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JAMES F. GI,ENN. M.D. April 8, 1993 Robert E. Rhoads, Ph.D. Louisiana State University Medical Center Department of Biochemistry and Molecular Biology 1501 Kings tlighway Shreveport, LA 71103 Re: Grant No. 3076R2 Dear Dr. Rhoads: The Council for Tobacco Research, USA, Inc. is pleased to award you a rcnewar grant in thc amount of $89,888.00 for the period from July 1, 1993 through June 30, 1994 for the study proposed in your application =Protcin Synthesis Initiation Factor 4E and Oncogencs." It is understood that this grant is made subject to acceptance by institutional authoritiks. Also, many applications to CTR indicate partial or complctc overlap with applications to other agencies. If the latter result in a~vards, we should bc promptly notified so that w~ can negotiate an appropriate adjustment of our award. Since this award will conclude the three years originally programmed for thc project, it is considered terminal for the project, and a comprehensive final report will be expected after its termination, as well as reprints of any current or future papers r.es~Iting from rcscarch supported by the award. Your attention is called to the enclosed =Important Procedural Information for Grantccs=. Plcasc fill in the attached =Notice of Rcscarch Project" and return it to me. Drs. Harmon McAllistcr, Scientific Director, and Vincent Lisanti, Associate Research Dircct0r, will scrvc as your primary contact with our scientific staff on matters concerning your grant. Picasc consult the staff as questions or problems arise, and kccp us informed about the progrcss of your study. As Principal Investigator, you should know that CTR recovers unexpended balances over $I,000 at the end of each grant )'ear (see attached "Financial Requirements" for specific details). Wc suggest that you personally keep an accurate record of expenditures and commitments to assure that you agree with your institution's official financial statement at the end of the grant year. Please note that year-end deficits cannot be recovered from CTR, even from funds awarded in a subsequent year. Finally, please be aware that our failure to receive timely annual financial statements from your grants officc will stop further grant payments. Appropriate forms will be sent to your financial officer at the end of the grant year. Administrativc problems arc relatively rare and wc hope that this statement of our fiscal procedures will obviatc any possible misunderstandings. Encis. cc: Mr. Ikd Muslow JFG:mm am~cs F. Glenn, M.D. bcc: Auditors, ROK, LP, FILE M/M, monitors 50326175

THE COUNCIL FOR TOBACCO RESEARCH - U.S.A., INC ~UI~PORTING I~IOI~EDICAL INV~$T|GA'EION Numb.er: 3076R2 9100 THZ~D AVENUE RiE,N!,EWA~L APPLICATFON for the Spring 1993 SAB rneet~ng Applicant: Institution: Louisiana State University,Shreveport, iA Area of c~cer Specialization: Title:,.Protein synthesis initiaticn factcr 4E and cnco~enesis" History: This program was considered as Case No. 2989 and encouraged by the Executive Ccmnittee. Applicaticn N. 3076 requested $92,223 for the first year of a three-year project. Budget estimates for the 2nd and 3rd years were $101,104 and $100,263 respectively. The request was approved at the April 1991 SAB ~eeting and f~nded ~or $80,000. Application No. 3076R1 requested $84,800 for the 2nd year of this 3-year prcgran. It was approved at the April 1992 SAB meeting and funded as requested. ~ Note thatREDAC.T.ED has .t~[s past sunder moved frun the University of }~_ntucky Medical Center in Lexington to hls new location in Louisiana. Request: This application requests $ 89,888 for the 3rd year of this 3 -year application. Documents Submitted: Application Dated: 11/..30/92 Progress Report: Yes (2 pages) Period Covered: 12/1/91 to 11/29/92 Biographical Sketches: Materials Reprints Manuscripts Number submitted Number that acknowlege CTR 2 2 2 Abstracts Other materials: Comments: As progress appears to be satisfactory, this application will be handled administratively and withheld from the bulky SAB Agenda for the SPRING, 1993 SAB meeting, unless you object. In this case your written comments will be appreciated end may be noted on the enclosed check-off sheet. Please return the check- off sheet at your earliest opportunity. If more than four reprints andlor manuscripts were included with this application, only four (4} of them have been submitted to you. Copies of only the abstract pages for any additional publications are included but If you wish the complete article, please inform me and I will forward it to you. F~DJ~CTED 50326176

