Formation of Cadaverine As an Effect of A-Difluoromethylornithine on Chick Embryo Fibroblasts Transformed by Rous Sarcoma Virus

(CANCER RESEARCH 45, 2159-2164, May 1985]

Formation of Cadaverine as an Effect of a-Difluoromethylornithine on Chick Embryo Fibroblasts Transformed by Rous Sarcoma Virus

Uriel Bachrach and Alalia Shtorch

Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel

ABSTRACT that DFMO does not interfere with the growth, DNA synthesis, and protein synthesis of normal and transformed cells. However, Chick embryo fibre-blasts grew normally in the presence of 1 DFMO leads to a significant reduction of cellular putrescine x 10~3 to 10 x 1CT3 M a-difluoromethylornithine (DFMO). This levels. This decrease is compensated by the accumulation of drug did not interfere with protein and DNA synthesis of normal cadaverine (1,5-diaminopentane) in the DFMO-treated cells. A fibroblasts and of cells transformed by Rous sarcoma virus. The preliminary account of this work has been given previously (5). morphological appearance of normal and transformed cells was not altered by DFMO, as determined by scanning electron mi croscopy. Flow microfluorometric analyses also confirmed the MATERIALS AND METHODS notion that normal or transformed cells were not blocked by Viruses and Tissue Cultures. A wild type of RSV-SR 17-A and its DFMO in the GÃ¬phase of the cell cycle. As expected, DFMO temperature-sensitive mutant, T5, were used as described elsewhere reduced cellular putrescine levels. This diamine, however, was (10,11). Primary cultures of CEF, prepared from 9- to 11-day-old chick replaced by the analogue cadaverine (1,5-diaminopentane), embryos, were seeded on 90-mm plastic plates (Nunc, Roskilde, Den which accumulated mainly in the transformed cells. The increase mark) at a density of 8.0 x 106 cells/plate. Cells were grown at 37 Â°Cor in cellular cadaverine levels was also demonstrated during the 42Â°Cin Eagle's minimal essential medium (Grand Island Biological Co., infection of chick embryo fibroblasts with a temperature-sensitive Grand Island, NY) supplemented with 20% tryptose phosphate broth mutant of Rous sarcoma virus under permissive conditions. (Difco Laboratories, Detroit, Ml), 5% inactivated calf serum, and 384 mg Under restrictive conditions (42Â°C),less cadaverine accumulated glutamine/liter. After 24 h, the growth medium was removed, and 1.0 ml of either fresh medium or RSV (1 to 2 x 106 focus-forming units) was in the infected cells. These findings suggest that diamines and polyamines are necessary for the transformation process and added to each plate. Incubation was continued for another 45 min, that blocking one pathway by DFMO leads to the activation of followed by the removal of the liquid overlay and the addition of 10.0 ml of fresh medium per plate. For subcultures, the medium was removed an alternative biosynthetic pathway. from 4- to 5-day-old primary cultures and washed twice with phosphate- buffered saline, and cells were dissociated by incubation with 0.25% trypsin. Approximately 1 to 2 x 106 cells were seeded on 90-mm plastic INTRODUCTION plates (in some studies, 50-mm plates were used). After 24 h, secondary cultures were infected with 1.0 ml of RSV. When the temperature- The naturally occurring polyamines putrescine, spermidine, sensitive mutants were used, cultures were incubated either at 37 Â°C and spermine are aliphatic bases essential for cell growth (3, 8, (permissive condition) or at 42Â°C (restrictive condition). Cultures were 27, 34) and differentiation (15). In general, polyamine levels are incubated in an atmosphere of 95% air/5% C02, and transformation was elevated in rapidly growing systems such as regenerating liver evident 4 days after the cells were infected with RSV. Subcultures were (30), embryonic tissues (7), and tumor cells (31). The increase in free of Mycoplasma when tested by culture or by electron microscopy. cellular polyamine levels usually corresponds to an increase in Polyamines. Cells were removed from the plates by scraping, sus the activity of ODC,1 the first rate-limiting enzyme in polyamine pended in phosphate-buffered saline, and extracted with perchloric acid synthesis (3, 31). Indeed, the activity of ODC rises when cells (final concentration, 3%). To 0.2 ml of the supernatant, obtained after are transformed by tumor viruses (4, 14, 22) or by carcinogenic centrifugation at 500 x g for 10 min, 18 mg of Na2CO3 and 0.6 ml of (6) or mutagenic compounds (31). DFMO, an enzyme-activated dansyl chloride (10 mg/ml in acetone; Fluka Chemical Co., Buchs, Switzerland) were added (32). After being kept in the dark for approxi and specific inhibitor of ODC (23), inhibits the proliferation of mately 15 h, the excess dansyl chloride was converted to dansylproline several types of cancer cell in vitro (22). More detailed studies by the addition of 0.05 ml of proline (10 mg in water). Thirty min later, demonstrated that DFMO was not very active when administered the dansylated polyamines were extracted with 2.0-ml quantities of as a single drug. On the other hand, a combination of DFMO toluene. The organic phase was removed after centrifugation and evap with other antiproliferative drugs such as methylglyoxal bis- orated. The dry residue was finally dissolved in 0.2-ml quantities of (guanylhydrazone) (17), 1,3-bis(2-chloroethyl)-1-nitrosourea (21, toluene and applied onto Silica Gel G plates (300 mm thick). Plates were 25), c/s-diamminedichloroplatinum (24), 5-fluorouracil (18), and run either in ethyl acetate/cyclohexane (%) or in Methylamine/Chloroform 1-0-D-arabinofuranosylcytosine (26, 33) was more effective. (Vio). Once identified, dansyl derivatives were scraped off the plates, extracted with 4-ml quantities of ethyl acetate, and assayed in a Turner The study reported here was designed to evaluate the effect of DFMO on the proliferation and polyamine content of normal Model 111 fluorometer at an excitation of 360 nm and an emission of 510 nm. The fluorescence corresponding to material in the scraped spot CEF as well as of cells transformed by RSVs. In this study, the wild type of RSV, Schmidt-Ruppin 17-A, and the temperature- was compared to that of known standards. Proteins were assayed according to the method of Lowry ef al. (19). sensitive mutant, T5, were used. The results reported here show Scanning Electron Microscopy. Normal (1-day-old) and transformed ' The abbreviations used are: ODC, omithine decarboxylase; DFMO, Â«-difluo (5-day-old) cells were washed with phosphate-buffered saline (pH 7.2, romethylomithine; CEF, chick embryo fibroblasts; RSV, Rous sarcoma virus. 300 mosmol) and fixed with glutaraldehyde (2%, in phosphate-buffered Received 8/1/84; revised 12/11/84; accepted 12/27/84. saline) for 1 h. Thereafter, cells were washed with phosphate-buffered

