Suotflies: Requests are made to cover expenses related to 32p. labelling/hybridization reageni~ and reagents for QFIGE experiments, cell culture supplies (medium, serum, CO2), generation and characterization of agents for testing, biochemical supplies and molecular biOlogieal probes/experiments. 6. APPENDIX: Place the appendix materials after the original and each copy of the applieatioa form as indicated in the Iasmmfions for New Applieafiom. a. Biographical Sketches of the professional personnel to be associated with the project Each sketch should be NO MOP.E THAN TWO (2) PAGES. The N'I]-I format is acceptable. The P.I. should include and indicate by an a.~terisk tho ~ (5) mo~ significmat publications whether or not they relate directly to tlfis application. b. Supporting material (such as letters ofcollaborationL c. Copies of not more than FD, q~ (5) ofth*, applicant's publication~ or manuscripts that are pertinent to the project 7..ad3STR.A.CTS of PUBLICATIONS : Only o*~e set ~ required. See M~ctio~ for New App~ca~o~. Sub~t ONE PHOTOCOPY of ~e abs~act page of ~aeh "pe~ent pub~fion" ~cluded m ~ appen~x (6 c.) above; For eac~ m~uscnpt, submit a single :om~si~ pa~e ~at includes augers, title, jo~, zbs~ct ~d pubh,~uon s~s ~fz.r ex~ple, '~ubm~ned for p~bhcatiz,n"). C 0 :,-F ID E: 50!,741 95
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SAanss. Give % vans ~:"~n ifno Billy W. Day, Ph.D. R. Balachandr~ Te~c~1 Supp~ Fringe Benefits B. Consumab1~ ~upplieg (by major category) Radioehemieals 1,000 Chemicals 500 Cell Culture 7,000 Chroma=ography supplies 500 Biochemicals 500 Compressed gases/cryogenics 500 C. Other Expergses (itemize) Printing & Publishing : i00 Mailing 50 Telephone/Fax 50 Space Rental 6,932 D. INDIRECT COSTS ( 15% ofA + B + C). 10% 5,545 75~ 30,000 13,320 A. Sa/aries Subtotal 48,865 B. Con~mmables Subtotal. 10,000 C. O~herExpenses Subto~.l. A+B+C Subtotal 65,997 7,132 D. ~direaCos~ 8,860 B. PermanemEquipment(itemize) Bio-Rad QFIGE apparatus E. Pormanemt ERuipm~nt Subtotal 5,000 Indi~te hero and on :Page 1. F .............................................................................................. : .............. TOTAL REQUEST. 79,857 Indi~to here and oa ]?age 1. 8. PROZECTED BUDGET AMOUNTS: BUDGET PERIOD Sa/arie~, Supplies and Oga~r exp~n~es 68,~98 70~952 9,235 9,603 *You may not use CTK P¢.=ds ~o purchase permanent eqmpment m the terminal grant yea:. TOTAL 77,733
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Anfitumor Mechanism of Dichloro- cis-diphenylcyclopropane Nitric Oxide-Induced Cell Injury: MoIecuIar Mechansims Predoctoral Training in Breast Cancer Biology and Therapy Free Radical Activation of VP- 16/ Tope ~ Interactions Role and Mechanism of Apoptosis in Breast Cancer Cells lxtduced by Novel Chemotherapeutic Agents ,VIH, 5 R01- 2A57288 NEH, 5 R01- ~qL51469 JSAMRDC MBS-BCRP amaer. Cane. ~oe.-DHP-E Pitt. Cane. Br. Cane. $243,553 $65,162 S754,400" $311,819" '.5 lst.$19,900 t. Available toYoa $78,473 $18,812 $189,200" $101,272" $19,900 Identify and describe any overlap of this app~i'eation with the above grants: 97101t31 98/06/30 98/08/3I 97106/30 96/06/30 The last listed is a seed money grant aimed at experiments described in the preliminary results of thi~ proposal. Otherwise, there are no cases of scientific or budgetary overlap. (*-Co-Investigator: all funds not available to B. W. Day) Indicate the total annual ftmds available to you this year for all research projects under your supervision. $ 129,385 PENDING OR PLANNED Title of Project New Mass Spectral Methods in Human Exposure Dosimetry Novel Anticancer Agents.. hatemational Training in Environmental Health ~P1300 LC/MS/MS S01ECeS (give grant numbers) ~ PA-HERL ] m. Cane. So ~ I/-I, Fogarty qlH, BRSIG Total Value of Grant (aireet costs) $345,364 ~.$319,406 $735,910" $350,000* Avg. Annual Amount Available to You $125,000 $112,500 $37,500* $0" Total Duration (give iaelusive ~htes) 95/09101 to 98/08/31 01/01]96 tO 12/31/98 09/01/95 tO 08/30/O0 07[01/96 tO 06130/98 Idemify and describe any overlap of this appIicat~on with the above project. Dr. BaIachandran is listed for 100% effort on the second propose. Otherwise, there are no cases vudgetary overlap. If both that ~nd the present proposal are funded, a ~tfitable replacement for him sill be found for the ACS grant. There are no cases of scien~ic overlap. CQ:£FFDE NTEAL ~'}3741 97
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BiographieaI Sketch Billy W. Day A~i~t2_nt Professor 09/10/61 Oklahoma Ci~, University, O';-da. Cip.,', OK B.S. Universi~, of Oklahoma, Okla. City, OK Ph.D. Massachusens Institute of Technol, Postdoc Cambridge~ MA FelIow 19S2 Chemis~'/Biolo~' 198S Medicinal Chem. I98S- Chemistry/ 1991 To.~Scology Positions: 1986 1986-1988 1988-1991 1991 - present 1991 - present 1992 - present 1991 - present Forensic Chemist, Serology and Drag Analysis Division, Oklahoma City Police Department, Oklahoma City, OK Adjunct Professor of Chemistry, Department of Science, Oklahoma City Community College, Oklahoma City, OK Postdoctoral Fellow, Department of Chemistry and Division of Toxicology, Massachusetts Institute of Technology, Cambridge, MA Member, Pittsburgh Cancer Institute, Molecular Carcinogenesis and Experimental Therapeutics Groups, Pittsburgh, PA Member, Drag Metabolism Group, Center for Clinienl Pharmacology, University of Pittsburgh, Pittsburgh, PA Affiliated Researcher, Div. of Toxicol., Dept. of Chem., and Harrison Spectroscopy Lab, Mass. Inst. of Technol., Cambridge, MA Assistant Professor, Departments of Environmental and Occupational Health and Pharmaceutical Sciences, University of Pittsburgh Graduate School of Public Health and School of Pharmacy, Pittsburgh, PA Hon ors: National Institutes of Health, MIT Hazardous Substances Management Program, and American Cancer Society Postdoctoral Fellowships, 1988 -91 John B. Bruce Memorial Outstanding Graduate Student in Medicinal Chemistry Award, University of Oklahoma Health Sciences Center, College of Pharmacy, 1988 American Institute of Chemists Outstanding Senior Chemistry Student, Oklahoma City University, 1988 National Science Foundation Undergraduate Summer Research Fellowship (Molecular Biology/Physical Biochemistry), Oklahoma State University, 1981 Selected Publications (total of 31 peer-reviewed publications): Gorbunov, N. V.; Osipov, A. N.; Day, B. W.; Zayas-Rivera, B.; Kagan, V. E.; Elsayed, N. M. Reduction of Ferrylmyoglobin and Fen-ylhemoglobin by Nitric Oxide: A Protective Mechanism Against Fcrryl Hemoprotein-Induced Oxidations. Biochemistry... 1995, 34, 6689-6699. Goldman, R., Stoyanovsky, D. A., Day, B. W., Kagan, V. E. Reduction of phenoxyl radicals by thioredoxin resuks in selective oxidation of its SH-grot~p~ to disulfides. An antioxidant function of thioredoxin. Biochemistry 1995, 34, 4765-4772. *Day., B. W., Jonnala.gadda, S. S. Synthesis af Z. and E-[2.3-2H2] and [233H2]-l,l-dichloro- 23-diphenyI©'clopropane (2H- and -~H-A nalog I[ and is Ira, isomer). J Labelled Com..r). Radiopharm. 1995, 36, 73-78.
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Day, B. W., Skipper, P. L., Zaia, J., Singh, K. and Tannenbaum, S. R. Enmnfiospecifi~q; of covalent adduct tbrrnafion by benzo[a]pyrene anfi-diol el:oxide with humma serum albumin. Chem..Re~. To×icol. 1994,'7, $29-835. Ryan, L. K., Jin, R., Boggs, S. S., KaroI, M. I-L and Day, B. W. A mouse model t'or assessing endotoxin involvement in the lung inflammation and cytoldne prc~:lucfion resulting from inhaled organic dust. Inhal. Toxicol. i994, 6, 485-499. *Kagan, V. E., Yalowich, J. C., Day, B. IK, Goldman, R., St<vanovs~, D. A. and Gantchev, T. Ascorbate is the primary reductant of tlw phe'~o.~'l radical of etoposide (VP-16) in the presence of thiols both in cell homogenates and in model systems. Bfochemist~, 1994, 33, 9651-9660. *Hossain, M. B., van der Helm, D., schmitz, F. J., Pordesimo, E. 0., Magarian, R. A., Meyer, K. L., Overacre, L. B. and Day, B. W. Molecular structures and conybrmational studies of triarylc3'ctopropyl a~d related nom'teroidal antiestcogens. J. Med. Chem. 1994, 37, 1670-1683. Day, B. W. and Singh, K. Fluorescence spectroscopy in the analysis of hemoglobin adducts. Methods Enzymol. 1994, 231,674-681. Jin, R., Day, B. W. and Karol, M. H. Toluene diisocyanate protein adducts in the bronchoalveolar lavage of guinea pigs exposed to vapors of the chemical. Chem. Res. Toxicol. 1993, 6, 906-912. Du, L., Hossain, M. B., Ji, X., van der Helm, D., Magarian, R. A. and Day, B.W. Structure of 1,1-dichloro-2-(4-methoxyphenyl)-2,3-diphenylcyclopropane. Acta Crystallogr. 1992 C48, 887- 891. Day, B. W., Sahali, Y., Hutchins, D. A., Wildschutte, M., Pastorelli, R., Nguyen, T. T., Naylor, S., Skipper, P. L., Wishnok, J. S. and Tannenbaum, S. R. Fluoranthene metabolism: human and rat liver microsomes display different stereoselective formation of the trans-2,3- dihydrodiol. Chem. Res. Toxicol. 1992, 5, 779-786. Day, B. W., Skipper, P. L., Rich, R. H., Naylor, S. and Tannenbaum, S. R. Conversion of a hemoglobin cz-chain aspartate(47) ester to N-(2,3-dihydroxypropyl)asparagine as a method for the identification of the principal binding site for benzo|a]pyrene anti-diol epoxide. Chem. Res. Tox~col. 1991, 4, 359-363. Day, B. W., Skipper, P. L., Zaia, J. and Tannenbaum, S. R. Benzo[a]pyrene anti-diol epoxide covalently modifies human serum albumin carboxylate side chains and imidazole side chain of histidine(146). J'. Am. Chem. Soc. 1991, 113, 8505-8509. *Day, B. W., Magarian, R. A., Pento, J. T., Jain, P. T., Mousissian, G. K. and Meyer, K. L. Synthesis and biological evaluation of 1,1-dichloro-2,2,3-triarylcyclopropanes as pure antiestrogens. .I. Meal Chem 199t, 34, 842-851. *Hossain, M. B., Wang, J. L., van der Helm, D,, Magarian, R. A., Griffin M. T. and Da?,.,, B. ~K Structural comparison of a gem-dichtorodiao'Icvclopr,:,pane antiesrrogen and three of its derivatives. Act~ CO'.~taltogr. I99t, B47, 511-521. Jankowiak, R_, Day, B. W., Lu, P., Doxtader, M. M., Skippcr, P. L, Tannenbaum, S. R. and Small, G. J. Fluorescence line aarro~ing s~ectral analysis of in vivo hemoglobin adducts of benzo[a]p;/rene diol epoxide: comparison ~o synlheticana!ogues. J. Am. Chem. Soc. I990, 5866-5869.
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THE COUNCIL FOR TOB.%CCO 19_ESE~%RcH-U.B..~., INC. April l0, 1995 Billy Day, Ph.D. University of PittsbuNh Dept of Environmental and Occupational Heaith 260 Kappa Drive Pittsburgh, Pennsylvania 15238 Re: 5567 Dear Dr. Day: Your recent inquiry concerning support by this Council of a proposed research project has been considered by the Executive Committee &the Scientific Advisory Board. The Committee expressed interest in examining a more detailed plan of the proposed investigations. I takepleasure in enclosing the necessary forms and relevant literature pertaining to the filing of a formal application. The next.deadline for submitting proposals is May 31, 1995. You understand, of course, that no co .mmitment is implied at this time concerning the final action of our full Seientifie Advisory Board on the complete proposal. Sincerely yours, Harmon McNlister HC~I s~ rrd~
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THE COUNCIL FOR TOBACCO RESEARCH NE~ YORK, N.Y. 1002-2: GEORGE A. HASHI~ PH.D. .- P~~ ~P~CA~ON ~UA~ON ':" DaLe: FEB. 14, 1995/ Reviewer 1: ~~.-/ . Reviewer 2: ' " / RE: Preliminary Application Number: 5567 Applieant's Name and Degree(s): Institution: Title: RI~'IF~ UNIVERSITY OF PITTSBURGH ROLE OF MECHANISM OF APOPTOSIS IN BREAST CANCER CELLS I. Please review this application and indicate your scoring below.. A return envelol~ is provided for your convenience. 2. We expect the top 30% of Preliminary Applications will.be encouraged to submit Full Applications. ~1 T~p 10% ~q~op 20% t-l~Top 30 %" Top 40% Top 50.%.. • ~low ~'o~ Reviewer's comments: " '-/ ~~.,,...~,,. ~ ,. ~.~,,,.r" " 5037~2@1 m IIII IIII [1~ 1 ............