THE COUNCIL FO~ TO'BAC.CO RESEARCH - U.S.A., INC FIRST RENEWAL SECOND RENEWAL [-~. DATE: 50 ~ove~er 199'2 PRINCIPAL INVESTIGATOR (Name, degrees and academic or professional title) Robert E. Rhoads, Professor and Head TITLE OF OR|GINALSTUDY Protein Synthesls PROPOSED START DATE Initiation Factor 4E and Oncogenesls FUND~ REQUESTED (CURRENT" YEAR) FUNDS TOTAL AMOUNT PERMANENT EQUIPMENT 89,888 0 REQ~JESTED ~OR NEXT YEAR (IF APPLICABLE) 1 July 1993 0 PRINCIPAL INVESTIGATOR FINANCIAL ADMINISTRATOR (do not indicate any co-P.I, on this page) (person to contact for budget information} NAME (LAST, FIRST M.I.) AND DEGREES: NAME: Rhoads, Robert E., Ph.D. Harold White DEPARTMENT: POSITION TITLE: Biochemistry & Molecular Biology Assistant Vice Chancellor INSTITUTION: Louisiana State gniverslt~/ Medical STREET ADDRESS: 1501 Kings Highway Cen£er CITY, STATE (OR COUNTRY) ZIP CODE: Shreveport, LA 71103 MAILING ADDRESSIFDIFFERENTFROM ABOVE: P. O. Box 33932 TELEPHONE NUMBERS: (318) 674-5160 INSTITUTIONAL OFFICIAL (accepting for the institution) NAME AND POSITION TITLE; Ikd Muslow, Vice Chancellor MAILING ADDRESS: P. O. Box 33932 Shreveport, LA 71130 TELEPHONE NUMBER: (318) 674-5400 I;z.A-FACF~ .LK K~cv 5r~ 1,72 MARLING ADDRESS; 1501 Kings Highway Shreveport~ LA 71103 TELEPHONE NUMBER: (318) 674-7655 I MAKE CHECKS PAYABLE TO; Harold White MAILCHECKSTO(NAMEANDPOSITION TITLE): Harold WhiTe, Asst. Vice Chancello~ MAILING ADDRESS: P. O. Box 33932 Shreveport, LA 71130 PPJNCIPAL INVES]IGA1 OR. I have r¢~ the Council's S~tcmenl of Polly agree to i~ ~ ~d condit~. Abe. I *c¢¢~ ~,~ihili~ for ~iemific ¢ond~l offfib proje¢l ~d will p~ovide pro¢~ r¢~ when ~qu¢~. I ~II ac~owledg¢ ~p~ by &e C'fKJNC[L FOR ~EARCil ~ publ~ re~ll~ from ~ ~SPON$11ILE INS IIR:~{ )NAI. O~TI~AL. 50326177

2 I. How have your results changed earlier specific research aims? The ~hree spedfi~ a~ms ~ u, noh~ng~l. 2. How have results changed earlier working hypotheses? In the original proposal, we hypothesized that natural tumors may adse by either an increase in protein level ofeIF-4E or an increased rate ofphosphorylation. Our results with ras and results from other laboratories now make it likely that it is phosphorylafion rather ~han changes in dF-4E lovels that are altered in the transformed phenotype. 3. Describe changes in personnel. Include biographical sketches of new key personnel. Ms. Carrie RJnker-Schaeffer has now obtained her Ph.D. and is a post-doctoral fellow at Johns Hopkins Medical School. Dr. Arrigo De Benedettihas advanced from Assistant Research Professor to Assistant Professor but is still in the same department as the P. I. and continues to work on the CTR project. Dr. Minicli (c.v. supplied in last year's report) continues to work on Specific Aim 1. To replace Dr. Rinker-Schaeff'er, we are currently recruiting a postdoctoral fellow who will bd on board before the beginning of the third grant year. 4. List publications or papers in press resulting from this or closely related work. See instructions regarding pa.ckagjog.of re, prints p,od/ogdnoIwscopts... s. v, mKer-~cnae~ter, ~. w., ~,usun, V., Zimmer, S., and Rhoads, R. E., ras Transformation of Cloned Rat Embryo Fibroblasts Results in Increased Kates of Protein Synthesis and Phosphorylation of Eukaryotic Initiation Factor 4E, d. Biol. Chem. 267, 10659-10664 (1992). 2. Joshi-Barve, S., DeBenedetti, A., and ghoads, R. E., Preferential Translation of Heat Shock mRNAs in HeLa Cells Deficient in Protein Synthesis Initiation Factors elF-4E and elF-4~,, J. Biol. Chem. 267, 21038-21043 (1992). 3. Rhoads, R. E., Regulation of Eukaryotic Protein Synthesis Initiation Factor Activity. Common Mechanisms Amongst Diverse Organisms, accepted for publication in The Journal of Biological Chemistry. CONFIDENTIAL 50326178