CANCER RESEARCH VOL. 45 MAY 1985 2159

saline and incubated with tannic acid (2%) and guanidine hydrochloride (2% in phosphate-buffered saline) for 1 h. Cells were washed again with phosphate-buffered saline for 15 min and incubated with osmium tetrox- ide (2%, in phosphate-buffered saline) for 1 h. Samples were then washed with bidistilled water and treated with ethanol in distilled water (increasing concentrations of 25, 50, and 75% and 3 washes with absolute alcohol). This was followed by washing with Freon 113 (TF) in absolute ethanol (increasing concentrations of 25, 50, and 75% and 3 washes with 100% Freon 113) for 10 min each. Fixed cells were coated with gold/palladium for 1 min and analyzed with a Philips 505 scanning electron microscope (13). Flow Microfluorometry. Cultures were incubated with 0.25% trypsin at 37 Â°Cto dissociate cells from the monolayers. Cells were sedimented by centrifugation and treated with a fluorochrome solution containing propidium iodide (0.05 mg/ml; Sigma Chemical Co., St. Louis, MO; 50 mg/ml) in 0.1% sodium citrate, supplemented with bovine pancreatic RNase (Sigma) to a final concentration of 27 units/ml (12). Triton X-100 at a final concentration of 0.5 to 1.0% was used as a fixative. Thymidine Incorporation. The incorporation of [1-14C]thymidine into trichloroacetic acid-insoluble material was studied as follows. Tertiary cultures (normal or transformed by RSV) were grown at 37 Â°Cfor 3 days. Thereafter, growth medium was removed, and fresh medium, supplemented with [1-14C]thymidine (0.2 //Ci/ml; specific activity, 60.4 MCi/mmol; Nuclear Research Center, Negev, Israel) was added to each culture. At various times, medium was removed, and cultures were washed 3 times with ice-cold phosphate-buffered saline. Macromolecules I8 22 were sedimented by adding perchloric acid at a final concentration of TIME (hr) 3%. Insoluble material was sedimented by centrifugation and finally Chart 2. Effect of DFMO on the incorporation of thymidine into the DNA of dissolved in NaOH. Aliquots were taken for determination of proteins transformed CEF. Tertiary transformed cultures were incubated with 0.2 pC'i [1- according to the method of Lowry ef al. (19), and the rest was resedi- 14C]thymidine in the presence or absence of 3 x 10~3 M DFMO. Incorporation was mented by the addition of ice-cold trichloroacetic acid at a final concen measured by precipitation of radioactive material with trichloroacetic acid. O, normal tration of 50%. The precipitate was finally collected by filtration through cells; A, DFMO-treated cells. a 0.45-jim Millipore filter and counted by means of a scintillation counter. All chemicals were of analytical grade. DFMO was kindly provided by 4.0 x EX x Dr. N. Seiler, Centre de Recherche Merrell International, Strasbourg, EM E E France. CD TRANSFORMED RESULTS fZa NORMAL CELLS 3.0 Preliminary experiments demonstrated that DFMO at final concentrations of 2 to 10 x 10~3 M had no significant effect on o> EnE E Â£ 2.0 40

