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7'rrr (;c)I xc [r. Tctn'1'tlr.cc c u I:Eat:,t [rc[[-[J.S.A.. ~ Fj ~jc,~~Igte marwwt!~ c# °~ 110 }:A::'P 3/~7 u ~'Cttt:1•:I' NF]11 lOltli. N.l'. 10022 -.~ JUl ~`'"73 (212) a21.k.yM. ~`j: Application For Renewal of,Research Grant u`~pWi G ~~} .~~~L ti~q r~ (Use extra poges as needed) i'1Il 1 0!4 First Renewal [] Second Renewal ~ Date: 6/24f 75 1. Principal Investigotor (give title and degrees): Daniel B. Rifkin, Ph.D. Associate Professor of Chemical Biology 2. Institution & address: The Rockefeller University 66th Street and York Avenue NeW York, NY 10021 3. Deportment(s) where research will be done or collaboration provided: Department of Chemical Biology 4. Short title of study: Proteases Produced by Mammalian Lung Tissue 5. Proposed renewal dnte: January 1, 1976 6. How results to dole have changed earlier specific research aims: Our results to date have not changed our earlier specific research aims. 7. How results to dmte have changed earlier working hypothesis: Our results to date have not changed our earlier working hypothesis.

8. Any additional facilities now required? Describe briefly: None H K822.69 075 9. Any changes in personnel? Append biographical sketches of new key professional personnel: None 10. Append obtline of experimental protocol for ensuing year. 11. List publications or papers in press resulting from this or closely related work. (append reprints or manuscipts not previously sent). , 1. Proteases and Biological Cor.trol , E. Reich, D. B. Rifkin, E. Shaw, editors, Cold Spring Harbor Press, Sept. 1975. 2. Pollack, R., Risser, R,, Conlon, S., and D. Rifkin"Plasminogen Activator Production Accompanies Loss of Anchorage Regulation in Transformation of Primary Rat IInbryo Cells by Simian Virus 40; PNAS 71:4792-L796 (1974). 3. Rifkin, D. B., Beal, L,, and Reich, E. s'Macromolecular Determinants of Plasminogen Activator Synthesis; to appear in Proteases and Biological Control, Reich, Rifkin and Shaw, editors, Cold Spring Harbor Press. 4. O'Malley, J. F. X ., and D. B.,Rifkin, "Plasminogen Activators from Cultured Human Embxyonic Lung" To be submitted to Laboratory Investiga- tion. 12. Summary progress report (append in standard form as separote document, unless recently submitted).

13. Budget fur the cor'ning year: A. Solorics (give nanres or stote "to bu recruited") Professional (givc °.o lime of invesrhgotor(s) even if no saiory requested) Daniel B. Rifkin Susannat) T. Rohrlich Technicol John F. Xo 0'Malley Staff benefits B. Conkumable sup'pl?es (by major.categories) tissue culture supplies radioactive isotopes chemicals glass ware C. Othor expenses (itemize) animal costs illustration costs $ervice contract -travel % time 50% !;XM6107F Amount 100N 12,000 100% 10,847 3,541 Sub•Total for A 26,188 3,300 2,000 1,300 1,100 Sub-Total for 8 7,700 ~ 600 tL 500 600 Sub-Total for C 600 2,300 Running Total of A + B + C 36,388 0. Permanent equipment (itemize) Fower supply for isoelectric focusing 1,000 Sub•Total for D 1,000 E -5.A8 E. Indirect costs (15% of A+B+C) Total request 4?,~8b6_^