3 Outline experimental protocol for ensuing year. Use only this page. Specific Aim A. Detect, purify and identify the putative elF-4E kinase. We will utilize the approach initially outlined, with the modifications stated in last year's progress report of using recombinant eIF-4E as well as elF-4F as substr.ates for the putative kinase. Another change is to use CHO-K1 cells instead of the HIR-3.5 cells initially proposed. The CHO-K1 ceils were given to us by Dr. Jonathan Whittaker, SUNV Stoney Brook, and contain a transfected human insulin receptor. They have the advantage over HIR-3.5 cells of growing faster and being capable of adapting to spinner cultures. Specific Aim B. Relationship ofras and elF-4E. We have obtained the interesting result (see Section 6 below) that expression of antisense RNA against elF-4E mRNA results in reversion of the transformed phenotype in ros-transformed continuous rat embryo fibroblasts. The onset oftumor growth in mice is greatly delayed in cells expressing antisense RNA. However, tumors eventually grow out. We have now developed cell lines from these tumors. In the next year, we will attempt to determine the mechanism by which these cells escape the effects ofantisense RNA. This could yield some very interesting results. For instance, the cells may now be overproducing elF-4E, or overphosphorylating the elF-4E which remains after antisense production, or somehow down-regulating the production of antisense RNA. Specific Aim C. Examination of naturally arising human tumors for perturbations in elF-4E. Our move to LSU Medical Center has broken offthe fruitful collaborations we had established with clinicians at the University of Kentucky. Hence, we are now establishing similar collaborations with clinicians at LSUMC. This should provide us a source of control and tumor tissue by the beginning of the third grant year. In addition, we now appear to have a monoclonal antibody against eIF-4E which should be more suitable than the polycional antibody we have been attempting to use up until now. CONFIDENTIAL 50326179

4 6. Brief summary ofp.rogress to date• Use only space provided on this page. ~ Nta~er 3075~I P: I. Name. R. E,. Rhoads Specific Aim A. Detect, purify and identify the putative eIF-4E kinase. The finding that extracts of HIR-3.5 cells failed to phosphorylate elF-4E (see last year's progress report) has led us to change our experimental approach. Three new lines of experimentation have been opened to circumvent this problem. First, we have changed to CHO-K1 cells as the experimental model. These were developed by Dr. Jonathan Whittaker, SUNY Stoney Brook, who transfected CHO cells with a vector expressing human insulin receptor. These can be grown in spinner culture which should provide us with more tissue for kinase isolation. Second, we have.used several methods to purify eIF-4F as a kinase substrate. Initial results look promising using rabbit liver, which is 1/100 the cost ofrabbit reticulocytes, the normal source for eIF-4F. Third, ~e have begun to investigate the finding that the eIF-4EAla'53 variant is phosphorylated, albeit more slowly than the wild type eIF-4Eset-53 [Joshi-Barve et al., J. Biol. Chem. 26~, 2979 (1990)].. This suggests that there are non-specific kinases for eIF-4E, which will complicate our identification of the correct kinase. We have made a vector encoding a truncated version ofeIF-4EAle-53 to enable us to isolate it free of wild type eIF-4ESer-53 and will determine the phosphorylation site(s) in eIF-4EAle-53. Specific Aim B. Relationship ofras and eIF-4E. As described in the accompanying manuscript (Kinker-Schaeffer e! al., submitted), we have now obtained evidence that eIF-4E is an obligatory link in the signal transduction pathway from activated p21ras to increased cell division and oncogenesis. Expression ofantisense RNA against eIF-4E mRNA causes reversion of the ras-transformed phenotype and retards tumorigenicity in nude mice. Furthermore, the results described in last year's progress report about overphosphorylation ofeIF-4E in ras-transformed cells have now been published (Kinker- Schaeffer et el., 1992; reprint attached). Finally, the effects on cells of decreasing eIF-4E levels by antisense RNA have been further investigated. It was found that, under such conditions, the predominant mRNAs that are translated are those of the heat shock proteins (Joshi-Barve et al., 1992; reprint attached). Specific Aim C. Examination of naturally arising human tumors for perturbations in eIF-4E. The human tumor and control tissues tested did not give consistent results due to the weakness of the polyclonal goat antibody used. Hence, we.undertook preparation era monoclonal anti-elF-4E antibody. To date, we have obtained two positive clones which react strongly with elF-4E by ELISA. We are currently testing these by the Western technique to determine specificity. END OF PROGRESS SUMMARY CONFIDENTIAL 50326180