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THE COUNCIL FOR TOBACCO RESEARCH- U.S.A., INO. .... ~UI~I~DgTI~O ]~IOMHDICAL IHVE3TIOATIO~ O~O~OE A. HASHIM, PH.D. PRELIM]NARY APPLICATION EVALUATION Dat~: FEB. 14, 1995 Reviewer 1: Reviewer 2: RE: Prclimin~y Application Numbcn Applicant's Name and Dcgrce,(s): Institu.tion: Title: 5567 UNIVERSITY 0F.-~ITTSBURGH ROLE OF MECHANISM OF APOPTOSIS BREAST CANCER CELLS 1. Please roviow this application and indicate your scoring below. -~ A return envelops is provided for your conveaieacc. 2. We expect the top 30% of P~cIiminaty Applications will be encouraged to submit Full Applications. Top 10% ~ Top 40% ~T oP 20% ~ Top 50~: op 3o~ IZI ~ow ~o~ Rcviswer's 60374202
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THE COUNCIL FOR TOBACCO RESEARCH • - NEw 7OR~, N.Y. 1002.2. GEORGE A. HA~HIM, PH.D. ' A3~OCIATE RESEARCH DIRECTOR p~~Y ~P~CA~ON HV~UA~ON : Dam: FEB. 14, 1995 Reviewer h DR. LYNCH Reviewer 2: DR. BRENI~AN 5567 BILLY DAY, PH.D. UNIVERSITY OF PITTSBURGH ROLE OF MECHANISM OF APOPTOSIS IN BREAST CANCER CELLS i. Please reviow this application and indicatv your scoring below. A mtum envelop{) is provided for your conve.ience. 2. We expect the top 30% of Preliminary Apph'cations will b~ encouraged to submit Full Applications. Top 10% Top 20% Top 30% Top 40~ Top 50%.. Bdow . 11eview~r's comm=nts: Signature: 50374203
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University of G~'~dz~zte ~b~'b t :, ! of P;dT/ic H~zI~b 412-£57-5~!3 February 13, 1995 Arthur D. Eisenberg, Ph.D. Associate Research Director The Council for Tobacco Research, Inc. 900 Third Avenue New York, NY 10022 (212) 42t-8885 Dr. Eisenberg, I have been studying the effects of xenobiotics on breast ceils for several years. Recently, my lab has noted the induction of apoptosis by an agent we have been studying in order to determine its molecular mechanism of action. We were intrigued to note that the interactions and roles of genes involved in regulation of apoptosis, particularly bax, have yet to be tully elucidated in breast cancer. We propose to perform studies that will allow us to elucidate the role of gene regulation in the apoptosis of breast cancer cells. Sincerel~y,. Billy W. Day, Ph.D. Assistant Not~ssor
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Cover Letter t. Name, De~ee5 and Acaderrfic Title: Billy W. Day, Ph.D., Asfistant Professor of MolecuIar Toxicology m'ad MedidnaI Chemi_~t~', Departments of Environmental & Ck:cupational Health and Pharmaceutical Sciences, Graduate Sctzool of Public Health and Sch,z~l of Pharmacy, University of Pittsburgh. 2. Title of Proje.e.t: Role and Mecb:misrn of Apoptosis in Breast Car~cer Cells 3. Phone Number: 412-967-6502 Facsimile: 412-624-1020 4. Duration of Project: Three (3) years 5. Estimate of the Direct Cost for the First Year: $74,000
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Role and Mechanism of Apoptosis in Breast Cancer Cells Prir, cipal Im'estigat~r: Pr,~ Bi~v W. D~;'. C~-In~:stigat_,_-,r: Dr. Ragk,~'an Balact.,..ar,~ra~ 1. SI~ecifie ~ims The objectives of this research are to define the role and mechartism of apoptosis in clonally different human breast cancer cells. Sped.tic-ally, we vddI t~t probe human breast canzer cell lines of differing hormonal sensitivity with agents studied in our lab that we have preliminarily shown to induce apoptosis in the cells. To be examined are estrogen receptor (-ER+)/¢stradiol (E2) re~qz~onsive, ER+/E2unrespon~ve, ~d ER negative fER-)/E2 unres13onsive human breast cancer cell lines MCF-7, MCF-7/LY2, and MDA-MB- 231. Concurrently ~e will study the interactions of, and elucidate the roles of, three specific genes, bax, bcl-2 and 1353, that have beenshown to regulate apoptosis. Bear and its protein product, ewen though recently shown tb plco' em integral role in apoptosis, have vet to be studied in breast cancer. The results frorn our studies will ~Iow us to elucidate the role of gen~ regulation in the apopto~s of breast cancer cells. Therefore, the specific aims of this project are: 1. To investigate the nature of the apoptosis in MCF-7; MCF-7/LY2 and MDA-MI~-231 human breast cancer cells. 2.. To identify the role of the p53, bet-2 and bz~x gene products in these cell lines and correlate their relative expressions with the apoptosis induced by xenobiotics. 2. Background and Significance Apoptosis and the genes involved. The role of apoptosis, programmed cell death, in cancer biology has recently gained both importance and widespread attention. Many toxicants reduce the proliferative potential of cancer cells and di~a-upt their passage though the cell cycle. A xenobiotic can initiate the signals necessary for induction of apoptosis. The e:dstence of a genetic program in the cell during a drug-induced death pathway suggests that modification in the expression of the responsible genes could have a'profound effect on the development of cancer. A sequential or concomit~t biochemical/genetic modulation that augments the initial steps of the cascade should fuel the apoptotie event induced by anfiproliferative xenobiotics. The genes bcl-2, bax, and p53, and their expressed protein products, have been linked to programmed cell death. The wild type (wt) p53 gene product induces apoptosis, while the bcl-2 gene product can inhibit apoptosis triggered by wt p53. These activities of both genes have been noted in breast, both clinically and in cell culture. The bcl-2 gone encodes for a mitochondrial protein that is localized to the luminal cells of normal breast, considered to be the origin of malignant breast disease. Several studies of breast tumors have shown that bcl-2 levels are positively correlated with ER and progesterone receptor (PR) positivity. The bci-2 product is generally associated ~vith small, ER(+), slowly proliferating, p53 negative tumors, and is not generally associated with large, ER(-), rapidly proliferating, p53 positive breast tumors. Hence, MCF-7 cells should be more resistant to apoptosis-inducing drugs than MDA-MB-231 ceils, Over-expression of the bax gene has been shown to counter the death repressor activity of bcl-2 via the formation of a heterodimeric protein. The bax gene product is thus a dominant inhibitor of the bcl-2 protein. The ratio of bcl-2 to bax determines the survival or death of a cell to an apoptotic stimulus. Bax is a relatively recently discovered and, to date, there have been no reports on the involvement of bear. in breast cancer. Although these three genes have been associated with the apoptosis process, their relationship to xenobiofic-induced cell death is not dear, especially in the case of breast cancer. The s~rreening of different human breast cancer cell lines with differing estrogen sensitivity for these genes during the, xenobiotic- induced apoptosis proces~ will provide useful information on cell death and cell proliferation of these ceils, and may provide important insight into the development and progression of breast c~mcer. 3. Preliminary Studies Generally, studies on apoptosis involve the use of chemic~ modifiers of cel/ular macromolecule synthesis or signal transduction, such as cytokines, interleukins, and/or bTowth factors. We have noted a chemically simple agent that induces apoptosis in breast car, oct cells, and propose to use it as the first of several initiating xenobiotics for biochemical elucidation of apoptosis. Novel anti-breast caacer agents i~uciag apoptosix. We have I~en studying Z- l, I-dichloro-cis-2,3- diphenylcyclopropane (1 }, a rigid analog of stilbenic estrogens/an.:iestrogens, fo,and to have antiestrogenic properties when mmlvzed b.v merotrophic and antiutero:rophic assays in immature female mice: ir had no estroge~sc activity and _qo~ses_.ed weak h,,- ,-.,~,- ~ _ ~ ..... ~ ,odu. ~ble zntimeron'ophic effects An intneuine prepcrb,
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no:cd for cc, mlso~nd I ~as that i~ had dmos~ no ~finiq; for ~¢ cI:s£¢J (~gh dfiniq;-low capaciu' - ~;?¢ r) ~t ute~ne es~ogen receptor, ~xifl~ a reladve b~d~g ~niD' les~ ~ 0.01% of ~at exhibffed by E2. Comp~~d i was later shown ~ ~"o ~erent sm~:s to have acfviB' cemp~ble to ~exifen ffA~ a~,~ ~. ~e ~o~one @rola:~)-degend:n~ 7,12-~m:~yi~nz[al~cene ~A)-~du:ed rat man~,, minor m~el in ~dvo, ~d to be more effective ~ T;~M ~ r~uc~g ~e ~cidence of new minors d~ng long te~ dosing. Compound I w~ ~so active ag~nst ~e D~A-4 me~mfic, ~spI~mble mt m~m~, minor line in ~Jvo. Addi~onN si~fie~t prop~es of ~ ~m~d include im low app~ent toxicity, e.g., ~e LD5:, in ~ce is 3683 mg&g 2~ h after a p.o. do~e, the idenficNIy dete~fined LD~0 in ra~ is ~so over 3 ~g, ~d ~e finding ~at it is not mutagenic to S. Gek'm~izm smain %~677 at concen~a6ons r~ging ~om 10 ~I to 1 ~M, bo~ wi~ ~d ~out a meta~H¢ ae~va~ng system. We have r~enfly noted ~at 1 ~d i~ ~jor breast c~cer ceH meta~te ae active agNnst breast cancer cell lines representing the fur spec~ of es~ogen sensifi~ not~ ~ human remora. Also, we have noted ~ @parent &ruction of @op~osix (fox,ion qf l SO bp DNA la~ers ~er 96 h e~osure ) ~' the compoun& in ~kese cell lines. We have Nso recently ~gun ~e study of apoptosis induc~ by xenobiofics i,n human ov~ ~ulosa cells. We have develop~ and utNized the t~h~ques necess~ tbr Mne~c anNysis of ~e genetic and ceRN~ steps ~ apoptosis ~rough these stu~es. Except to note the t~ct that we have seen siva ho~onN ka]uences opera,rig in ov~ and breast c~cer ceils, we will not describe ~ese studies here. Note that we ~e not proposing to develop 1 or my o~er cheMcNs in this submission -- our obsen, ations x~th it have, however, piqued o~ scientific curiosity so that we wish to define the bioche~cN mechanisms of apoptosis in ~e breast cancer cell lines. 4. Experimental Design and Methods The induction of apoptosis, determined from both the ability to cause DNA laddering and early cleavage of DNA into large (50-700 kb) fragments will be determined in MCF-7, M_DA-MB-231, MCF- 7/LY2, in vitro, the latter by quantitative field inverMon gel electrophoresis, hybridization with the 32p_ labelled aIu I probe, and phosphoimaging techniques. Beginning with I, selected compounds will be studied to correlate their induction of apoptosis with their inhibition of proliferation ofthe cells, as xvell as with their cytotoxicities by clonogenic assays. The -k_inedcs of these processes will in turn be correlated with levels and expression of p53, bax and bcl-2, measured by PCR immunological and techniques. MCF-7 (28th passage), MCF-7/LY2 (both and obtained from Dr. Marc Lippman of the Lombardi Cancer Center at Georgetown University), and lVIDA-M_B-231 (obtained from ATCC) cells are routinely grown as monolayers in DiEM (no phenol red) with 5% heat inactivated fetal calf serum and 4 mlU/mL human insulin in 175 cm2 flasks at 37 oC in a 5% COx-enriched humidified atmosphere. The complete medium is sterilized by filtration through a 0.20 gm filter. The cells receive an additional media change prior to the next treatment. Cells are routinely screened for mycoplasma contamination. QCtostatic activity of the test compounds is measured by determination of growth curves for the three ceil lines. Cells (10,000 per well) are plated in triplicate in 6 well plates in IMEM with 5% fetal calf serum (dextran-coated charcoal-stripped in the case of E2-responsive cells). After a 24 hour attachment period, the cells are grown in the presence of the test compound (in > 999:1 medium-DMSO) for 0 to M4 hours. The concentrations of drugs used is 0 and 10-9 to 104 M. At the time points 0, 6, 12, 24, 48, 72, 96, 120 and 144 hours, medium with nonadherent cells is removed and adherent cells are detached by a stream of medium (expelled from a syringe) or with trypsin. It should be noted that in the cases of MCF-7 and MCF-7/LY2 cell lines, detached cells are a common occurrence even in healthy/untreat.e~, cultures, and are usually not a good indicator of on-going apoptotic processes with these lines. Remaining adherent cells are gently scraped from the plate witla a teflon spatula. The medium and dislodged/scraped cells are combined, centrifuged, resuspended in HBSS, and an aliquot is mixed with an equal volume of 0.08 % trypan blue in HBSS. After a 10 minute peried to allow for dye uptake in nonviable cells, dye exclusion by viable cells is determined mi~o~opic:dly using a hemozytometer. Viable celt number is cMculated by multiplying total cell number by the fraction of cells excluding the dye. Cytotoxic activiv.v is determined by a clonogenic assay. Cells in HBSS, which were. not used for counting from the above experiments, are plated in IMEM in wdth 10 % fetal c~If ser0m in triplicate in 6- ~cll pla:es at limiting dilution (5C~3-1C~)tX) ~iablc cells per well ~. The concentration of test drug for cytotoxicity evMuation is typically chosen from a concentTation that shows a wok< (10-40%) antiproliferadve at time points of 96 hours and beyond. It is somer.imes necessary, to add 2000- II)ClX/ gamma-irradiated and .hilted' seedi~ g' cell s at thi; point to promote attachment. After foar ~o four~een davs of grove:h, depending on the ~owth rate of the specific cell line, cells are tixezl aith 25 % mc~hanol, the~ sta:.~ed with cn'stal v{o!et. Colonie~ 05 > 50 cells are counted by rrdcroscop.v. The c'.onegcnic ability' of the 5 0 ~, 7 4 2 0 7
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untreated cells is d~tc.~d~ Resul~ of ~e ~2g ~e~en~ on clonogenic cgpxci~~ ~e e,:pr~:s~d ~ ~cfion of~e clonogenic percentage of ~eated cells vs. un~mt~ cells. Cells undergoing apeptosis ~ow a sp~Nfie DNA ~enmfion pa~em when ~flyzed by ag~se gel elec~oph~resis. DNA ~iI1 ke an~v~d ar vmc,~,~ ~e pin~ ~ter ~e cells ~e ~eated wifl~ ~e che~c~ of ~t~est, inclu~ng to~ DNA. Adh~ent ~d non-~erent cells xe remov~ ~ p~ ~e cytostasis ~say protoN, w~hed 3x wi~ PBS or ~SS, ~d pelleted by cenMNga~on. The ce~s ~e lysed in either 0.75% (-)-n-~'l-~-glucop3~o~de, 1% N~-40 detergent, or 0.5G Na lauD, l sxcosine in 5 mM Tfis, pH 7.4, con~ning 5 ~M gDTA ~d 50 g@~ prominase K t~r 0.25-I h on ice. ~e ce~I d~bNs rind un~a~ent~ (actu~y, relatively un~ent~ DNA is pellet~ by cen~gafion, and the su~ematant is incubated ~ 5~ unim/n~ R~XrAase A for 0.5 h at 37 cC, 1 h at 50 ~C. ~e DNA in ~e sapemamnt is ~Nyzed by elec~ophoresis on 1.8-2% ag~ose cont~dng e~i~um bro~de in 0.5% ~E (89 mM Tfis, 50 mM ~fic aci~ 2 ~,I EDTA). 2~e gels ~e des~ned ~d ~e DNA is ~su~ed by UV m~s-illumina~on and photo~phed. A st~d~d DNA m~ker ladder (1(~ or I80 bp) is included in each gel for laddefing comp~son. For qu~fimfive field ~version gel elecmophoresis (QFIGE) stuNes, h~ested cells ~e suspended in 10 ~M Tfis ~uffer (pH 7.2) cont~g 20 ~ NaC1 ~d 50 mM EDTA at 50 oC. An equal volume of 2% InC~rt ~ose and solidifi~ plugs contNning 10s cells ~e made at 4 oC in Nsposable molds. The plugs ~e ~cubat~ at 50 oC overnight in 4.8 ~M Na dcoxvcholate, 1% Na la~,l s~cosine, I~ mM EDTA, pH 8.0 ~d 0.5 m~ proteinase K, then washed ~x wi~ 50 mM EDTA and 20 mM Tfis, pH 8.0. QFIGE is peffo~ed on 5 x 105 celts per lane on a FIGE Mapper Elecmophoresis System in 1.5% pulse field ceNfied ag~ose in 0.5% TBE. The elec~ophorefic p~ameters ~e: 12 rain continuous fo~md pulse at 150 V, followed by 30 h at 150 V using a 21% ~p from 0.9 to 30 see in the fonv~d direction and 0.3 m 10 sec in the reverse ~recfion at 10 oC. Gels ~e stain~ Mth ethidium bromide then visuNized by UV tmns-illu~nafion and photo~aphed. The DNA is nicked wi~ UV light and mansfe~ed to a nylon membrane wi~ alkNi 0.4 N NaOH, 1.5 mM NaC1, 2-2.5 days) by capill~ action. The membrane is neumalized in 0.5 M Tfis, pH 7.0 and washed 2x with 0.15 NaC1, 15 ~ Na cimate. The ~ansfe~ed'DNA is hybfi~zed for 1~24 h at 42 ~C wi~ 3zP-a-d~-la~lled alu I sequence pro~, ~e membranes are washed with buffered detergent, ~d ~e anNyzed on a PhosphorImager. Resulting lu~nescence is cMlected and m~sfo~ed to 16-bit da~ ~d ~Nyz~ by image ~Nysis qu~fificafion softw~e. Tr~slafionN products 0f p53, bmx ~d ~1-2 will ~ det~ted us~g co~erciNly and collabomfively avNlable ~fi~Nes to ~e proteins by i~ohist~he~cN me~ods. The ~anscfipts of these genes w~l be detected ~d.qu~fifi~ by hybfi~zafion ~d dot or no,hem blot anNysis. RNA will ~ isolated by hot phenol or gu~niNne tNcyanate.metho~, ~A Mll be denat~ in fo~adlehyde-fomaa~de-MOPS buffer ana sep~ated on ag~ose gel ~d m~sfe~ed to niffocellulose. ~e ~ters will ~ pro-hybridized and hybfi~Z~ in st~d cock~ls with a nick-manslated pro~. The filters ~e ~en washed ~d aumradi6~aphs Will be prep~ at -70 oC. ~-Acfin will sere as the internN conmol. Low copy num~rs ~I1 be detected by a~ptafion of RT-PCR me,otis presently mnn~g in o~ labs. Appropriate primers ~ either avNlable (Clonet~h) or Mll be synthesized at ~e Univ. Pit~b~gh DNA Syn~esis FaNlity ~ter ~Nysis ~d location of appropriate s~uences using the OLIGO primer design softw~e ~om NationN Biosciences. Isolated ~A w~ be reverse ~scfi~d wi~ AMV reverse ~s~ptase at 42 0C t~r 30 ~n in the presence of d~oxynucleotides and 3'-primer oligonucleofide, eDNA • ~ gg)is mix~ in 50 gL PCR buffer with 5 and 3 primers and dNTPs, overlaid with mineral oil, and suNected to 30-35 cycles of AT (94 oC for 1 rain, 55 oC for 1 rain, then 72 oC for 1 min). Noducts will be anNyzed by 10% PAGE ~ter hybfi~fion wi~ 3ZP-la~Hed pro~s, eDNA conmol ~plificafions MI1 ~ peffo~ on ~-acfin with NI ~A prep~afions. Translat~onal Potential of The Proposed Project . Bcl-2 overexpresNon represents a mechanism tbr the supeivN ofc~cer cells. We wffi dete~fine if breast cancer cells with differing ratios of bcl-2 to bmx show ~N~rent sensitivities to xenobiotics, reflected by ~e~ relative abilities to undergo apoptois. Cells ovcrexpressing bax can undergo apoptosis following an inifi~ stimulus. Since bmx counters the effect of ~I-2, o~ results c~ be used t~m~el and identify stcpwisc apoptosis and ceil proliferation in breast cancer. 50374206