4. N!(0261 07? 14. Other sources of financial support: List financial support from all sources, including own institution, for this and related research projects. Title of Project CURRENTLY ACTIVE Source (give grant numbers) Amount Inclusive Dates Surface Proteins of NIH CA-13138 242,672 2/1/75- 1/31/80 Normal and Transformed Cells Plasminogen Activator in Fibroblasts amon Runyon 8,000 /1/75-3/31/76 for 1 year only PENDING OR PLANNED Source Inclusive Title of Project (give grant numbers) Amount Dates none It ic understood that the investigator and institutional officers in applying for a grant have read and accept the Council's "Statement of Policy Containing Conditions and Terms Under Which Project Grants Are Made." Principal investigator Typed Name Daniel B. Rifkin Signature bi~^s'' 13" ~ Date /O 43 i Telephone 212-360-1922 Area Code Number Ealeation Checks payable to Responsible officer of institution The Rockefeller University T ped Name Frederick Seitz Mailiiig address for check: y Title President Mr. Adam Alberico, Manager of Accountin~ Si nature i - ' Date 6/24/75 g Services, The Rockefeller Un4versity Telephone 212-360-1234 Area Cxle Numb.r Eahnuon

NXQ2261078 Item 10 I. Preparation of dntibodies to Human lunp cell Plasminogen Activator (FA) A. The purified activator will be analysed for homogeneity by the following methods: 1. SDS-polyacrylamide gel electrophoresis -- one single. protein should be present. 2. PA will be labeled with 32P-DFP, electrophoresed in agar, stained and autoradiography performed to insure that the 32P-DFP which acts at the active site of PA corresponds to the visible protein band. B. Preparation of antibodies using purified PA: 1. Rabbits and goats will be injected with PA irn a polyacryl- emide gel. This technique has been shown to be extremely effective in producing antibodies since the acrylamide acts as an adjuvant. The HEL PA is active in polyacry'iamide. 2. The Y-globulin fraction will be purified by DEAE-chromoto- graphy a.nd pevicon block electrophoresis. 3. The antibodies will be checked to determine that they inhibit HEL PA and that any immune complex seen on Ochterlony corresponds to the position of 32P-DFP plasminogen activator. C. Fluorescent a;.ti-ra*.ibi t.ibit a.d anti-goat antibodies will be prepared. D. Use of antibodies to plasminogen activator for analysis of lung tissue for plasmi.nogen activator production: 1. Biopsy material will be fixed and stained with antibodies to determine what cells contain plasminogen activator and to determine the development of this activity as aa function of time. 2. Other cells: and tissues will also be tested to determine if tnis activator is unique to lung cells or is more general in its distribution. E. Human sera from individuals will be s.sssyed for the potential to inhibit subsequent antibody-activator interactions as a means of determirfiing whether this may serve as a tool to quantitate circulating levels of PA. II. Control of Human Lung cell plasminogen activator synthesis. A. Effects on HEL of drugs which depress PA production in other systems, 1. Steroid Hormones. 2. Base atialogues. 3. Anti-imflamatory agents. 4. Changes in oxygen tension. B. Chicken lung cells 4ill be infected with tumor viruses,both wild type and temperature sensitive,to determine: l. Does transformation affect PA production. 2. If PA prodt.iction is affected, is its control similar to that seen in chicken fibroblasts, i.e., see appended manuscript(/,~,-'Ildlx I ),IK

H K6,2261 079 C. Analysis of elastolytic activity of transformed cells: 1. 125I-elastin Sepharose wi11 be placed in cultures of normal and transformed cells andd release of soluble elastopep- tides assayed. 2. The reaction will be checked under conditions where serum is present or absent: a. No serum present. b. Serum deficient in protease inhibitors c. Complete serum present. D. Analysis of activity in other cells: 1. 2. 3. 4. Macrophages. Neutrophils. Smooth muscle cells. Lung fibroblasts. VI. Completion of studies on surface properties of skin and lung fibro- blasts. It has been reported that lung cells make heparin. is well known that heparin activates antithrombin III into a It potent antiplasmin. This may serve as a mechanism protecting the cells from the proteases which they generate. This will be tested in the following manner: A. 1. Lung and skin cells will be grown in S35sulfate. 2. Homogenized cell extracts will be run on polyacrylamide 3. gels. Gels will be assayed for protein and S35. 4. The distribution of S35 will demonstrate if these cells 5. produce sulfated glycosaafihoglycans. Heparinase will be used to establish if any of these molecules are heparin. B. If lung cells make heparin, cells wi11 be grown in the presence of heparinase to determine if the capability of cells to make heparin makes them resistant to plasmin. C. Cells will be grown in the presence of certain inhibitors of glycosaminoglycan synthesis (i.e. DON) and analysed for surface changes related to plasmin activity.