7a. ,BUDC~ T: Stat~ ~mncs or ~o be re~]t~d~. P.I. Name. Robert E. Rhoads Pm~ionalPe~onnelindudingPfincipallnvesti~tor Robert E. Rhoads, P. 1. Waldemar B. Minich, Postdoctoral Fellow Postdoctoral Fellow, T. B. A. 5% 6,600 100% 28,600 100% 28,292 Technical Support A. Salaries Subtotal B. Consumablesuppl~s ~y m~orcmego~) Affinity and chromatography media Sera, antibodies, retlculocyte lysat@ Tissue culture media, supplies, CO2 Reagents,lenzymes Radioisotopes, film 787 2,809 3,356 1,685 3,034 B. CdnsumablesSubtotal C. Other Expenses 0temize) ., . ...... . .... Equipment Maintenance, service contracts 2,000 Publication costs 1,000 C. OtherExpenscs Subtotal 63,492 11,671 3,000 D. INDIRECT COSTS ( 15% of SUBTOTAL FORA + B + C) E. Permanent Equipment (itemize) SUBTOTAL FOR A + B + C D. INDIRECT COSTS -- 78,163 11,725 SUBTOTAL FOR PERMANENT EQUIPMENT. 0 TOTAL REQUEST. 89,888 7b. ESTIMATED FUTUREREQUIREMENTS (This budget is for my third year.) BUDGET PERIOD Salaries. Supplies and Permanent Indirect TOTAL Other expenses Equipment Costs Year 3: N.A. - 0* - "You may not use CTR funds to purchase permanent equipment in the terminal grant ye~. CONFIDENTIAL 50326181

Regulation of Eukaryotic Protein Synthesis Initiation SOURS (give grant numbers) Total Value o f Grant NIGMS GM 20818 709,262 Identify and de'scribe any overlap of this application with the ............~.,.~r',.~ml Anthill" Amount Available to You 153,820 above grants: Date of Temaination of Grant 31 Dec 92 As explained in the initial submission of the OTR proposal, the specific aims of the NIGMS and CTR grants do not overlap. Indicate the total annual funds available to you this year for all rese,'u'ch projects under your supervision. (NIGHS & CTR) $ 227,950 PENDING OR PLANNED Title of Project Sources Total Value Average Total Duration (give grant of Grant Annual (give inclusive numbers.') .... Amount dates) 913,49_5 168,656 Regulation of Eukaryotic Protein Synthesis Initiation identify attd describe ~ay overlap of this application with NIGMS GH 20818 tbe above project. I Jan 93- 31 Dec 97 No overlap. CONFIDENTIAL 50326182