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BiographicaI Sketch Billy W. Day Education Assistant Professor Oklahoma Cit3' University, Oklahoma City, OK University of Oklahoma, Okhhoma City, OK Massachusetts Institute of Technolo~,~ Cambridge, ~Uk Research and/or Professional Experience Positions: B.S. 1982 Chemislxy/Biology Ph.D. 1988 Medicinal Chem. Postdoe ] 988- Chemistry/ Fellow 1991 ToMcology 1986 1986-1988 1988-199t 1991 - present 1991 - present 1992 - present 1991 - present Honors: Forensic Chemist, Serology and Drag Analysis Division, Oklahoma City Police Deparm~ent, Oklahoma City, OK Adjunct Professor of Chemistry, Department of Science, Oklahoma City Community College, Oklahoma City, OK Postdoctoral Fellow, Department of Chemistry and Division of Toxicology, Massachusetts Institute of Technology, Cambridge, MA Member, Pittsburgh Cancer Institute, Molecular Carcinogenesis and Experimental Therapeutics Groups, Pittsburgh, PA Member, Drug Metabolism Group, Center f~w Clinical Pharmacology, University of Pittsburgh, Pittsburgh, PA Affiliated Researcher, Div. of Toxicot., Dept. of Chem., and Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, Assistant Professor, Departments of Environmental and Occupational Health and Pharmaceutical Sciences, University of Pittsburgh Graduate School of Public Health and School of Pharmacy, Pittsburgh, PA Nadonal Institutes of Health, MIT Hazardous Substances Management Program, and American Cancer Society Postdoctoral Fellowships, 1988-91 John B. Bruce Memorial Outstanding Graduate Student in Medicinal Chemistry Award, University of Oklahoma Health Sciences Center, College of Pharmacy, 1988 American Institute of Chemists Outstanding Senior Chemistry Student, Oklahoma City University, 1988 National Science Foundation Undergraduate Summer Research Fellowship (Molecular Biology/Physical Biochemistry.), Oklahoma State University, 1981 Sdected Publications (total of 29 peer-reviewed publications): Goldman, R., Stoyanovsky, D. A., Day, B. W., Kagan, V. E. Reduction of phenoxyl radicals by thioredo.,dn results in selective oxidation of its SH-groups to disulfides. An antioxidant function of thioredoxin. Biochemistry_ 1995, 34, in press. Hopp, L., Bunker, C. H., Day, B. W. Quinine sensitive changes in cellular Na+ and K+ homeostasis of COS-7 cells caused by a lipophilic phenol red impurity. In Vitro 1995, in press. Day, B. W., lonnalagadda, S. S. Synthesis of Z- and E-[2,3P-He] and [2,3-3H~]-I ,l-dichloro- 2,3- diphen>'lcyclopropane. IzH- and 3H-Analog 1I and its u-:ms isomcrl. J. Labelled Comp Radiopham~. 1995, 36, 73-78. Banni, S., Day, B. W., Evans, R. W., Corongiu, F. P., komb:~rdi, B. Liquid chromatography-mass spectrcvnek,-ic ac~:il:, ~is ofconj~gatcd dieae fatty acids in a p:ctially hy~rogena:ed fat. J. Am Oil Chem. Soc 1994.71. 1321-1~25
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Day, ]3. W., Skipper, P. L., Zaia, J., Singh, K. z~d Tannenbaum, S. R. Enan~o~pecil2city of covMent adduct t~fion by ~enzo[a]pb~rene ~-~ol eFo:dde ~th hum~ ~e~ ~hu~n. ~' ~ Cn~m. Re~. ToxicaL 1994, ~, 829-S35~ Ryan, L. K., Jin, R., Boggs, S. S., Karol, M. H. and Day, B. W. A mouse model for assessing endotoxin i.p. volvement in the lung inflammation and c;¢otfine production resulting from inhaled organic dust. Inhal. Toxicot. t994, 6, 485-499. " Kagan, V. E., Yalowich, J. C., Day, B. W., Goldman, R., Stoyanovsky, D. A. and Oantchev, T. Ascorbate is the primary reductant of the phenoxyl radical of etoposide (VP-16) in the presence of thiols both in celI homogenates and in model systems. Biochemist" 1994, 33, 9651-9603. Hossain, M. B., van der Helm, D., Schmitz, F. J., Pordcsimo, E. O., Magarian, R. A., Meyer, K. L., Overacre, L. B. and Day, B. W. Molecular structures and conformational studies of triarylcyclopropyl and related nonsteroidal antiestrogens. 1. Med. Chem. 1994, 37, 167(1-1683. Day, B. W. and Singh, K. Fluorescence spectroscopy in the analysis of hemoglobin adducts. Methods Enzymol. 1994, 231,674-681. Tannenb'aum, S. R., Skipper, P. L., Wishnok, J. S., Stillwell, W. G., Day, B. W. and Taghizadeh, K. Characterization of various classes of protein adducts. Environ. Health Perspect. 1993, 99, 51-55. Jin, R., Day, B. W. and Karol, M. H. Toluene diisocyanate protein adducts in the bronchoalveolar lavage of guinea pigs exposed to vapors of the chemical. Chem. Res. Toxicol. 1993, 6, 906-912. Du, L., Hossain, M. B., Ji, X., van der Helm, D., Magarian, R. A. and Day, B. W. Structure of 1,1- dichloro-2-(4- methoxyphenyl)-2,3-diphenylcyclopropane. Acta Cry_ stallogr. 1992 C48, 887-891. Day, B. W., Sahali, Y., Hutehins, D. A., Wildsehutte, M., Pastorelli, R., Nguyen, T. T., Naylor, S., Skipper, P. L., Wishnok, L S. and Tannenbaum, S. R. Fluoranthene metabolism: human and rat liver microsomes display different stereoselective formation of the trans-2,3-dihydrodiol. Chem. Res. Toxicol. 1992, 5, 779-786. Day, B. W., Skipper, P. L., Rich, R. H., Naylor, S. and Tannenbaum, S. R. Conversion of a hemoglobin 0~-chain aspartate(47) ester to N-(2,3-dihydroxypropyl)asparagine as a method for the identification of the principal binding site for benzo[a]pyrene anti-diol epoxide. Chem. Res. Toxicol. 1991, 4, 359-363. Day, B. W., Skipper, P. L., Zaia, J. and Tannenbaum, S. R. Benzo[a]pyrene anti-diol epoxide covalently modifies human serum albumin earboxylate side chains and imidazole side chain of histidine(146). J. Am. Chem. Soc. 1991, 113, 8505-8509. Day, B. W., Magarian, R. A., Pento, J. T., Jain, P. T., Mousissian, G. K. and Meyer, K. L. Synthesis and biological evaluation of 1,1-dichloro-2,2,3-triarylcyclopropanes as pure antiestrogens. J. Med. Chem. 1991, 34, 842-851. Hossain, M. B., Wang, J. L., van der Helm, D., Magarian, R. A., Griffin M. T. and Day, B. W. Smactural comparison of a gem-dichlorc~dia.rylwclopropane antiestrogen and three of its derivatives. Acta .Crystallogr. 1991, B47, 5II-521. Jankowiak, R., Day, B. W., Lu, P., Doxtader, M. M., Skipper, P. L, Tannenbaum, S. R. and Small, G. Fluorescence line narrowing spectral analysis of in vi~ o hemoglobin adducts of benzo[a]pyrene diol epoxidc-: comp:mson to synthetic analogue_~. J. Am. Chem. Soc 199 112, 5,_q66-5869. Day, B. W.~ Naylor, S., Gan, L-S., Sahali, Y., Skipper, P. L, Ngu:,'en, T. T., Wishnok, J. S. and Tannenbaum, S. R. Molecular dosimeu-y of polycyclie mromaric ?'.,'drocarbon cpoxide.s ~md diol epo×ides via he::v)globin adduc:_,. Cancer Res. 1990, 50, 4611-461 g077a21
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Other Support -- Billy W. Day, Ph.D, Active -1. Title, b. d. e. Source and identifying no. NIH, 5 R01-CA57288 P.I. Billy W. Day, Ph.D. Antimmor Mechanism of Dichloro-cis-diphenylcyelopropane, Role Principal Investigator, 20 % Effo~ Dates and costs of entire project 94i02/01 to 97i01/31 ; $243,553 direct Dates and costs of current year 94/02:'01 to 95i01/31 ; $75,553 direct Specific aims of project 1. Characterize growth inhibitor3' action of tide compoundin MCF-7, MCF-7/LY2 and MDA-MB-231 cells 2. Synthesize radio- and heavy atom labeled versions of the dtle compound 3. Determine smacrares of metabolites of tide compound in cell culture and microsomal systems 4: Determine ff covalent adducts of tide compound are t'on:ned in vitro 5. Determine tissue dis~budon of tide compound and its metabolites in mice Scientit~c and budgeta_rT overlap None Adjustments None o ao Title b. C. fo Tide b. C. d. e. Source and identifying no. NIH, 5 R01-HL51469 Nitric Oxide-Induced Cell Injury: Molecular Mechansims Role Co-Investigator (P.I. of consortium), 5 % Effort Dates and costs of entire project 94/07/01 to 98/06/30 ; Dates and costs of current year 94/07/01 to 95/06/30 ; P.I. Douglas R. Spitz, Ph.D. $ 586,629 direct (consortium costs: $ 65,162 direct) $157,869 (consortium costs: $18,812 direct) Specific aims of project 1. Determine if exogenous reactive nitrogen species (RNS) results in quantifiable injury to ceils in culture which correlates in a dose-response or time-dependent fashion with oxidative damage to lipids, proteins and DNA. 2. Determine if enymatically-generated RNS results in the same activity as per aim 1. 3. Determine if cellular resistance to oxidative injury confers resistance to RNS. 4. Determine if manipulation of cellular antioxidants alter cell injury and oxidative damage caused by RNS. 5. Determine, in cell culture, if exposure to low levels of RNS is capable of inducing an adaptive response. Scientific and budgetary overlap None Adjustments None Source and identifying no. Univ. Pittsburgh Central Development Res. Fund P.I. Billy W. Day, Ph.D. Metabolically Activated Antitumor Agents Role Principal Investigator, 5 % Effort Dates and costs of entire project 93t07/01 to 95/06/30 ; $13,000 Dates and costs of current year 93/07/01 to 94/06/30 ; $13,000 Specific aims of project 1.1"o synthesize several analogues of Z-l, l-dichloro-2,3-diphenylcyclopropane for enhancement of tubulin polymerization inhibitor3, and a~ticancer activity. This is a seed money ~ant. Scientific and budgetax-)' overlap None Adjustments None
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~I~ b. C. d._ e. Title b. C, d. e. Sonrcc and identi~dn~ no~ USAMPd~C ~A:BS-BCPd? P.I. ~. S. L~o, Phi. ~cdozto~A TrMni~ in Br~t. C~cer B~r,'o~.~_~ =. ~d Th~mpy Role P~cipafing Fac~,, conca~ent eflbn Dates ~d costs ofen~e pr~ec~ 94j09j0I to 98/08131; $ 754,~ dkect Dates ~d costs of c~ent yea 9410910I to 9-/0f431; $189,2(~ direct Spedfi¢ ~s of proj~ 1. Re~t qu~ed prMoatorN students to th~ w~dng pm~. 2. Educate students in the Nndamen~ principles of breast c~cc~a~obiolo~, ~a ~e~py. 3. EvNuate the progcss of ~e studenm. 4. EvNuate the pro~am ~d seek ad~onM ~nds ~d resources. ScienCe and budge~' overlap None Adjus~ent~ None Source and identit~,ing no. American Cancer Society, DHP-125 P.I.J.C. Yalowich, Ph.D. Free Radical Activation of VP-16/Topo II Interactions Role C0-Investigator, 10% Effort Dates and co~,ts ofentire project 94/(t7/0I to97/06130; $ 311,819 direct Dates and costs of current year 94/07/01 to 95106/30; $101,272 direct Specific aims of project 1. F .u,rther characterize the topo II inhibitory activity of flee radical activated VP-16 in human leukemia K562 cells. 21 Ide.nfify activated forms of VP-16 in the presence of exogenous flee radical initiators. 3. Identify and characterize direct chemical interaction between activated VP-16 and topo II. Scientific and budgetary overlap None Adjustments None Pending Title b. C. d. e. go 2. a. Title b. C. Source and identifying no. Environmental Protection Agency P.I.B.W. Day, Ph.D. New Mass Spectral Methods in Human Exposure Dosimetry Role Principal Investigator, 20% Effort Dates and costs of entire project 95/07/01 to 98/06/30; $ 345,364 direct Dates and costs of current year 95/07/01 to 96/06130;; $110,637 direct Specific aims of project 1. Prepare isotope-labeled internal standards of human serum 'albumin (hSA) covalently modified with 2,4- and 2,6-toluenediisocyanate, benzo[a]pyrene anti-diolepoxide and fluoranthene anti-diol-epoxide. 2. Use the isotope-labeled internal standards to study ion geam and pneumatically-assisted eleetrospray mass spectral techniques for detection and quantitation of hSA adducts of the electrophiles in aim 1. 3. D~velop internal standardization methods for MALDI-LAMMA analysis of hSA adducts. 4. Develop laser and pneumatically assisted electrospray MS techniques for detection and quantitation of.intact hSA adducts. Sdentific and budget~y overlap None Adjustments None Source-and identifying no. NCI -- SPORE in Breast Cancer P.I. Ken McCart3,, M.D., Ph.D. SPORE in Breast Cancer (BDay subproject: QSAR/Synthe_~is/Evaluation of Anti-Breaker Cancer Agents / Role P.I. of subproject. 25% Effort Dates and costs of entn-e project 9.5,"09/01 to 98/08/31 ; Dates and costs of current 95/01/01 to 392,878 direct subproject COS[5 16-+,_70 direct ~ubproject COSts
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Sg.ecific ~ms of project I. To deveIop es~ro~enid~-,/~fie~rogeniciq, qc~inogenidB"/anfi-bre~ c~c~r cornF QS~ t~ing ~ets. 2. Comp~e results from ~ I to exisifing compumfion~ da~bases. 3. ~edict -the s~c~ ~d ae~fies of, synopsize or ob~, ~d test agNnst humm brat c~cer ce~s ~ cul~e new che~c~ entities for bre~x c~c~ gea~en~ Scientific ~d budge~' overlap None Adjus~ents None Title b. C. d. fo Source artd identifying no. Nil-I, 5 R01 ES0565I P.I. Meryl H. Karol, Ph.D. Chemically-Induced Chxonie Allergic Lung Disease Role " Co-Investigator, I0% Eft'oft Dates ~d costs of entire project 95/08/01 to 99/07131 ; $1,124,350 direct Dates and costs.of current year 95/08/01 to 99/'07131 ; $ 267,455 direct Specific aims of project 1. Identify adduction sites of toluene diisocyanate on extravasated serum proteins in lungs of animals exposed to the chemical. 2. Identify in vivo adducts in lungs of animus by electron microscopy. 3. Determine the nature of th~ allergenic TDl-protein conjugates. 4. Test the hypothesis that oxidative processes are involved in the sensitization to TDI. Scientific and budgetary overlap None Adjustments None
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Biographica! Sketch Balachxndran, Raghavan Education Research 0 Sll 6!45 Kerala University, India Kerala Universit3,, India Jawaharlal Nehru Univ., India Jawaharlal Nehru Univ., India Positions: B.S. 1967 Botam,, & Zcolog3' M.S. 1969 GenelJcs & Plant Br& Ph.D. 1978 Life Sciences Postdoa 1978-79 Biology 1970-1972 1978-1979 1979-1980 1980-1983 1983-1986 1986-1994 1994-present Publications: Teacher, Thimphu Public School, Thimphu, Bhutan Post-doctoral Fellow, Jawaharlal Nehru University, India, Biolog3, . Research Associate, Department of Radiology, Washington University, St. Louis, MO Visiting Fellow, Laboratory of Cellular and Molecular Biology, Nadonal Cancer Institute, Bcthcsda, MSD Visiting Associate, Laboratory of Biochemistry, National Cancer Institute, Bethesda, Research Associate, Department of Infectious Diseases and Microbiology, GSPH, University of Pittsburgh Research Associate, Department of Environmental and Occupational Health, GSPH, University of Pittsburgh Aaronson, S.A., Storch, T.G., Balachandran, R., and Reddy, E.P. Different hematopoietic target cells for transformation by replication-competent routine leukemia viruses. In Marchesi, V.T. and Gallo, R.C. (Eds.): Differentiation and Function of Hematopoietic Cell SurPaces. New York, Alan R. Liss, Inc., 1982, pp. 251-261. Balaehandran,'R., Reddy, E.P., Dunn, C.Y., Aaronson, S.A., and Swan, D.C. Immunoglobulin synthesis and gene rearrangements in lymphoid ceils transformed by replication-competent Rauscher nmrine leukemia virus: transformation of B cells at various stages of differentiation. EMBO J,, 3:3199-3207, 1984. Gupta, P., Balachandmn, R., et al. Detection of human immunodeficiency virus by reverse transcriptase assay, antigen capture assay, and radioimmunoassay. J. Clin. Mierobiol., 25:1122-1125, 1987. Balaehandran, R., Thampatty, P., Rinaldo, C.R., and Gupta, P. Use of cryopreserved normal peripheral blood lymphocytes and isolation of human immunodeficiency virus from seropositive men. J. Clin.. Microbiol., 26:595-597, 1988. Gupta, P., Balachan&an, R., Ho, M., Enrico, A., and Ri~aldo, C.R. Cell-to-cell transmission of human immunodeficiency virus in the presence of azidothydine and neutralizing antibody. I. Virol., 63:2361- 2365, 1989. Melder, R.J., Balachandran, R., Rinaldo, C.R., Gupta, P., Whiteside, T,L., and Herberman, R,B. Cytotoxic activity ag:dnst H1V infected monc, cytes by recombinant interleukin 2 activated natural Miler cells. AIDS Rcs.&HumanRctroviruses 8:1(t1I-1(115, 1990. Gupta, P., Brdach~dran, R, Thampatty, P., Rinaldo, C., and Ho, M_ Oxophena_rsine, and antisyphillis drug inhibits HIV-I specific protein synthesis in acutely aad persistently infected lymphocytes. AIDS Res. & ttuman Retroviruses. 6:1417-1a23, I990.