I.O â€¢lisa X EOEEE IEI X o^ftJWÃ•OÂ« â€” eme E o 30 OWMWO'0 CE Lu o.

TIME (days)

o Chart 3. Effectof DFMO on the protein content of normal and transformed CEF. u_ 20- Experimental conditions were those given in the legend of Chart 1 except that the o protein content of the cells was determined 8 days after seeding. (E LU m Z the growth rate of either normal or transformed CEF. Quantitative 2 results were obtained by counting the number of cells grown in the presence or absence of the drug as follows. Secondary lO subcultures were prepared by seeding 1.8 x 106 cells onto 90- 02 3 5 8 IO 2 3 5 8 IO mm plastic culture plates and allowing them to grow for 6 days. mM DFMO was added to some cultures immediately after seeding. Chart 1. Effect of DFMO on the number of normal (H) and transformed (O) CEF. Secondary cultures were grown for 6 days in the presence or absence of DFMO At various times, growth medium was removed, monolayers (0 to 10 mM). The number of cells was determined by counting in a hemocytometer. were treated with 0.25% trypsin, and the number of single cells

CANCER RESEARCH VOL. 45 MAY 1985 2160

The effect of DFMO on cellular proteins was also studied. Secondary cultures of CEF were prepared by seeding 1 x 106 cells onto 90-mm plastic culture plates. After 24 h, some cultures were infected with RSV, and 2 x 10~3 or 3 x 10~3 M DFMO was added to some cultures. After additional incubation for 8 days, cellular proteins were determined colorimetrically (19). It may be seen (Chart 3) that the drug did not reduce cellular proteins of normal and transformed cells. Flow microfluorometry demonstrated (Chart 4) that 3 x 10~3 V ^"MwMAM**. M DFMO did not cause an arrest in the GÃ¬phase of normal K LU tertiary CEF cultures exposed to the drug for 24 h. Transformed OD cells behaved similarly (Chart 5), and no arrest in G, phase was observed after treating the cultures with 3 x 10~3 M DFMO for 24 h. Similar results were obtained when cultures were treated with DFMO for 48 h (results not shown). Scanning electron microscopy demonstrated that the mor phology of normal CEF (Fig. 1) or transformed culture (Fig. 2) was not affected by 3 x 10~3 M DFMO. When polyamines were extracted from DFMO-treated cells FLUORESCENCE ABSORBANCE and analyzed by thin-layer chromatography (as dansyl deriva Chart 4. Flow microfluorometry of normal CEF. Tertiary untreated cell (a) cul tives), the following picture emerged: (a) putrescine levels tures were exposed to 3 x 10~3 M DFMO for 24 h (ti). Cells were analyzed by flow dropped significantly (Chart 6), as did cellular spermidine (Chart microfluorometry as described under "Materials and Methods." 7); (b) cadaverine, which was present in small amounts in both normal and transformed cells, accumulated in the presence of 3 x 10~3 M DFMO. Results were more dramatic when transformed CEF were analyzed (Chart 8). The identification of the diamine as cadaverine was confirmed by running the thin-layer chromato-