,CTR P~OGRESS RE~ORT Nameoflnvestigator: Robert E. Rhoads Title of Original Grant: Protein Synthesis Initiation Factor 4E and Oncogenesis Abstract of the Specific Aims as stated in the original application: Our central hypothesis is that phosphorylation ofeIF-4E is an important molecular switch which mediates the anabolic and proliferative action of a number of mitogens and hormones. Furthermore, observations in various signal transduction pathways may increase cell growth by specifically stimulating the kinase, as yet unidentified, which is responsible for in vivo phosphorylation ofeIF-4E. We propose to study the link between elF-4E activity and the rate of protein synthesis and cell growth by three independent approaches. First, we will attempt to detect the kinase which is responsible for eIF-4E phosphorylation by fractionating extracts of insulin-stimulated HIR 3.5 cells and assaying for an increase, relative to unstimulated control cells, ofa kinase activity that uses eIF-4E as substrate. Second, we will test whether eIF-4E phosphorylation occurs through a ras-rclated signaling pathway in two different systems:. continuous rat embryo fibroblasts transformed with activated ras gene from the human T24 bladder carcinoma, and Xenopus laevis oocytes into which activated ras protein has been microinjected. Third, we will determine whether elF-4E levels are elevated in naturally occurring tumors from brain, breast and colon. If so, we will attempt to correlate this with the presence of activated ras protein or other oncogene products. List any publications (author, title and journal) resulting front the current CTR award that contain a printed ackno~vledement of CTR support For manuscripts, the designation "in press" means you have a preprint: otherwise use "accepted for publication by ....", "submitted for publication" or "in preparation". Do not list publications that do not acknowledge CTR support. 1. Rinker-Schaeffer, C. W., Austin, V., Zimmer, S., and Rhoads, R. E., ras Transformation of Cloned Rat Embryo Fibroblasts Results in Increased Rates of Protein Synthesis and Phosphorylation of Eukaryotic Initiation Factor 4E, J. Biol. Chem. 267, ! 0659-10664 (i 992). 2. Joshi-Barve, S., DeBenedetti, A., and Rhoads, R. E., Preferential Translation of Heat Shock mRNAs in HeLa Cells Deficient in Protein Synthesis Initiation Factors elF-4E and eIF-4y, J. Biol. Chem. 267, 21038-21043 (1992). 3. Rhoads, R. E., Regulation of Eukaryotie Protein Synthesis Initiation Factor Activity. Common Mechanisms Amongst Diverse Organisms, accepted for publication by The ,Journal of Biological Chemistry. 4. Rinker-Sehaeffer, C. W., Graft, J. R., Zimmer, S. G., and Rhoads, R. E., Decreasing the Level of Translation Initiation Factor 4E Causes Reversal of Ras-mediated Transformation and Tumorigenesis of Cloned Rat Embryo Fibroblasts, submitted for publication. Report of Progress (no more than four (4) pages, please.) (continued) 50326183

REPORT OF PROGRESS Specific Aim A. Detect, purify and identify the putative elF-4E kinase. The finding that extracts of HIR-3.5 cells failed to phosphorylate elF-4E (see last year's progress report) has led us to change our experimental approach. Three new lines of experimentation have been opened to circumvent this problem. First, we have changed to CHO-K1 cells as the experimental model. These were developed by Dr. Jonathan Whittaker, SUNY Stoney Brook, who transfected CHO cells with a vector expressing human insulin receptor. These can be grown in spinner culture which should provide us with more tissue for kinase isolation. Second, we have used several methods to purify eIF-4F as a kinase substrate. Initial results look promising using rabbit liver, which is 1/100 the cost of rabbit reticulocytes, the' normal source for eIF-4F. Third, we have begun to investigate the finding that the eIF-4EAla-s3 variant is phosphorylated, albeit more slowly than the wild type eIF-4ESer-53 [Joshi-Barve et al., J. Biol. Chem. 265, 2979 (1990)]. This suggests that there are non-specific kinases for eIF-4E, • • which will complicate our identification of the correct kinase. We have made a vector encoding a' truncated version ofcIF-4EAla-53 to enable us to isolate it free ofwild type eIF-4Eser-53 and will determine the phosphorylation site(s) in cIF-4EAla-53. Specific Aim B~ Relationship ofras and elF-4E. As described in the accompanying manuscript (Rinker-Schaeffer et ai., submitted), we have now obtained evidence that elF-4E is an obligatory link in the signal transduetion pathway from activated p21ras to increased cell division and oncogenesis. Expression ofantisense RNA against elF-4E mRNA causes reversion of the ras-transformed phenotype and retards tumorigenicity in nude mice. Furthermore, the results described in last year's progress report about overphosphorylation of elF-4E in ras-transformed cells have now been published (Rinker- Sehaeffer et al., 1992; reprint attached). Finally, the effects on cells of decreasing elF-4E levels by antisense RNA have been further investigated. It was found that, under such conditions, the predominant mRNAs that are translated are those of the heat shock proteins (Joshi-Barve et al., 1992; reprint attached). Specific Aim C. Examination of naturally arising human tumors for perturbations in elF-4E. The human tumor and control tissues tested did not give consistent results due to the weakness of the polyelonal goat antibody used. Hence, we undertook preparation of a monoelonal anti-elF-4E antibody. To date, we have obtained two positive clones which react strongly with elF-4E by ELISA. We are currently testing these by the Western technique to determine specificity. 50326184