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BaIachandran, R., Thampxtty, l:'.~ Enrico, A., I;:.ir~aIdo, C., and Gupta, P. Human immur~edetleiency isolates frc, m asl, mptomati¢ homosexual m:r~. and from AIDS patient~ have d~.ct bio!o~e arid gen.v.tic propertie_a. Vir,~logy, 180:229-238, 1991. Farmdo, M.R., BaIaehandran, R., Gupta, P., and Wolin~k'y, S.M. Analysis of alternatively spliced human immunodeficiency ~qxus t.~l~e I (NIXr- 1) mP, NA species one of which encodes a novel tat-env-fusion protein. Virology; 185:258-270, 1991. Shankarappa, B., Balachandran, R., Gupta, P., and Ehrlich, G. Introduction of multiple restriction enzyme sites by in vitro mutagenesis using PCR. PCR Metho&, & AppI., 1:277-278, 1992. Balachandran, R. and Gupta~ P. Active nuclear transport of proviral DNA is important for HIV-I pathogenesis. Abstract IXth International Conference_on AIDS, Berlin, 1993. Grovit-Ferbas, K., Balaehandran, R., and Gupta, P. HSV-1 and HIV-1 interaction induces apoptosis in CD4+ T cells. Abstract The First Natianal Conference on Human Re~oviruses and Related Infections, Washington, DC, December, 1993. Sova, P., Gupta, P., Balachandran, R. and Volsk3', D.J. Genetic variability of the human immunodeficiency virus type 1 (HIV-1) vif gene in vivo. J. Vm-,l., t995 (in press). Balachandran, R. and Gupta, P. A defect in nuclear translocation of proviral DNA is responsible for lack of ~owth in T cell line of human immunodeficiency virus isolates from asymptomatie homosexual men. Virology, 1994 (submitted). Balachandran, R. and Gupta, P. Incomplete reverse transcription and lack of integration are responsible for the reduced expression of human immunodeficiency virus isolates from asymptomatic individuals (in preparation).
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OIher Suppor~ -- Razh~van B~chaniran. Ph.D. -- Co-lnvcsd~a~or Active Non~ Pending a. Soarce and identifying no. NIH P.I. Donald R. Mattison, M.D. Title Role of Apoptosis in Ovarian Toxicity b. Role Research Associate, 100% Effort c. Dates and costs of entire project 07-01-95 to 06-30-00; $1,117,033 direct costs d. Dates and costs of current year 07-01-95 to 06-30-96; $ 238,224 direct costs e. Specific aims of project 1. Anatyze whether PAHs induce apoptosis in human ovarian cells. 2. Identify the role of c-myc, c-myb, c-los, bcl-2 and o53 in ovarian toxicity. 3. Elueldat~ the roles of transcription, translation, cellular proteases, endonucleases and topoisomerases in ovarian toxicity. f. Scientific and budgetary overlap None g. Adjustments It" the NIH grant is funded, a suitable replacement for Dr. Balachandran will be identified and hired for the Research Associate Position under Prof. Day. 5037=2i6
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where rsq~stcd oa ~ll self-addressed, stumped enveIope. 1.. Ca~gor.~' and Suh¢lass{H~.-a0a, n (see Pages ILl-Ill: Type )ha: fi',e-characIer 199~ .~kCR ABSTRACT FORM FO ] C:< ] O:,:~. FOE O~.--ICE USE DeA~afiv~ of Z-l,l-dichloro-2J-diphen.,dcycIopropane (Analog II) lhat ~uppresa brea~l cancer ceil growth by inhibiting tubulin pol.,,merizafion. .lonr, alagadda., 8.S., "~Harnel, E., ~r I-Ia.~, E_ Day, B.W. Depralment of Envk'onmcntaI & Occ~pation~ Heahh, Uniter.dry oF Pit~b~h, PA 15238 (SSJ, E~, B~) ~d ~te~tcC,' of MoI~cul~ Nafion~ C~.cer I~fi~te, Bcthas~ ~ 20~92 Z.l,l-~cNer~2,3-diphenyle>cIo~rop~e (£:a a~og g) is ~ effective aiti- brat c~cer agent in roJen~ ~g ~ cell cNl~e. We recently datelined that it has we~ ~buUn ~olymeH~fion inhibitor' acfivi~, OC~ 10-1a g~r). In tNs study we began to dcveIop a s~cmre-acti~JW NIationship of,£chlomdiphenylc~'clopr@~es tubuIin polgm~cn ~hibito~, V~oas dcNvatives of i~og H were prepaed and t~ted for thek i~hibifion of tubul~ polyme~ation. Of the compounds teste~ Z-I,1- dichlom-2-O-me~ox}Thenyl)-3-~enylcyclo~mp~a, Z- l,I-~chlom-2-(4-fl~omphenyD- 3-phenylcycloprop~e and Z-I,I-dichIoro-2-(4-chlorophenyl)-3-phenylcydopropane ~cre tb~nd to be acti~,e with IC~o vNuea of 3-5 gM. E-l,l-diebloro-23-die%yl-2,3- diphenyIc~cloprop~.e wm; equipotent win diethyIsdlb~ol (DES) (IC:~:, = i0 Other ~Nogs, such ~ 4-Mme~oxy, 2,5-ditluoro, 2.4-ditluom and 4A'-dimethoxy exhibited some efl?ct on the polyme~zafion r~ction by delaying its onset. The cytQs~tic acti~4ty of ~ese cempeun~ in humm bre~t c~cer cells is being evNuated. For example we have found ~at these com~unds have effect on NICF-7 ceils at I0a -10"~ M. (Supposed by ~ g~ant CA 57288). Type abstract within blue linen. See sample 2. -kb~'lraet is SPONSORED b.~: 77¢~ Member No. S. Ab~Ira~( i, ~o be PRESENTED (See Direcm~ of Members tbr Member No.I Ill an AACR Billy W. Day Name 260 Kappa br~ve .~d,ve~ 260 gmDPa Drive \Icmb~,r Pittsburgh, PA C ~>'.Sm~e Pittsburgh,. PA ..... Zip/ Zip/ 15238 . Po~tal Cude USA CounI~" 1523S Po,.{al Co, de __ 41 P - q 67 - fi ~ 02 Telcphon: N,z 4 12-967 -650 7 412-~24-i020 FAX No. ~ 12-624-I020 A~sc, ciatc M, mb~r i¢ :F,:~ , i- ~r.J f--' -~r,-- (2:: f':;j):/: , aF;,I.,, ,: ,,_, ~ :1:,- : >.'.:~Tvr2 ,:- ('.2c J ' FAX 6. EIigihiti.ty f~r Travel Av~ards ~_.~A. The PRESENTER ofthi~ physician in trainings, or postdoctoral to}low 5 0,~,7a2 i 7
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Category nmt Suhclassil~cation code in the bl~ks provided, for *xampb, 1995 .~kCR .~BSYRACT FORM ~o ; c~-: FOR OTFICE U~E ONLY Analysis of the "ant[prollferativ~ -action "-o£ - Z-I,l:dichIor6:2,3. diphenylcyclopropane in br~st tum~r.celIs. ~er H~ E. ~t Day, B.W. Deponent of Envkonmen~ Unlvcrsi~" of Pit~=gh, Pitch.h, PA 1323~ Z-IJ-Dichl~ro-2,3-~henylcydop:@~(1) has lon~ ~fim~o~ophic ~ ~ce, ~fiprolffemdve in m~ m~mm~' D~mor m~l:, ~,I ~o ha~ c a LDS0 ~ r~en~ of >3g, kL S~ce I h~ a low e~ogen r~e;tor ~) ~finib', ~ve have t~tcd i~ ~fiFrol~cmfive ~paci~y ~ ~ee ~fercnt bre~X t~mor M~-7 ~R+, es~ogen re~onsive), MCF-7~Y2 ~R+, es~ogen h~A-~-231 ~R-, es~ogen umes~onsive). We found that 1 inhibited pr~lffcrafi~n ~d ~e clonogeni¢ capazit:: of ~[ ~:e ceil Ikms, ~cating mech~ffm is inde~ndent from ~e es~ogen receptor. Memt~Ec s~es have shown • at &e major mem~te of 1 ~t~ ~cubadoa wi~ &e ~ee t, re=[ tumor cell Z-cblor~h~cone. ~en we tested &e ~d~m~emave ~d ~tictoaogenic c~acity of this mcmbolite ze found ~at it was [0 dme~ more p~tent th~ 1 in the ~ce cell lines. Because ~e mcch~ism of action of 1 seems to be ~depend,:nt from es~ogen ~eptor, I ~d Z-chlor~h~cone were tested tbr bin~g at ~e t}~ ~ nucl~ bin~ng ske. Bo~ ¢om~un~ were ~so tested for Iubulin ~lymefizafion inhibition. Only 1 inhibited Iub~in ~lym~r~fion at I0 ~.I in vitro. A F~ssible deto~icarion route tTr I ~d Z~hlor~h~con¢ w~ stu~ed by ~yzing ~¢ mem~Iic faro of 1 ~d Z-chlor~h~cone in ~e presen¢¢ of glum~ione. In~tead of tb~ing ~ adduc~ wi~ glum~ione, 1 was 8olvolyzed imo Z-2-chloro-2.3-diphenyl-2-propen-l-ol. and subs¢quently rapidly oxidized ~d d:chlofinated into ¢~s-ch~cone, a command. (Suppled by h~H g~t CA57288) Type abstract within blue lincg. See sample abstract. 5. Ab:.tract is to be PRESENTED b.~: Member No. ,If an AACR Ernst tar Haar Name (Plca~¢ P~nt) 260 Kao~adrive 2. Abstract is SPONSORED I,.v: 6 7 1 Member No. ~See Director" of Members for Member No.) Mary Conner, Ph.D. h'~me University of Pittsburzh Pi~tsbur.~h PA City.State Zig/ 15238 Po-~tal C,~dc U. (/, 12 ) 9 6 7-6~ C~.l 2 ) 624-1020 3. As the SPONSOR of this abstract and on behaIf of all the bdicate my support for fl-r~ dam co:rained h~rein awJ ~:r~fcr its cop:.d~ht ~~,, Signature of 5027=.2" "
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S.~nlhesis of Z- and E-[2,3--~Hzl and [2,3--aHzl-l,l-Diehloro-2,3- diphen)lcyclopropane (ZH- and 3H.Analog II and its trans Isomer) Billy W. Day" and Sastry S. ,lonnalagadda Departments of En~ironm~.ntaI & Occupational Health and Pharmaceutical University of" Pittsburgh 260 Kappa Drive Pittsh~_wgh, Penns)'Ivania 1523S USA SOMMARY 2H and 31t labeled Z- and E.l,l-dichloro-2,3-diphenylcyclopropane (1 and2) were n3nthesized starting fr~:~n~ NaB2H4 and NaB.~ H4 reduction of benziL The resMting glycols were" tr a~formed to the 1,2-I~eIed Z- and E-stflbencs by thermolyMs of their cyclic thionocarbonates in trime~hylphosphite. The atilbenes were reacted with plvase tra~rfer-generated diehlorocarbene to form the title compounds. Tl~e &'deuterio isomers were separated by.tractional crystaIlizalion in ytelds of 60 and48 %. Each was greater than 99 % geometrically and 98 % isoropicalfy pure. The ditdtio isomers were separated by C-18 HPLC. The radio~hemical ylelds, on a molar basis using benzil as the limiting reagent, were 42 % and 23 %, each with specific activity of 88.5 mCilmmol and radiochemlcal pur~ty of > 95%. Key words: 1,1-dichloro-2,3-diphenylcyclopropane, deuterium, tritium, Analog INTRODUCTION Z-1,1-Dichloro-2,3-diphen~,leycloprop~e (I), also "known a~ Analog lI (1), is a novel anti- breast cancer agent, effective in vivo against carcinogen-induced a~d tr~plantable rat m~a'tmary tumors (2,3) as well as in vitro against estrogen-dependent and -independent human breast tracer ceils grown in ¢altur~ (4,5). The mechanism(~) of antiproliferative and cytotoxic action shown by 1 is (a.re) urAr~own. We are 6arrentl;,, stud~,ing tl~e metabolism and maeromoleeule binding of this agent both in vireo and in vgvo, and our studies required Ic~oth high speckt2e activity rad~olabeled 1 for bicdistrib~tiorgr-~iotraccr experiments and stabI~ isotope heavy-a~om ladled I for use as an Latem~l Thi~ communication dc~cribos the ~yathe~i~ of high ~;pecific acd,,4ty [2,3-~H2]-la~l~ ~d high
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THE COUNCIL FOR TOBACCO RESEARCH - U.S.A., GE0X~E A. HASHIM, PH.D. A~SOClATE RESEARCH DIRECTOR D~: FEB. I~, 1995 NEW YORK, N.Y. 100"_-2. (212) 421-85~$ ---- .; Reviewer I: DR. LYNCH Reviewer 2: DR. BRE~II~A~ RE: Preliminary Application Number: 5567 Applicant's N~rne and Degree(s): Insfitu.tion: Title: BILLY DAY, PH.D. UNIVERSITY OF PITTSBURGH ROLE OF MECHANISM OF APOPTOSIS IN BREAST CANCER CELLS 1. Please review this application and indicate your scoring below. ~ A return envelolx) is provided for your e,,onvemienc~. 2. We eXlXX:t the top 30% of Preliminary Applications will.be encouraged to submit Full Applications. Top 10% Top 20% Top 30% ~ Top 40% 13 Top Reviewer's commonm: Copic~ to: Dra Etae b:rg, pt-r~2.do¢ rc~07.1 i 9. McA11i~:cr Signature: 5037~220
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Universit3 of Pi D~;~_~;,~r_ . ::FE~ ..... ~:r,:,'~;~*~ ......... zt ~,d Occ,~2 ,~.~' ~,.d F2~: 412-~24-1 ~23 February 13, 1995 Arthur D. Eisenberg, Ph.D. Asc~'~ciate Research Director The Council for Tobacco Research, Inc. 900 Third Avenue New York, NY 10022 (212) 421-8885 Dr. Eisenberg, I have been studying the effects of xenobiotics on breast cells for several years. Recently, my lab has noted the induction of apoptosis by an agent we have been stud)4ng in order to determine its molecular mechanism of action. We were intrigued to note that the interactions and roles of genes involved in regulation of apoptosis, particularly bax, have yet to be fully elucidated in breast cancer. We propose to perform studies that will allow us to elucidate the role of gene regulation in the apoptosis of breast cancer cells. Sincerely, , /'") Billy W. Day, Ph.D.-- Assistant Professor