"DFMC" 200r dlTRANSFORMEDm.DFMO

CELLSDFMO-0iDFMONORMAL

te. E IOO- r-r'-i Ili CD 3 //. J DFMO DFMO 3mM'//n ' 2mM3mM1 Mdl456 2m 1nh mm456E1Ã•M 456n 456DAY456 456

Chart 6. Effect of DFMO on cellular putrescine levels. Putrescine was extracted from secondary normal CEF or from cells transformed by RSV. To some primary cultures, DFMO was added as indicated. Treatment with DFMO was continued FLUORESCENCE ABSORBANCE throughout the experiment.

Chart 5. Flow microfluorometry of transformed CEF. Secondary cultures were transformed by RSV and examined as tertiary cultures. Untreated cell (a) cultures DFMO-0 were treated with 3 x 10~3 M DFMO for 24 h (b). Cells were analyzed by flow microfluorometry as in Chart 4. 200-

was determined by counting in a hemocytometer. It is evident DFMO from Chart 1 that DFMO at concentrations of 2 x 1CT3to 10 x 25mMâ€”]1--I[;DFMO;rii2m^MDf2 10~3 M hardly affected the number of the cells (normal or trans IOO- formed) grown in the presence of the drug for 6 days. i1 The effect of DFMO on the incorporation of [1-14C]thymidine r-DFMO2-nDFMO-0lM â€¢"â€¢ into cellular nucleic acids was next studied. Chart 2 shows that Fl DFMO did not reduce the rate of thymidine incorporation into ;.',,â€¢ tertiary transformed CEF when analyzed as described under v'/AVÂ¡'MOn "Materials and Methods." Similarly, no significant reduction in 456 456 456 456 456 456 the incorporation of [14C]thymidine into nucleic acids was ob DAY served when normal CEF were grown in the presence of 3 x Chart 7. Effect of DFMO on cellular spermidine levels. Experimental conditions 10~3 M DFMO (results now shown). were those given in legend to Chart 6, except that spermidine was analyzed. W. normal CEF; D, transformed cells.

CANCER RESEARCH VOL. 45 MAY 1985 2161

CÂ°5S? DFMO- DFMO DFMO DFMO DFMO like noninfected controls and contained traces of putrescine. 100o.CiE13mMâ„¢-i-_~~~PI~~i;g^1?.'/*/x\sÂ¡7OmM 2mM 2.5mM 3mM OmM DFMO caused the appearance of cadaverine (instead of putres cine) in the infected cells incubated at 42Â°C.