H KE226108t7 III. Anslysic of cell aosociated fibrinolytic inhibitors: A. Purification: 1. Cells will be grown and extracted With triton, which solubilizes inhibitor. 2. Inhibitor will be purified by standard techniques of chromatography and electrophoresis. It will be assayed by its ability to suppress the fibrinolytic reaction of PA, plasminogen and 125I-fibr•in. 3. Purified inhibitor will be related to other known protease inhibitors both by physical characteristics and action. 4. A determination will be made of whether this inhibitor is a i i serum proteirn which sticks to cells or if it is syn- thesized by celbs. This will be done by growing cnlis in radioactive hiedium and ar.aly,zing the cell associate3 inhibitor from these cultures for incorporated radio- B. activity. Antibodies will be made by a fluorescent indirect technique to determine if these inhibitors are limited to decreate regions of IV. cells•or are randomly distributed. Analysis of RDS serum. This will eventually be phased out since the availability of antibodies to PA will allow more direct assays. However, in the interim, the following approaches will be made: 1. Quantitation of serum plasminogen -- done by Rocket immuno- electrophoresis. 2. Quantitation of serum protease inhibitors -- done by Rocket immunorlectrophdresis. 3. Quantitatiod.of serum PA a. Protease inhibitors will be eliminiated by acid treatment. b. Plasminogen will be removed by mierocolumns of lysine-Sepharose, if necessary. c. This serum w2.ll be tested for ability to activate exogenous plasminogen, thus indicating that PA V. was present. Development of a radioactive assay for elastase and an analysis of cells A. and tissues for elastolytic activity: Preparation of 125I-elastin substrate 1. 2. 3. 4. lodination of soluble elastin. Purification on Sephadex of 1251-elastin. Coupling of 125I-elastin to Sepharose. Determination of specific activity. B. Analysis of proper assay conditions: 1. Dete.nmination of proper elastin:Sepharose ratios with 2. purified elastas;e. Determinatioh of levels of sensitivity with purified elastase. 3. Determination of serutn effects on enzyme assay. Determina- tion of effect of various assay. serum protease inhibitors on 4. Analysis of what proteases are capable of hydrolysing the radioactive elastin.

t! h.k`,;92 16 10 fs 1 cTR Grant rro. 9a-a pROGRESS REPORT NO. 2 (January }-, 1975-June 24, 1975) Daniel B. Rifkin, Ph.D. Associate Professor of Chenical Biology The Rockefeller University