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Universit3 of 412 ~24-1020 Februa.ry 13, 1995 ,arthur D. Eisenberg, Ph.D. Associate Research Director The Council for Tobacco Research, Inc. 900 Tkird Avenue New York, NY 10022 (212) 421-8885 Dr. Eisenberg, I have been studying the effects of xenobiotics on breast cells for several years. Recently, nay lab has noted the induction of apoptosis by an agent we have been studying in order to determine its molecular mechanism of action. We were intrigued to note that the interactions and roles of genes involved in regulation of apoptosis, particularly bax, have yet to be fully elucidated in breast cancer. We propose to perform studies that will allow us to elucidate the role of gene regulation in the apoptosis of breast cancer cells. Sincerely, ,, ~ Billy W. Day, Ph.D.~ Assistant Professor ~,537a222
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TRC pr~Trc, t:osa.T F.~g~~ 2 Cover t. Name, De~ees and Academic Title: Billy W. Day, Ph.D., Asfistant Professor of Molecular Toxicolo~" and M'v/idnal C.hen~istr3,, Departments of En~.q_roamental & Occupational Health ;rod Ph;wmaceutical Sciences, Graduate School of Public Health and School of Pharrnacy, University of Pittsburgh. 2. Title of Prqiect: Role and Mechanism of Apoptosis in Breast Cancer Cells 3. Phone Number: 412-967-6502 Facsimile: 412-624-1020 4. Duration of Project: Three (3) yea.rs 5. Estimate of the Direct Cost for the First Year: $74,000
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RoIe an¢l Mechanism of Apoptosis in Breast Cancer Ceils Princi;M l~m~n'gat~r: Fr@ BiI~v W. D~', C~-tm'eso~g~r: Dr. Rag~ar:an BaI,2ct~. ,ndrat~ 1. Specific ~ims The objectives of this research axe to define the role and mechar'a~-m of aFoptosis in cIonaIIy different human breast cancer ceils. -Spe~ficalIy, we ~LI first probe human breast cancer cell lines of differing hormonal sensiti'dty with agents studied in ore" lab that we have preliminarily shovm to induce al~ptosis in the ceils. To be examined are estrogen receptor fER+)/estradiol (E2) respon~ve, ER+/'E2unresponfive, and ER negative (ER-)/Ez unresponsive human breast cancer cell lines MCF-7, MCF-7/'LY2, and MDA-MB- 231. Concm'renfly we v,411 study the interactions of, and eh:cidate the roles of, three specific genes, bax, bel-2 and p53, that have been-shovaa to regulate apopmsis. Box and its protein product, even though recently shown rb play an integral role in apoptosis, have yet to be studied in breaz't cancer. The results from our studies will allow us to elucidate the role of gene regulation in the apoptosis of breast cancer cells. Thbrefore, the speci£:c aims of this project axe: 1. To investigate the nature of the apoptosis in MCF-7; MCF-7/LY2 and MDA-MB-231 human breast cancer ceils. 2.. To identify the role of the p53, bcl-2 and bax gene products in these cell lines and correlate their relative expressions with the apoptosis induced by xenobiofics. 2. Background and Significance Apoptosis and the genes involved. The role of apoptosis, programmed cell death, in cancer biology has recently gained both importance and widespread attention. Many toxicants reduce the proliferative potential of cancer ceils and disrupt their passage though the cell cycle. A xenobiofie can initiate the signals necessary for induction of apoptosis. The existence of a genetic program in the ceil during a drug-induced death pathway suggests that modification in the expression of the responsible genes could have a'profound effect on the development of cancer. A sequential or concomitant biochemical/genetic modulation that augments the initial steps of the cascade should fuel the apoptotic event induced by anfiproliferative xenobiofics. The genes bcl-2, bax, and p53, and their expressed protein products, have been linked to programmed cell death. The wild type (wt) p53 gene product induces apoptosis, while the bcl-2 gene product can inhibit apoptosis triggered by wt p53. These activities of both genes have been noted in breast, both clinically and in cell culture. The bcl-2 gene encodes for a mitochondrial protein that is localized to the luminal cells of normal breast, considered to be the origin of malignant breast disease. Several studies of breast tumors have shown that b¢1-2 levels are positively correlated with ER and progesterone receptor (PR) posifivity. The bel-2 product is generally associated with small, ER(+), slowly proliferating, p53 negative tumors, and is not generally associated with large, ER(-), rapidly proliferating, p53 positive breast tumors. Hence, MCF-7 cells should be more resistant to apoptosis-inducing drugs than MDA-MB-231 cells. Over-expression of the bax gene has been shown to counter the death represser activity of bel-2 via the formation of a heterodimeric protein. The bax gene product is thus a dominant inhibitor of the bcl-2 protein. The ratio of bel-2 to bax determines the survival or death of a cell to an apoptofie stimulus. Bax is a relatively recently discovered and, to date, there have been no reports on the involvement ofbax in breast cancer. Although these three genes have been associated with the apoptosis process, their relationship to xenobiofic-indueed cell death is not clear, especially in the case of breast cancer. The screening of different human breast cancer cell lines with differing estrogen sensitivity for these genes during the xenobiotic- induced apoptosis process will provide useful information on cell death and cell proliferation of these cells, and may provide important insight into the development and progression of breast cancer. 3. Preliminary Studies Generally, studies on apoptosis involve the use of chemical modifiers of cellular macromolecule synthesis or signal transduction, such as cytokines, interleuldns, and/or gro,~¢h factors. We have noted a chemically simple agent that induces apoptosis in breast cancer cells, and propose to use it as the first of several initiating xenobiotics for biochemical elucidation of apoptosis. No~'el anti-breast cancer agents inducing apoptosis. We have lxen studying Z-I, l-dichloro-cis-2,3- diphenylcyclopropane (1), a rigid analog of stilbenic estrogens/antiestrogens, found to have antiestrogenic properties when analyzed by utero~ophic ~d m~tiuterotrophic assays in immature female mice: it had no esuogenic activity and possessed weak but reproducible antiuterotrophic effects. An inmguing properW
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noted for tempotrod I was Ii~ i~ hzd c~no~ no af~--nnity fo~ d~ cl~ici (~H~h ~lri~y-~ow D mt ute~e e~ogen recep:or, ~i~ a reh~ve b~g ~;' Ie::s ~ 0.01% of Com~und I ~as lat~ shown ~ ~'o ~nt sm~es to have ac~, compmble to ~o>~n ~M) ag~st Ne heine (prolac~)-deNndent 7,12-~me~yI~[N~cene ~X~Al-~d~ced mt ~or m~el in %4vo, ~d to be more effective ~ T~XI ~ r~c~g Ne incidence of new mmc)rs dating long te~ dos~g. Compound 1 w~ Nso active ag~st ~e D~A-4 me~mfic, ~I~mble mt - m~ ~or ~e in %~vo. Ad~fion~ g~c~t gop~es of ~s communal include i~ low app~nt toMNty, e.g., ~e ~0 ~ ~ce is 3683 m~kg 24 h ~ter a p.o. dose, the idendc~y dete~in~ LDs,? in mrs is N~ over 3 ~g, and ~e ~g ~at it is not mutagenic to S. O'p~m~i~ sm~ ~677 at concenmadons ~g from 10 gM to I ~M, ~ ~i~ ~d ~out a metallic activating system. We have recently not~ ~at 1 ~d its ~jor ~t c~cer cell meta~Hte ~ active agNnst bm~t c~cer ceil Nnes repmsen~g ~e full spec~m of esmogen sen~d~V not~ in hum~ ~m. Also, we ~e noted t~ apparent induc~on of ~opwsis ~o~tion of 180 bp DNA la~ers ~er 96 h e~osure) the compoun& in t~se cell lines. We have Nso recen~y ~gun ~e study of a~pmsis ~duced by xenobiodcs ~ h~ organ ~osa cells. We have develop~ ~d u~ ~e t~hNques necess~, for ~efic anNysis of ~e genetic ~d cellul~ steps in a~ptosis ~ough these stu~es. Except to note ~e Net t~t we have seen s~l~ ho~onaI ~fluences opem~g in ov~ ~d b~st c~cer cells, we will not des~ ~ese sm~es here. Note ~at we ~ not propos~g to develop 1 or ~y o~er che~cNs in this submission - our obse~afions wi~ it have, however, piqu~ o~ scientific c~osity so ~at we wish to de~e the bi~he~cal mechanisms of apoptosis ~ ~e breast c~cer cell ~es. 4. Experimental Design and Methods The induction of apoptosis, determined from both the ability to cause DNA laddering and early cleavage of DNA into large (50-700 kb) fragments will be determined in MCF-7, MDA-MB-231, MCF- 7/LY2, in vitro, the latter by quantitative field inversion gel electrophoresis, hybridization with the 32p. labelled alu I probe, and phosphoimaging techniques. Beginning with 1, selected compounds will be studied to correlate their induction of apoptosis with their inhibition of proliferation ofthe cells, as well as with their cytotoxicities by clonogenic assays. The kinetics of these processes will in turn be correlated with levels and expression of p53, bax and bcl-2, measured by PCR immunological and techniques. MCF-7 (28th passage), MCF-7/LY2 (both and obtained from Dr. Marc Lippman of the Lombardi Cancer Center at Georgetown University), and MDA-MB-231 (obtained from ATCC) cells are routinely grown as monolayers in IMEM (no phenol red) with 5% heat inactivated fetal calf serum and 4 mIU/mL human insulin in 175 em2 flasks at 37 oC in a 5% CCh-ertriched humidified atmosphere. The complete medium is sterilized by filtration through a 0.20 gm Falter. The ceils receive an additional media change prior to the next treatment. Cells are routinely screened for mycoplasma contamination. C)tostatie activity of the test compounds is measured by determination of growth curves for the three cell lines. Cells (10,000 per well) are plated in triplicate in 6 well plates in IMEM with 5% fetal calf serum (dextran-coated charcoal-stripped in the case of Ea-responsive cells). After a 24 hour attachment period, the cells are grown in the presence of the test compound (in > 999:1 medium-DMSO) for 0 to 144 hours. The concentrations of drugs used is 0 and 10-9 to 104 M. At the time points 0, 6, 12, 24, 48, 72, 96, 120 and144 hours, medium with nonadherent cells is removed and adherent eells are detached by a stream of medium (expelled from a syringe) or with trypsin. It should be noted that in the eases of MCF-7 and MCF-7/LY2 cell lines, detached cells are a common occurrence even in healthy/untreated cultures, and are usually not a good indicator of on-going apoptofic processes with these lines, t~emaining adherent cells are gently scraped from the plate with a tefl~n spatula. The medium and dislodged/scraped ceils are combined, centrifuged, resuspended in HBSS, and an aliquot is mixed with an equ~ volume of 0.08 % trypan blue in HBSS. After a 10 minute period to allow for dye uptake in nonviable cells, dye exclusion by viable ceils is determined microscopically using a hemocytometer. Viable cell nthmber is calculat'ed by multiplying total cell number by the fraction of celts excluding the dye. Cytotoxic activity is determined by a clonogenic assay. Ceils in HI3SS, which were not used for counting from the above experiments, are plated in hMEM in with I0 % fetal calf serum in triplicate in 6- well plates at limiting dilution (500-10000 viable cells per well). The concentration of test drug for cytotoxicity evaluation is typically chosen from a concentration that shows a weak (10-40%) antiproliferative at time points of 96 hours and beyond. It is sometimes necessary to add gamma-irradiated and killed 'seeding' cells at this point to promote attachment. After four to fourteen days of growth, depending on the growth rate of the specific cell line, cells are fixed vdth 25 % methanol, then stained with cry. stal violet. Colonies of > 50 cells aye counted by microscopy. The clonogenic ability of the
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fraction or" ~ clonog~nic p~ccnmg¢ of m~ted c~H~ vs. un~t~ Ce~s undergo~g apop:o~s show a ~e~c DNA ~enufion pa~em ~fien ~Mb~zed by ag~ose gel el~mophor~is. DNA ~ffi ~ ~vzed a~ v~ous time ~m ~¢r ff~ ~Hs ae m~ted ~i~ ~e of ~IeresL ineI~g to~ DN~ A~ren¢ ~d non-aGh~ent cells ae remov~ as per ~ c:,,tos~Js prot~oI, w~h~ 3x wi~ PBS or ~SS, ~d ~Het~ ~ cen~gafion. ~e cells ae lysed ~ either 0.75% (-)-n-~'l-~-glucop}~o~de, 1% N~-40 detergent, or 0.5% Na la~,,1 sacosine in 5 mM Tfis, pH 7.4, ¢on~n[ng 5 ~M EDTA and 50 p~ proteinase K for 0.25-1 h on ice. ~¢ cell debris ~d un~a~m~ (actu~Iy, ~Iafively unffa~ented) DNA is ~He~ed by cen~ugafion, ~d the superna~t is incubated ~ 5~ uni~/mL RNAase A for 0.5 h a~ 37 oC, I h at 50 cC. ~e DNA ~ ~e supema~[ is ~alyz~ by el~mophoresis on 1.8-2@ agaose cont~g ~i~um bm~de ~ 0.5% ~E (89 mM Tfis, 50 ~I ~fic acid, 2 ~I EDTA). ~e gels ae des~n~ ~d ~ DNA is ~u~ed by ~ ~s-fllum~fion ~d'pho}o~phed. A st~dad DNA maker ladder (1~ or 180 hp) is ~eluded in each gel tbr laddefing comp~son. For qu~fi~five field ~version gel eleemophoresis (QNGE) stu~es, h~ested cells ~e suspend~ in 10 ~ Tfis buffer (pH 7,2) coning 20 ~ NaC1 and 50 mM EDTA at 50 oC. An ~uM volume of 2~ hC~ ag~os, ~d soli~fi~ plugs coming 1~ cells ~ made at 4 ~C in ~sposable molds. The plugs ~e incubat~ at 50 oC overnight in 4.8 ~ Na deoxycholate, I% Na la~l s~cosine, 1~; mM EDTA, pH 8.0 ~d 0.5 m~ protein~e K, ~en w~h~ 4x wi~ 50 ~ EDTA ~d 20 ~M Tfis, pH 8.0. QFIGE is peffo~ed on 5 x 10~ cells per l~e on a ~GE Mapper El~c~ophoresis System in 1.5% field ce~fied ag~ose ~ 0.5% ~E. ~e elec~ophor¢fic p~etem ~e: 12 ~ continuous fo~ pulse at 150 V, followed by 30 h at 150 V using a 21% r~p from 0.9 to 30 sec ~ ~e fo~d ~ecdon ~d 0.3 to 10 see in ~e reverse ~ecdon at 10 oC. Gels ~e st~ wi~ e~ bro~de ~en visuMized by ~ gans-~lu~afion and photo~aphed. ~e DNA is nicked ~ ~ light ~d ~st~ed to a nylon membrane wi~ ~i (0.4 N NaOH, 1.5 ~ NaCl, 2-2.5 days) by capill~ action. ~ membY~ is neuvalized in 0.5 M Tfis, pH 7.0 and washed 2x wi~ 0.15 NaC1, 15 ~M Na ci~ate. The w~sfe~ed-DNA is hybfi~z~ for 1 &24 h m 42 oC with szP-~-d~-la~ed Mu I s~uence pro~, ~ membr~es me washed ~th buffer~ detergent, ~d ~e analyz~ on a Phosphor~ager. Resulting lu~nescence is co~¢ct~ ~d m~sf0~ed to 16-bk dam ~d ~yz~ by. image ~Mysis qu~fificafion Tr~slafionM pr~ucts of p53, b~ ~d ~F2 ~ ~ det~t~ us~g commercially and colla~mdvely avMlable ~fi~s to ~ proteins by i~unohist~he~cM me~. The m~s~pts of these genes will detect~ ~.qu~t~.by hybfi~mdon ~d dot or no,hem bMt ~Mysis, ~A ~ ~ isolm~ by hot phenol or ~idin¢. ~i~ate.meth~s, ~A will ~ dena~ ~ fo~adlehyde-fo~d¢-MOPS buffer ~d sep~ated on ag~ose gel, .~d ~sfe~ed to ni~ellulose. ~ filters ~ ~ pm-hybfi~zed ~d hybfi~ in st~d c~kmils with a nick-~slated pro~. ~e ~tem ~e ~en wash~ ~d autora~o~phs ~11 ~ pr¢p~ at -70 oC. ~-Acfin ~1 s,~e as ~e ~t,rnM convol. Low copy num~rs ~11 ~ det~ted by a~pmfion of RT-PCR ~ presently ~ningin o~ labs. App~opfiat~ pfime~ ~e ei~ avMlable (CMnet~h) or w~ ~ syn~esized at ~e Univ. Pittsb~gh DNA SyntNesis Fa~ alter ~Mysis ~d Mcafion of appropfime s~uences us~g &e OLIGO primer design sofi~¢ from Nation~ Biosciences. Iso~t~ ~A ~1 ~ r~verse ~s~ ~ ~IV vans~pmse at 42 bC for 30 ~ ~ ~e pr6sence of deoxynucleofides ~d 3'-primer oHeonucl~ofid~ eDNA (0.3 ~g)4s mLX~ m 50 ~ PeR buffer w~ 5 ~d 3 primers ~d ~s, oveflmd ruth ~neml off, ~d subject~ to 30-35 cycles of AT (94 oC for 1 m~, 55 oC for 1 ~n, ~en 72 oC for I ~). ~ucts w~ ~Myzed by 10% PAGE alter hybfi~afion ~ ~2P-la~Ned pro~s, cDNA congol ~pl~cafions ~11 peffo~ on ~-ae~ .~ Ml RNA prep~fions. Translational Potential of The Proposer Project • . BcI-2 overexpression represen~ a mech~sm for ~e s~,ivM of c~cer cells. We will detc~ne if breast c~cer c~s with ~ffe~g ratios of ~I-2 to b~ show ~ferent sensitivities to xenobiofics, reflect~ by ~e~ relative ab~fies to undergo a~ptosis. C,lls ovemxpress~g bmx c~ ~dergo apoptosis following ~ ~ifiM stimulus. Since bmx co~ters ~e eftWct of ~I-2, o~ resuln~ c~ be used to m~el ~d identify stepwise apoptosis ~d cell prolit~mfion in br~st c~cer,
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Biographical Sketch Billy W. Day Education As=~%tant Professor 09110/6I Oklahonm City Universib,, Oklahoma City, OK University of Oklahoma, Oklahoma City, OK Massachusetts Institule of Technology, Cambridge, MA Research and/or Professional Experience B.S. 1982 Chemistr3qBiology Ph,D. 1988 Medicinal Chenz Postdoc 1988- Chemistry/ Fellow 1991 Toxicology Positions: 1986 1986-1988 1988-1991 1991 - present I991 - present 1992 - present 1991 - present Honors: Forensic Chemist, Serology and Drag Analysis Division, O "ldahoma city Police Department, Oklahoma Cit3,, OK Adjunct Professor of Chemistry, Department of Science, Oklahoma City Community College, Oklahoma City, OK Postdoctoral Fellow, Department of Chemistry and Division of Toxicology, Massachusetts Institute of Technology, Cambridge, MA Member, Pittsburgh Cancer Institute, Molecular Carcinogenesis and Experimental Therapeutics Groups, Pittsburgh, PA Member, Drug Metabolism Group, Center for Clinical Pharmacology, University of Pittsburgh, Pittsburgh, PA Affiliated Researcher, Div. of Toxicol., Dept. of Chem., and Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, MA Assistant Professor, Departments of Environmental and Occupational Health and Pharmaceutical Sciences, University of Pittsburgh Graduate School of Public Health and School of Pharmacy, Pittsburgh, PA National Institutes of Health, MIT Hazardous Substances Management Program, and American Cancer Society Postdoctoral Fellowships, 1988-91 John B. Bruce Memorial Outstanding Graduate Student in Medicinal Chemistry Award, University of Oklahoma Health Sciences Center, College of Pharmacy, 1988 American Institute of Chemists Outstanding Senior Chemistry Student, Oklahoma City University, 1988 National Science Foundation Undergraduate Summer Research Fellowship (Molecular Biology/Physical Biochemistry), Oklahoma State University, 1981 Selected Publications (total of 29 peer-reviewed publications): Goldman~ R., Stoyanovsky, D. A., Day, B. W., Kagan, V. E. Reduction of phenoxyl radicals by thioredoxin results in selective oxidation of its SH-groups to disulfides. An antioxidant function of thioredoxin. Biochemist. 1995, 34, in press. Hopp. L., Bunker, C, H., Day, B. W. Quinine sensitive changes in cellular Na+ and K+ homeostasis of COS-7 cells caused by a lipophilic phenol red impurity. In Vitrq 1995, in press. Day, B. W., Jonnalagadda, S. S. Synthesis of Z- and E-[2,3-2H21 and [2,3-3H2l-I,l-dichloro- 2,3- diphenylcyclopropane (~H- and 3H-Analog 11 and its u-ans isomer). J. Labelled Comp. Radiopharm. 1995, 36, 73-78. Banni, S., Day, B. W., Evans, R. W., Comn~u, F. P., Lombardi, B. Liquid chromatography-mass spectrometric an~ysis of conjugated diene fatty, acids in a partially hydrogenated fat. J. Am. Oil Chem So¢. 199~,, 7l, 1321-1325.
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Ryan, L. K., Jin, R., Boggs, S. $., Kaml, M. H. artrt Day, B. W. A mouse model for assessing endoto.'~a involvement in the lus-ag inflammation and cytokine production resulting from inhaled organic dust. Inha]. Toxic01. 1994, 6, 485-499, Kagan, V. E., Yalowich, J. C., Day, B. W., Goldman, R, Stoyanovsky, D. A. and Gantchev, T. Ascorbate is the prirnaxy reductant of the ,p, henoxyl radical of e~,oposide (VP-16) m the presence of thiols both in cell homogenates and in model systems. Biochemistry 1994, 33, 9651-9660. Hossain, M. B., van der Helm., D., Schmitz, F. J., Pordesimo, E. O., Magarian, R. A., Meyer, K. L., Overacre, L. B. and Day, B. W. Molecular sm~ctures and conformational studies of triarylcyclopropyl and related nonsteroittal anfiestrogens. J. Med. Chem. 1994, 37, 1670-1683. Day, B. W. and Singh, K. Fluoreseence spectroscopy in the analysis of hemoglobin adducts. Methods Enzymol~ 1994, 231,674-681. Tannenb'aum, S. R., Skipper, P. L., Wishnok, J. S., Stillwell, W. G., Day, B. W. and Tagbizadeh, K. Characterization of various classes of protein adducts. Environ. Health Perspect. 1993, 99, 51-55. Jin, R., Day, B. W. and Karol, M. H. Toluene diisocyanate protein adducts in the bronchoalveolar lavage of guinea pigs exposed to vapors of the chemical. Chem. Res. Toxicol. 1993, 6, 906-912. Du, L., Hossain, M. B., Ji, X., van der Helm, D., Magarian, R. A. and Day, B. W. Structure of 1,1- dichloro-2-(4- methoxyphenyl)-2,3-diphenylcyclopropane. Acta Crystallogr. 1992 C48, 887-891. Day, B. W., Sahali, Y., Hutchins, D. A., Wildschutte, M., Pastorelli, R., Nguyen, T. T., Naylor, S., Skipper, P. L., Wishnok, J. S. and Tannenbaum, S. R. Fluoranthene metabolism: human and rat liver mierosomes display different stereoseleetive formation of the trans-2,3-dihydrodiol. Chem. Res. Toxicol. 1992, 5, 779-786. Day, B, W., Skipper, P. L., Rich, R. H., Naylor, S. and Tannenbaum, S. R. Conversion of a hemoglobin o~-chain aspartate(47) ester to N-(2,3-dihydroxypropyl)asparagine as a method for the identification of the principal binding site for benzo[a]pyrene anti-diol epoxide. Chem. Res. Toxicol. 1991, 4, 359-363. Day, B. W., Skipper, P. L., Zaia, J. and Tannenbaum, S. R. Benzo[a]pyrene anti-diol epoxide eovalenfly modifies human serum albumin carboxylate side chains and imidazole side chain of histidine(146). J. Am. Chem..Sot.. 1991, 113, 8505-8509. Day, B. W., Magarian, R. A., Pento, J. T., Jain, P. T., Mousissian, G. K. and Meyer, K. L. Synthesis and biological evaluation of 1,1-dichloro-2,2,3-triarylcyclopropanes as p~re antiestrogens. J. Med. Chem. 1991, 34, 842-851. Hossain, M. B., Wang, J. L., van der Helm, D., Magarian, R. A., Griffin M. T. and Day, B. W. Structural comparison.of a gem-dichlorodiarylcyclopropane antiestrogen and three of its derivatives. Acta Cr3'gtallogr. 1991, B47, 511-521. Jankowiak, IL, Day, B. W., Lu, P., Doxtader, M. M., Skipper, P. L, Tannenbaum, S. R. and Small, G. 1. Fluorescence line narrowing spectral analysis of in vivo hemoglobin adducts of benzo[a]pyrene diol epoxide: comparison to synthetic analogue;. J. Am. Chem. Soc. 1990. 1 I2. 5866-5869. Day, B. W., Naylor, S., Gan, L-S., Sahali, y.,,Skipper, P. L, N,g, uyen, T. T., Wishnok, L S. and Tannenbaum, S. R. Molecular dosimetr3' of l~b cyclic aromatic h~,drocarbon epoxides and dioI epoxides ~Sa hemoglobin adducts. Cancer Res. 1990, 50, :~611-4618.