1l~U2261 ©l32 Item 12 Sumrury Proeress FteFort During thc past year our work has emphasized three aspects of plasminogen activator bioloo•. These are: 1) Purification and characterizia-• tion of the enzyr..e, 2) control elements in the synthesis of plasminogen activator, 3) analysis of' sera from control and RDS infants. A brief summar,v of this work is described in the reDort below as well as in the supplemental material. J. Purificatioh and Characterization of the plasminogen activator from human embryonic lung (HEL) cells. We have chosen a purification scheme which consists of the fbllowing sequence of operations: 1) Serum-free conditioned medium is collected from cultures of HEL cells. This solution contains soluble plasminogen activator. 2) The serum-free conditioned medium is passed through an arginine methyl-este'r-Senharose colu.~nn. The PA is retained and is then eluted with arginine, 3) The plasmiinogern activator is then precipitated in a 4Q-60% ammonium sulfate fraction. 4) The dissolved PA is then chromato- graphed on Seph adex G-:00 and concentrated by vacuum dialysis. 5) The activato- is then further purified by isoelectric focusing. 6) This material is then put onto an SDS-polyacrylamide gel. The plasminogen activator band can then be directly injected into rabbits while still in the gel. The acrylanide gel appears to act as an excellent adjuvAnt, as this system has been used effectively to prepare antibodies in several systems. We are at present processing 20 liter batches of serum-free conditioned medium to obtain a sufficient amount of material for in,jection.i The characterization of the IML plasminogen activator has relied upon the ability of this enzyme to convert plasmino~en to plasmin and the ability of the plasmin generated to release 12 I-fibrinopeptides from an insoluble 1z5I-fibrin film on a petri dish. Using this assay we have been I able to characterize the HEL P4 in e variety of ways (see enclosed manuscript). The p3asminogen activator appears to be a serine protease as Judged by those molecules which inhibit it, i~e., DFP and NPGB. The plasmin formed can be inhibited by Soybean :rypsin inhibitor, a-macroglobulin, and a1-antitrypsin.' These three molecules, however, are either ineffective or poorly effective in inhibiting the activation reaction. Since as mentioned in an earlier report, the enzyme is st able to 0.1% SDS, active enzyme can be recovered from SDS- ' polyacrylamide gels. By this te:chnique, two enzyme species can be seen; one with a molec ular weight of 30,000 and one with a molecular weight of 60,000. If the HEL PA is inactivated by (3H) DFP, run on a polyacrylamide gel, and assayed by autoradiography; only one peak can be seen with a molecular weight of 60,000. The sensitivity of the HEL PA to DFP appears to differ from the urokinase activator. This would imply that there are structural differences betweero these molecules. This is an important observation because it demonstrates that a variety of plasr.unogen activators exist and are pr obably tissue specific. A more detailed description of this ). work can be found in the apYerided manuscript( iw a;-}c~J.x I lzl~

;:i hL2261037) II. Control of Plasminogen activator synthesis I a. A series of experiments was conducted in collaboration with Dr. 2. Friedman of N.Y.U. Medical School to determine if the production of plasminogen activator by WI-38, a human embryonic lung cell strain, was dependent upon the division number of the cells. These cells have been used as an in vitro model for aging since they cease dividing afterr a defined number of cell divisions. While our data indicated that the specific activity of the plasminogen activator in these cells decreased as a function of cell number, the decline,was quite small and did not reflect the decrease in division time. Thus, we concluded that there was no major arrest of PA synthesis as WI-38 bells age and that the rate of production of FA in these cells is a reflection of the rate of protein synthesis which continues at approximately the same rate throughout aging. b. Control of Plasminogen activator synthesis in chick cells infected with a virus temperature sensitive for transformation. As described earlier we have been interested in those elements which control the synthesis of PA in normal and transformed cells. One mf the systems we have examined rigorously is the chick embryo fibroblast system and in particular cells infected with a temperature sensitive mutant of Rous sarcoma virus. Cells infected with this virus appear to be transformed when grown at 36° but when shift-a to 41° rapidly convert into normal cells by all criteria. At 36° these cells synthesize and secrete large amounts of PA while at 41° there is no detectable PA associated with the cells. We have found that when the cells are shifted from 36° th L1°, the loss of PA is very rapid with a half time of approximately 1 hir. However, if actinomycin D is added at the time of the temperature shift, the synthesis of PA continues at high rates for periods up to 18 hr. Other inhibitors of DNA dependent RNA synthesis such ae dauncmycin and bromotuber.:idin produce a similar effect. If RNA synthesis is reinitiated at any time after the 36° + 41° shift, PA synthesis rapidly halts. Not only is RNA synthesis required, but also protein synthesis appears to be necessary. If at the time of temperature shift, cycloheximide is added to the cultures, the level of PA in tkie cells rapidly decreases and quickly reaches zcro. During this ~riod normal cellular RNA synthesis takes place. If after four hours at 41° in the presence of cycloheximide, the medium is removed and replaced by medium containing only Actinomycin D, i.e., the cycloheximide is removed and protein synthesis initiated, the plasminogen activator synthesis is reinitiated even though the cultures are at 41°. This implies several things. 1) The message for PA is stable. 2) RNA synthesis is not enough to eliminate the message for plasminogen activator. Only when protein synthesis and RNA synthesis take place is the message for PA destroyed. 3) The RNA whichh must be made to suppress PA synthesis must turn over rapidly since RNA synthesis takes place in the first four hours of this experiment.