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Other Support -- BiIIy W. Day, Ph.D. Active Title b. d. e. Source and identi.fb~g no. Nrltt, 5 ROI-CA57288 P.I. Billy W. Day, Ph.D. Ant/tumor Mechanism of Dichloro-cis-diphenyleyclopropane Role Principal Investigator, 20 % Effort Dates and costs of entire project 94/02/01 to 97/01131 ; $243,553 direct Dates and costs of current year 94/02/01 to 95/0t/31 ; $75,553 direct Specific aims of project 1. Characterize growth inhibitor, action of title compound.in MCF-7, MCF-71LY2 and MDA-MB-231 cells 2. Synthesize radio- and heavy atom labeled versions of the title compound 3. Determine stnactures of metabolites of t/fie compound in cell culture and microsomal systems 4. Determine if covalent adducts of title compound are formed in vitro 5. Determine tissue distribution of tide compound artd its metabolites in trice Scientific and budgetary overlap None Adjustments None Title b. C. Title b. C. d. e. Source and identifying no. NIH, 5 R01-HL51469 P.I. Douglas R. Spitz, Ph.D. Nitric Oxide-Induced Cell Injury: Molecular Mechansims Role Co-Investigator (P.I. of consortium), 5 % Effort Dates and costs of entire project 94/07/01 to 98/06/30 ; $ 586,629 direct (consortium costs: $ 65,162 direct) Dates and costs of current year 94/07/01 to 95/06/30 ; $157,869 (consortium costs: $18,812 direct) Specifie aims of project 1. Determine if exogenous reactive nitrogen species (RNS) results in quantifiable injury to cells in culture which correlates in a dose-response or time-dependent fashion with oxidative damage to lipids, proteins and DNA. 2. DeCermine if enymatically-genemted RNS results in the same activity as per aim 1. 3. Determine if cellular resistance to oxidative injury confers resistance to RNS. 4. Determine if manipulation of cellular ant/oxidants alter cell injury and oxidative damage caused by RNS. 5. Determine, in cell culture, if exposure to low levels of RNS is capable of inducing an adaptive response. Scientific and budgetary overlap None Adjustments None Source and identifying no. Univ. Pittsburgh Central Development Res. Fund P.I. Billy W. Day, Ph.D. Metabolically Activated Ant/tumor Agents Role Principal Investigator, 5 % Effort Dates and costs of entire project 93/07/01 to 95/06/30 ; $13,000 Dates and costs of current year 93/07/01 to 94/06/30 ; $13,000 Specific aims of project 1. To synthesize several analogues of Z-l,l-dichloro-2,3-diphenylcyclopropane for erthancement of mbulin polymerization inhibitory and anficancer activity. This is a seed money grant. Scientific and budgetary, overlap None Adjustments None
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b. d. fo Title b. C. d. ~cdc~to~ Tr~r~g ~ Brc~t ~cer Bio!o~" ~ ~empy Role F~dpafing Fac~: continent cffo~ Dates~dcos~ofcn&eproject 9~/0%'0I to9S~'0~/31; 5754,4~ect Dates and cos~ of c~ent y~ 94~09~0I to 95~'0~)31; $1~9,2~ ~ect S~c ~s of pr~ie~ I. Re~it qu~ed pr~tor~ students to the ~ing pro~. 2. Edu~te stu~n~ in the f~en~ p~ples of bre~t cmce~a~oNolo~, 3. Ev~uate ~e pro~ess of ~e student. 4. E~uate ~e pro~ ~.d seek ad~6c, n~ furxds ~d teso~ces. ScienCe ~d budge~' overlap None A~us~en~ None So,co ~d identifying no. Amefcan Cancer S~iety, D~-125 P.I.J.C. Yalo~ch, Ph.D. Free R~ie~ Activation of ~-16ffopo ~ ~temcfions RoIe ~whvesfigator, 10% Effo~ Dates and cos~ of en~e project 94/07/01 to 97/~/30; $ 311,819 direct Dates ~d cos~ ofcu~nt ye~ 94/07t01 to 95]~]30; $101,272 direct S~c ~s of proj~t 1; F~er ch~ct~ ~e topo ~ ~hibito~ acfi~ of ~ee radic~ ae6vated VP- 16 ~ h~ le~e~a K562 cells. 2. Ide~y activated fo~s of VP-16 ~ the presence of exogenous ~ee ra~c~ i~fiato~. 3. Iden~ ~d ch~actefize ~ect che~c~ ~teracfion ~tween acfivat~ VP-16 Scientific ~d budge~ overlap None Adjus~en~ None Pending Title b. C. d. e. Title b. Source and identifying no. Environmental Protection Agency P.I.B.W. Day, Ph.D. New Mass Spectral Methods in Human Exposure Dosimetry Role Principal Investigator, 20% Effort Dates and costs of entire project 95/07/01 to 98/06/30; $ 345,364 direct Dates and costs of current year 95/07/01 to96/06/30;; $110,637 direct Specific aims of project 1. Prepare isotope-labeled internal standards of human serima "albumin (hSA) covalently modified With 2,4- and 2,6-toluenediisocyanate, benzo[a]pyrene anti-diolepoxide and fluoranthcne anti-diol-epoxide. 2, Use the isotope-labeled internal standards to study ion geam and pneumatically-assisted elec.trospray mass spectral techniques for det~tion and quantitafion of hSA adducts of the electrophiles in aim 1. 3. Develop internal standardization methods for MALDI-LAMMA analysis ofhSA adduets. 4. Develop laser and pneumatically assisted electrospray MS techniques tbr detection and quanfitafion of.intact hSA adduets. Scientific a~d budgetary overlap None Adjustments None Source-and identifying no. NCI -- SPORE in Breast Cancer P.I. Ken McCarty, M.D., Ph.D. SPORE in Breast Cancer (BDay subproject: QSAR/Synthesis/Evaluation of Anti-Breast Cancer Agents) Role P.I. of subproject, 25% Effort Dates and costs of entire project 95/IN/01 to 98/08/31 ; $ 392,878 direct sabproject c_r)st~ Dates and costs of current year 95/01./0l to 95/1~3I ; $16,4,270 direct subproject COSTS
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Sped~¢ ~ of QS~R le~ing 2. Comp~ resets from ~ 1 to e~i~g compum~onfl ~mb~. 3. ~e~ct ~e ~=~es ~d activities of, s~m~e~ or ob~, ~d test ag~nst h~ bre~t c~cer ce~s ~ c~e new che~c~ entities tbr bre~I e~eer ~a~en~ S~en~e ~d budge~, overhp N~ne Adjustments None Title b. d. e. Source and idenfi .fying no. NIH, 5 R01 ES05651 P.I. Mei3,1H. Karol, Ph.D. Chemically-Induced Chronic Allergic Lung Disease Role " Co-Investigator, 10% Effort Dates and costs of entire project 95/08/01 to 99107/31 ; $1,124,350 direct Dates and costs of current year 95/08/01 to 99t07/31 ; $ 267,455 direct Specific aims of project 1. Identify adduction sites of toluene diisocyanate on extravasated serum proteins in lungs of animals e~osed to the chemical. 2. Identify in vivo adducts in lungs of animals by electron microscopy. 3. Determine the nature of the allergenic TDI-protein conjugates. 4. Test the hypothesis that oxidative processes are involved in the sensitization to TDI. Scientific and budgetary overlap None Adjustments None
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Biographical Sketch Balachandran, Raghavan Education Re~m-ch As.~-'iate 08116/45 Kerala University, ~dia Kerala University, ~dia Jawaharlal Nehru Univ., India Jawaharlal Nehru Univ., India Positions: B.S. 1967 Botany & Zo3IoD' M.S. 1969 Genetics & Plant Brd. Ph.D. I978 Life Sciences Postdoc 1978-79 Biology 1970-1972 1978-1979 1979-1980 1980-1983 I983-1986 1986-1994 1994-present Publications: Teacher, Thimphu Public School, Thimphu, Bhutan Post-doctoral Fellow, Jawaharlal Nehru University, India, Biology Research Associate, Department of Radiology, Washington University, St. Louis, Visiting Fellow, Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD Visiting Associate, Labomtog,, of Biochemistry, National Cancer Institute, Bethesda, Research Associate, Department of Infectious Diseases and Microbiology, GSPH, University of Pittsburgh Research Associate, Department of Environmental and Occupational Health, GSPH, Universit3, of Pittsburgh Aaronson, S.A., Storch, T.G., Balachandran, R., and Reddy, E.P. Different hematopoietic target cells for transfo .rmation by replication-competent murine leukemia viruses. In Marchesi, V.T. and Gallo, R.C. (Eds.): Differentiation and Function of Hematopoietie Cell Surfaces. New York, Alan R. Liss, Inc., 1982, pp. 251-261. Balachandran, R., Reddy, E.P., Dunn, C.Y., Aaronson, S.A., and Swan, D.C. Irnmunoglobulin synthesis and gene rearrangements in lymphoid cells transformed by-replication-competent Rauscher murine leukemia virus: transformation of B ceils at various stages of differentiation. EMBO J., 3:3199-3207, 1984. Gupta, P., Balachandran, R., et al. Detection of human immunodeficien~y virus by reverse transcriptase assay, antigen capture assay, and radioimmunoassay. J. Clin. Microbiol.., 25:1122-1125, 1987. Balachandran, R., Thampatty, P., Rinaldo, C.R., and Gupta, P. Use of cryopreserved normal peripheral blood lymphocytes and isolation of human immunodeficiency virus from seropositive men. J. Clin: Mierobiol., 26:595-597, 1988. Gupta, P., Balachand~an, R., Ho, M., Enrico, A., and Rihaldo, C.R. Cell-to-cell transmission of human immunodeficieney virus in the presence of azidothydine and neutralizing antibody. J.. Virol., 63:2361- 2365, 1989. Melder, R.J., Balachandmn, R., Rinaldo, C.R., Gupta, P., Whiteside, T.L., and Herberman, R.B. Cy~otoxic activity against HIV infected monocytes by recombinant interleukin 2 activated natural killer cells. AIDS Res. & Human Retroviruses, 8:1011-1015, 1990. Gtapta, P., Balachandran, R., Thampatty, P., Rinaldo, C., and Ho, M_ Oxophenarsine, and antisyphillis drug inhibits H1V-1 specific protein synthesis in acuteIy and persistently infected Iyrnphocytes. AIDS Res. & Human Retroviruses, 6:I417-1423, 1990.
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Balachandran, R., Tnampat~.", P., Enrico, ~., P.in:i/do, C., :rod G~p~.~. P. H~man immuncdefiden~.v isolates.from a~anptonmfic homo~xu-'.d m~n and from .~h]DS p~fien~ hav~ distinct b~olo~c ~d gen~c pralines. Virology, 150:229-23S, 1991. Furtado, M.R., Balachandran, R., Gapta, P., and Wohn~kT, S.~L Analysis of alternatively ~Iiced human immunc~teficien~,, virus .type I tHIV-1) mRNA spedes one of ~a, hich encodes a novel tat-env-fusion protein. Virology; 185:258-270, 1991. Shankaxappa, B., Balachandran, R., Gupta, P., and Ehrlich, G. Introduction of multiple restriction enzyme sites by in vitro mulagenesis using PCR. PCR Methods & Appl. 1:277-278, I992. Balachandran, R. and Gapta, P. Active nuclear transport of proviral DNA is important for HIV-1 pathogenesis. Abstract FXth International Conference on AIDS, Berlin, 1993. Grovit-Ferbas, K., Balachandmn, R., and Gupta, P. HSV- I and HI'V- 1 interaction induces apoptosis in CD4+ T cells. Abstract The First National Conference on Human Retrovimses and Related Infections, Washington, De, Decemlx~r, 1993. Sova, P., Gupta, P., Balachandran, R. and Volsky, D.J. Genetic v,~ability of the human inmmnodcficiency virus type 1 (HIV-1) vif gone in vivo. ~, 1995 (in press). Balachandran, R. and Gupta, P. A defect in nuclear lranslocadon of proviral DNA is responsible for lack of growth in T ceil line of human irnmunodeficiency virus isolates from asymptomadc homosexual men. Virology, 1994 (submitted). Balaehandmn, R. and Gupta, P. Incomplete reverse ~anscription and lack of integration are responsible tbr the reduced expression of human immunodeficiency ,~ irus isolates from asymptomatic individuals (in preparation).
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Other Support - Raghavan Balachandr~, Ph.D. - Co-Inve~figatc, r Active None Pending a. Source and identifying no. NI]-t P.I. Donald R. Mattison, M.D. Title Role of Apoptosis in Ovarian To.~icitb, b. Role Research Associate, 100% Effort c. Dates and costs of entire pr~ect 07-01-95 to 06-30-00; $ I,i 17,033 direct costs d. Dates and costs of current year 07-01-95 to 06-30-96; $ 238~224 direct costs e. Specific aims of project 1. Analyze whether PAHs induce apoptosis in human ovarian cells. 2. Identify the role of c-myc, c-myb, c-fos, bcl-2 and p53 in ovarian toxicity. 3. Elucidate the roles of transcription, translation, cellular proteases, endonueleases and topoisomerases in ovarian toxici .ty. f. Scientific and budgetary overlap None g. Adjustments If the NIH grant is funded, a suitable repIacement for Dr. Balachandmn be identified and hired for the Research Associate Position under Prof. Day.
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copies.) Ac~mo~led~e~t Card 1. CategoD and Suhdassifk'atfon Isce Pages 10-tlh Typ, the five-character c~d~ in dw. h[oc~ f;rm,[ded, tbr example. I CO NOT FOLD [ o:,L', DeMvafi~ of Z-l,t-~i~Iaro-2~-diph~Ic)d~pr~pane (Analag ~ that br~ ~ncer ceil g~ by ~h~i~g tuhulin palyme~=ti~n, J~a~ad~ S.S., ~m:l, E., ~t ~, E., Day, B.W. Dep~m~nt of Envkonm~n~ & Occ~pa~o~ H~, Univevjty of Pi~b~h, PA I5~ (SSJ, E~, B~) ~ ~bcmtcU of ~,IoIcc~ Na~onN ~c~ ~mte, B~es~ ~,~ 20S92 ~. Z- I,l~chlorc-2,3-~henylc~ cl~prop~e (~ ,~Nog g) is ~ effec~ve ~ti- b~t c=:~ ~gent in rcdan~ ~d ~ ceR c~t~e. We recently detem~ad t3at we~ ~bu~n polyme~=~on inhibiteD' acfivi~ gC~ I0-14 ~, In INs s~dy began to develep a s~cmre-acti~ity re~onship of ~chIomdiphenylc~cl~m~es tubulin polyme~cn ~hibito~. Vm~o~ derivatives of AnMog II '~ere prepped and tested for the~ inhlN~on of tubulM polyme~ation. Of ~e compounds tested, Z-IA- ~chlom-2-(&me~ox~henyl)-3-~henylcyclopmp~e,Z-l,l-~chlom-2-(&~uomphenyl)- 3-phenylcyelaprop~e and Z-IA-dichlaro-2.(4-chlomphenyl)-3-phenylcyclopro~ane were fo~d to be active with IC~o vMues of 3-5 ~.I. E-l,14ichIoro-23-dia~yl-2,3- • phenylcycIopmp~e w~ equipotcnt wi~ d~ethylstitbes~al (DES) (IC~ = I0 gM). Other ~ogs. such ~ 4-Mmeflmxy, 2,Sdifluom, 2,4-dR]uom and 4,4"-dimethoxy exhibited some effect on the polyme~ation r~ction by delaying its onset. The cytos~tic acridly of ~ese compoun&~ in hum~ 3re~t c~cer cells is 3eing evMuated. For example we have found ~at these comNunds have effect on MCF-7 cells at 104 -10"~ M. (Supposed ~y ~t ~nt CA 57288). Type abstract v, ithin blue lines. See sample abstract. 2. Abstract is SPONSORED by: 7 7 6 6 Member No. $. Abstract is to be PR~ENTED by: (See DirectoD' of Members tbr Member No ) Billy W, Day 260 Kappa Drive M,2mber N,I. Ill'an AACR .Mem~er.t Name Address 260 }~p,p,{, Drive .Aaere,,~ Pittsburgh, PA City.State Pitt shurg.h, PA CiLv. Sta~e 15 2 3 8 _ P~:~tal Code USA h ] P-067--~502 ...... Telephone No. 412-967-6507 6 [ 2-6 2~-1020 FAX No. 4 i2-6 24-I020 FAX ~,~ 3. As the SPONSOR of fftL,; abstract and on behalf of all the autb:,rs. I indicate my s~pp,:~ for the data comai~ed hereto and trm~s)'er i~ to the American Asso,:h~eer R~e~ch, D~. 4. fC~,m;bre ordy ~fSPONSOR iS ~ Ass~c~te Member~ L the A~od~I~' ~lcm~er i~ ~'p,:,n,,w ~n,J presenter, IS~< ~uh:,,:.n~ AF, pI>,n 6. Eliglhility f~r Trnvel Awards ~A. The PRESENTER of t~m abstract is a medi~:al or graduate ~tudenl. physician in training, or ~docrom~ fdlow. ~ B. ~-~ PRESENIER m;~ts ~= zd:e~a in Bux A aSa~e and ~., al,) a !5
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~ Ofi~nzl fo~ ~I~ 2 c~mplelely and ~ A ~¢IGaddres~e~ I. CategoO' and Subelussificalion (s~ Pages I0-111: T~pe ~e fivceh~¢ter c~de in ~be blo~k~ provided, for example, 2, Abstract is SPONSORED by: ~rv 1995 AACR ,,IBSTRACT FOl~5,I ~. ~:::z: Analysis " of th~ -antiproliferative~action-'or - Z-I,I-di~Mo~0~2,3. diphen~IcycIopropane in breast tumor celIs. ter H~, E, ad Day, B,W. Deponent of Envkonmen~ Univ~sig of Pk~b~gh, Ht~gh, PA 15238 Z-l,l-Dk~or~2,3-diF~enyl:ycloFrcp~e(I) has Ion~ been ~u~cro~phi~ ~ mice, ar~proM~fi~e in mt m~m~' minor m~e~, ~d to have a LD50 m r~en~ of >3g&g, Since 1 h~ a low ~ogcn r~eFtor ~) ~fini~', ~e have MCF-7 ~R+, ea~ogcn =s~nsive), blCF-7~Y2 ~R+, ~ogen ~cs~nsive) N~A-N~-231 ~-, es~ogen u~es~onsive), We found Nat 1 inhibited pro~cra~on ~fl ~e cI~nogcn/c capacity of NI ~e ce~ mcch~m is indeNndent ~om ~e es~ogcn receptor. Mcm~c sm~es have shown ~at ~e major memento cf 1 ~ter incubation wi~ ~e ~ee bre~t tumor ceil ~nes is Z-chlor~hNcone. ~en we ~stcd ~e mfiprolffemfive ~d ~ficlonogenic capacity of ~is mcmbolile we found ~t k was 10 times more potent th~ I in ~hc ~ee cell Iines. Because ~e mechanism of ac~en of I seems to es~gcn ~ap:cL 1 ~d Z-chlcr~h~cone were t~ed for bin~g at ~e ty~ g nucl~ bin~ng site. Bo~ com~un~ w~e ~so tested for ~ubulin ~lymefizafion inhibi~un. ONy I inhibited mbut[n ~lyme~fion at 10 ~ O~ ~'itro. A ~ssz~le deto~ica~on mute for 1 ~d Z-chlor~hNcone w~ stu~ed by ~Nyzing Z-chlor~h~cone in ~e presence of gtu~ione. Nstead of foxing glum~ione, 1 was solvolyzed into Z-2-chloro-2,3-~phenyl-2-propcn-l-ol, and subsequently rapidly oxidized ~d dechlodnated into g~s-chNcone, a non-to~c communal. (SupN~ed by ~H g~t C~7288) Type abstract within blue lines. See sample abstract. 671 Member No. (See Director)' of Members for Member Conner~ Ph.D. Name (Plc~c Print} U n±versity o~ Pi~tsbu~ah .... • 260 K~drive Pie tsburgh PA Zip/ 15238 Posul C~d~ U.S,A. (/~ I2) ~-6538 ~,~ 2) 624-1020 City.State .Counm.,, Tdepl~one No. F.~X No. 3. As the SPONSOR of thi~ ab:;lract and on behalf of all &c ambx_~. 1 Mreby indicate m) ~upp,:n for the data c~mairted herein and ~o.~t~r 5. Abstr,~ct is to be PRESENTED by: Ernst ter Haar (If an AACR Member) --ih%gaeraity ef Pi~-e=_~'argh 260 KaDnadrive Member No. Name (Please Print I Address 15238 . P~sI~/Code U. S .&. ~ountD- .... (6 !2) 967-6507 (~12) 624-1020 FaX No. E~gigility for Trowel 17.
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Synthesis of Z- and E-[2,3-2H:z] and [2,3-3Hz]-l,l-Dichloro-2,3- diphenylcyclopropane (2H- and 3H-Analog H and its trans Isomer) Bill**" W. Do.v" and Sastr.~ $, Jonnalagadda Departments of Environmental & Occupational Health and Pharmaceutical Sciences University of Pittsburgh 269 Kappa Drive Pittsburgh, Pennsylvania 15238 USA SUMMARY ZH and ~H labeled Z- and E-l,l-dichloro-2,3-diphenylcyclopropane (1 and 2) were ~3'nthesized starting from Na132H4 and NaB3H4 reduction of benzil. The resMtir~g glycols were transformed to the 1,2.1abeted Z- and E-stilbenes by thermob'sis of their ~'clic th~onocarbonates in trimethylphosphite. The stilbenes were reacted with tTha~re trar.rfer-generated dichlorocarbene to form t~e title corr~youn~. The dideuter~o isomers were separated by fractional crystaIfization in yields of 60 and 48 %. Each was greater than 99 % geometrically and 98 % isotopically pure. The ditritfo isomers were separated by C-18 HPLC. Th~ radiochemfcal yields, on a molar basis using benzil as the limiting reagent, were 42 % and 23 %, each with specific activity af88.5 mCilramol and radiachemical purity qf > 95%. Key words: 1,1-dichloro-2,3-diphenylcyelopropane, deuterium, tridum, Analog II INTRODUCTION Z- 1,1-Dichloro-2,3-diphenylcyclopropane (1), also known as Analog II (1), is a novel anti- breast e~cer agent, effective in vivo against car~q.nogen-indaced and War~splantable r~t mammary tumors (2,3) as well as in vitro against estrog~-dependent and -independent human breast cancer cells grown in culture (4,5). The mechanism(s) of anfiproliferative and eytotoxie action shown by 1 is (are) unknown. We arc currently storying the metabolism and macromolecule binding of this agent b~th in vireo and in vivo, and our studies required b~th high specific aetlvity mdiolabeled 1 for biodistribudow'radiotracer experiments and stable isotope heavy-atom labeled I for use as an internal standard in mass spectrometric determinations, and as a probe for metatx~lie reacfien mechardsms. This communicatien describes the synthesis of high specific aclivi~ [2,3-~H.7]-labeled ~cl high iso:op~c purity [Z3-ZHzl-labeAed L Tt~e syrtdaetlc procedures utilized al~o yHded appxeciable amotmts of rite labeled versior~s of the biologqcally inactive E isomer 2. CCC 0362-4~ S 02. ~/!) i 13073-!36 1'3'34
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DISCUSSION ~rc~=~re~ ~ o~!d ~ ~ppI~bl: ~o ~: ~fi~ Ia~IM ~mFo~n~ ~d to dete~n= if ~: versions e~ 1 to ~y disc~mable e:¢mnu Ben~ w~ reduced wi& 1.2 mol~ equiv~e~ of N~2~ ~d the rc~uI~g ~<,renate c~I~x w~ decomposed ,M~ aqg~o~ HCI to yd~Id [I~-~H~-I,2- diphcnylgly¢ol 3 ~ q~v¢ )deld. ~¢ #y¢ol w~ ~sfo~¢d to a 1:I ~xt~ of" ~ Z- and E- ~ionoc~nate 4 by reaction ~vi~ one mol~ equivMent of ~¢ ~d=oI: derivative of ~iophosgcn: in boiling toluene. ~ea deoxygenated in ~fling ~methylphoap~t~. ~c ~deut:~o sfil~nes were ~fied ~ a ~x~re by fl~h SiOz chmmato~aph], ff~ subjected to ph~, ~sfer ~hlo~Ucbpmp~afion using ~CI~, 33% NaOH, =$ benzy1~e~yla~oMum chlo~de ~ the ca~yst (1,8). ~ reacdon ~e was exmct~ ~d subjectM to flash Si~ ¢~to~phy to pufi~, ~e cycloprop~es as a t:1 mixture. The Z isomer [2~.2H~-I was isolated by ~sml~=fion wi~ p,woleum ether conmin~g a w~e of ~mlute EtOH, ~en ~oyst2~M from pewoleum ether (60 % yield from 5). The E isomer [2#~H~-2 was obtMn~ from the mother liquor by ~stMliza~on from absolute E:OH (48 % yield ~m ~. Capfll~ GGMS ~d proton ~IR ~alyses indicated tMt oach w~s > 99% pure geome~c~y, ~d that ~e isotopic purity at C-2 ~d C-3 was ~ 98 ~ ~e syn~esis of ~e ~do ccmpo=~ was peffo~ed u~ng the s~e synthetic roam. U~ of a 1.2:1 mol~ rado of N~3H~ to ~n~ did ~ot ~eld eomplem convemion to ~e #ycol ~ o~ h=~. ~H*NaOH -- ~n O~ CH~H$. 5 2) H~ (c}I~OhK a ~ + c~*~, N*~H ~
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1":'0,000- 100,000 80,000' ~0,000- 40,000 - 20,000- 0 0 ~ "10 "15 20 Time (rain) Figure 1. Upper: Representative OV (214 yen) d~tected chromatograra of the reversed pAase C-18 HPLC purilTcation ~I'[2,3dFlz]-2 (retention time 14.4 rain) a~d [2,3-stI,j-I (retention time 1Z2 m~n). Lower: Rad~bchr,)mato~ram of t~e one m~a.t?ac~i,~ns collected from the upper rrac,~'. T~: 50374239
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EXPERIMENTAL pre~s~e). Ma~ aNecm w~e dete~ in eI~cn ioa~on (70 eV) mcd~ ~gh liquid c~e~o~phy (~LC) was pe~;:,~ed ca a Hewlett Paced I~ LC equipped ~i~ a I040 di.:d~ ~y, detecter ~ a 3L~) Chems~a~e~. Compou~& were se;m~ed on a 7.8 x F~cp Nova-P:~ HR C18 CO,~ G ~ rever~e-~h~e cdumn (~fil~) ~g a He-spigot mobile p~e of 7:3 ~CN-HzO. NaB3~ was purchased ~om New Engl~d Nuetex. reagent:, c~cma~o~ph~c mated~:~, ~d ~,I~'en~ ~'~e pu~h~ed from Al~ch Chem~cM Co. ~d l.~-d~ehenv~-l.Z.ethanedio~ fi,Z-~H~I ffl,Z.z~ Benzil (2.1 g, 10 retool) was di~olved in 30 ~ CH3OH, s~, ~d ~¢at~ poNon-Mse wilh N~ ~ 98 % aH isotopic p~, 485 rag, 12 retool) over a 2 h Nfiod. ~e ~xmre w~ s~ ~ ad~fionN 1 h at r~m ~em~emtme, fl~cn poured onto 1~ ~ ef 10 mM H~ ~d s~ ~ ad~fion~ 0.5 h. The solution was a~iusted to pH 8 M~ NaOH, ~en exacted ~lh EtbAe (4 x 75 ~), ~e oN~ie layem were combin~, washed Mth HzO, ~ea ~IgSO4), filter~, ~d ¢oncen~t~ on a rol~, evaporator the pr~uct as a white solid (2.16 g, 1~ % Neld) which, when ~NyzM by GC-MS, applied to 100% pure, and w~ us~ without Nnher purification or ch~etefizafion. EI-MS, ~z (%): 108 (i00, (C~s~OH)+), S0 (~0), 78 (42), 5~ 1.2-DiDhenvl-l,~-ethanenvrothioqgcarbonat¢ rl D~dcut~o compound [1,2-~H~]-3 (2.16 g, 10 retool) w~ dissolv~ ~ 90 ~ tolu~a~ comaining 1,1'-~i~boayld~midazol~ (90 % P~W, 1.98 g, c~ 10 ~ol) ~d h~at~d to ~flux wilh scrag for 2 h. ~ ~olvent was r~moved on a ~o~, evaporator ~d ~ resulting ~row~i~h ~o~ was purified by flash SiO2 chromatography (2:1 ~2Cl2-p~oIeum ~the0 to giv~ [1,2-2H2]~ as a foul-smelling, w~t~, ~j~line solid (2.5 g, 97 % )4eIfl). A v~id m~l~ng point could ~ot ~ ob~cd du~ to th~ hy~oscopi~ ~d ~e~olab~o n~t~r~ of~o pr~c~ El-MS, ~z (%): 258 (7, M+.), (1~). ~-~IR, d: 7.2-7.07 (m, 6H), 7,03-6.8g (m, Z- and E-SiliCone [i.2-0i-2~~]-5 and 1,2-~ Tho didcutono th~o~opy~oc~nale [1,2.2H~]-4 (2.5 g, 9.7 retool) was dissolved in 100 ~ of (CH3OhP (stench!) ~d hea~cd to reflux ~4th ~fi~ng for~3 h. ~e ~olu6on was c~l~d ~d R~c ~olvcnt wa3 removed on a ro~' evaporator. The r~ulti~g brown oil w~ puttied by flash Si~ (yetcc~Ieum ~d~er t~:,llowed by 9:1 ~e~,?Ieum eS-~ec-~zClz) ~o give a 1 :I rak~t~r¢ of ~h~ products ( 1.73 g, 96 % >.-re!d) ~xs ~ ~a,,~ oil EI-MS (the ma~ ~pec~ of [I~2.aH~]-3 a~d [1,2-~H~1-6 equivalccl0, m]z (%): IS2 (I00, M~.). 167 (28), I53 (8), 90 (I2),77 (:g), 51 (5).
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yieM from ~ Z-~fil~ne). EI-MS, m/z (~): 265 0.5, ~7~7~1 -M*.), 266 (5, 3~C137CI-M*.), 2~ (~.5, ~CP~Cl-M*.), 231 (2~), 22~ (60), 193 (66), 16S (13), 153 (35), 1S1 (1~0). tH-NMR, 7.255-7.2 (~, ArH, 6~, 7.055-7.0 (m, ArH, 4H), 3.296 (~, ~idu~ ~3¢Iopropyl Th~ t~naI moth~r l~quor wa~ ~cat~d with EtOH to ),i~Id [2,3-ZH~.2 a~ a white solid, rap: 41-43 (602 rag, 48 % y[elfl ~om the E-s~ne). EI-MS, ~z (%): 268 (1, 37C137~-M*0, 266 (3.5, 35C137CI-M+.), 2(~ (5, 35C13sCI-M+-), 231 (20), 229 (58), 193 (50), 153 (38), 151 (lC0). ~H-NMR, 6:726 (br s, ~H, 10~, 3.225 (s, residufl eyelepmpyl ~H, 0.08 H). Z- and E-I.l.U~ehloro.2.3.d~henvlevclonrovane ~~ BonN1 (1.4 rag, 7 greet) w~ dissolved in 1~ of ~3OH, a 1:1 ~xmre of N~3~ (t 3.9 gruel, 5 mCi) and N~ ~H4 (13.9 ~oI) in 103 ~ of 0.1 N NaOH w~ added, ~d solution was st~ed at morn tempemt~e for 24 h. The ~xt~e was adjusted m approxi~tely pH 2.0 with I N HCI and Ne soludon was evaporated to ~n~s N vac~. ~e residue was ~ssolved ~ 2.0 n~ of toluene ~d 5.0 mg of I, l'-tNoc~nyl~m~d~ole w~ added. ~N ~xt~e was gently refluxed for 2 hmd evaporated m ~ness fn vats. To Nis residue, (~3013P (2 ~) was add~ and ~e ~t~e w~ heat~ to reflux ~d s~ed under ~ for 30 h. ~e (~30)~ vac~ ~d the ye~ow, oily residue was app~ed to ~ 5 ~ ~d. x 50 ~ Si~ onus. ~e sfil~nes w~c p~aHy p~ed by elu~g the colu~ by ~4ty flow wiO a ~adient of ~oIeum e~ to CHzCIz to CHCI3. ~e combin~ fmc~ons we~ concen~t~ in vac~ ~d ~e residue eon~ning the 1,2-~fiated sfilbenes w~ used in ~e next step without f~er eh~aetefi~tiom ~e 1,2- dtNfiated sfil~nes were ~ssolved in CHCI3 (1.75 ~) coning benzylNe~yl~oNum chloride (1.5 rag). To this ~xta~, 35% aqueous NaOH (2.5 ~) w~ addM &opwise while s~ng and the reaction was ~owed m proceed for 24 h at morn tempmmre. To ~e resulting tw~phase n~xt~e, CHaCIz (10 ~) was added. The oN~ie layer w~ ~p~ated,-washed ~ H~O (10 x 10 ~) to remo~'e the ca~yst m~d ~aces of NaOH, ev~erat~ to ~mess, ~d ~ssolved ~ of 7:3 ~CN-HzO. C-18 ~LC ~alysis of tNs ~xt~e in,cared the pro:once of compoan~ in a Z-to-E ra~o of 9:5. ~e two compound; were purified on a se~-p~p~afive u;ing c~. 113% c.f ~e ~de reaction prMuct for each f~LC ~p~c,n. Each id~nt;~
Page 65: 50374242
ACKNOWLEDGEMENTS Instigate l~w~.s~ Car..:er Initiative ~.:I the Urqversiv.,' ef Ntt~bargh Cer~wa.l Develop~--.n~ Recta'oh F~d. REFERENCES Magarian R. A. and Benjamin E. J. - J. Pharm. Sci. ~5,t: 1626 (1975). King M. M., ,.In,arian R. A., Terao 1. and Bmeggemann G. L. - L Nail, Cancer Inst. 7'4: 447 (19~5~. King M, M., PenIo J. T,, Magafian R. A. amd Braegge~-~mn G. L. - N~t~. Cancer 7:239 (I985). Day B, W., Magafian R. A., I:ento J. T., Join P. T., Mo~sissian G. K. and Meyer K. L. - J. Med. Chem. 34:842 (199I). ter Haar E. and Day B. W. - Prec. Amer. Assc~. Cancer Res. 3~4:345 (1993). Griffin M. T., Pento J. T., Magarian R. A., Mqusi~ian G. K. and Basmadjian G. P. - Endocrine Res. 16:269 (1990). Corey E. J. and Winter R.A.g. - .L Amen Chem. $~e. 85:2677 (1963). Dehmlow W. V. and $chonefeld J. - Liebegs Ann. Chem. 744:42 (1971).

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