60o.UJzCADAVER DISCUSSION

Polyamines and diamines are required for the growth of eu- karyotic and prokaryotic cells (3, 8, 20). Putrescine, which is the 8DFMO most common diamine, also serves as the precursor for the synthesis of the naturally occurring polyamines spermidine and 45678 45678 45678 45678 45678 45678 spermine (8). In some systems, including bacteria, cadaverine TIME (days) can replace putrescine (3). The synthesis of this diamine is Chart 8. Effect of DFMO on cellular cadaverine levels. Experimental conditions catalyzed by lysine decarboxylase which occurs in bacteria such were those given in legend to Chart 6, except that cadaverine was analyzed. Ãœ, as Escherichia coli and Salmonella strains (3). In putrescine- normal CEF; D, transformed cells. depleted bacteria, cadaverine is the dominant diamine (9). Tfiis diamine is further converted into polyamine analogues, namely, A/-3-aminopropyl(1,5-diaminopentane) and A/,A/-bis(aminopropyl)- 1,5-diaminopentane (9). Pegg and McGill (28) claimed that a highly purified preparation of ODC also catalyzed the formation of cadaverine from lysine and that the activities of both ornithine and lysine decarboxylases were lost to a similar extent by treatment with DFMO. Persson (29) also came to a simiar conclusion while studying the formation Cadaverine Putrescine of cadaverine by mouse kidneys. Our findings, that cadaverine Spermidine was synthesized in the presence of DFMO, suggest that lysine Spentane decarboxylase and ODC are either 2 distinct enzymes or consti tute one enzyme with different specific active sites. Recently, Alhonen-Hongisto ef al. (1) reported that cadaverine and its aminopropyl derivatives replaced polyamines in Ehrlich ascites carcinoma cells grown in the presence of DFMO. Sub sequent studies indicated that cadaverine can be formed by Mycoplasma spp. which are resistant to DFMO (2). It has not been excluded that the cadaverine-containing Ehrlich ascites carcinoma cultures were contaminated with Mycoplasma and that this contaminant contributed to the appearance of the new diamine.2 Mycoplasma could not be detected in our cultures. HÃ¶lttÃ¤etal. (16) have recently confirmed our previous findings 37Â°C42Â°C370C37Â°C42Â°C42Â°C that CEF transformed by RSV had higher putrescine levels than N N T T+D T T+D did the respective uninfected cells (16). They also reported that Fig. 3. Polyamine content of CEF. Cells were infected with the temperature- sensitive mutant RSV T5 and incubated at either 37Â°Cor42Â°C.Tosome cultures, transformed cells had higher ODC activities compared to the 3 x 10"3MDFMOwas added. Polyamineswere extracted from secondarycultures, normal ones. Those findings were in agreement with published and dansyl derivatives were separated by thin-layer chromatography using ethyl data (11). HÃ¶lttÃ¤efal. (16) also observed that 3 x 1Qr3 M DFMO acetate/cyclohexane (%) as solvent. 0, DFMO-treated; T, infected with RSV T5; W,normal cells. did not affect the maintenance of the transformed state of CEF; neither did the drug interfere with the transformation process. They suggested that "the transformation-associated pattern of gram in several solvents (results not shown). The best evidence for the replacement of putrescine by cadaverine was obtained polyamines in chick embryo fibroblasts is not a prerequisite for by infecting CEF with a temperature-sensitive mutant of RSV at morphological transformation of these cells." Unfortunately, permissive and restrictive conditions as follows. Secondary CEF HÃ¶lttÃ¤efal. (16) did not analyze the polyamine patterns of DFMO- were infected with the temperature-sensitive mutant 24 h after treated cells and thus overlooked the possibility of substitution seeding in the presence and the absence of 3 x 10~3 M DFMO. of certain diamines or polyamines by their analogues. Data Cultures were incubated either at 37 Â°Cor 42 Â°C.After additional presented in this paper suggest that the conclusion drawn by incubation for 5 days, polyamines were extracted from the HÃ¶lttÃ¤efal. (16) should be interpreted with great care. It appears respective cells and analyzed by thin-layer chromatography. Fig. (Fig. 3) that diamine and polyamine accumulation may be a 3 shows that normal cells contained less putrescine than did the transformed ones when grown at 37 Â°C(permissive tempera prerequisite or, alternatively, be associated with the transfor ture). In the presence of 3 x 1(T3 M DFMO, cadaverine (and not mation of CEF by RSV. Moreover, if one pathway is blocked, an putrescine) accumulated. CEF infected with the temperature- alternative pathway is activated to satisfy this requirement. sensitive mutant under restrictive conditions (42 Â°C)behaved 2L. Alhonen-Hongisto,personal communication.

CANCER RESEARCH VOL. 45 MAY 1985 2162

18. Kingsnorth, A. N., Russell, W. E., McCann, P. P., Diekema, K. A., and Malt, R. REFERENCES A. Effects of n-difluoromethylornithine and 5-fluorouracil on the proliferation of 1. Alhonen-Hongisto, L, Seppanen, P., Holtta, E., and Janne, J. ReplaÅ“ment of a human colon adenocarcinoma cell line. Cancer Res., 43: 4035-4038,1983. natural polyamines by cadaverine and its ammopropyl derivatives in Ehrlich 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein ascites carcinoma cells. Biochem. Biophys. Res. Commun., 706: 291-297, measurement with the Polin phenol reagent. J. Biol. Chem., 793: 265-275, 1982. 1951. 2. Alhonen-Hongisto, L., Veijlainen, P., Kommonen, E. C., and Janne, J. Polya 20. Luk, G. D., Civin, C. I., Weissman, R. M., and Baylin, S. B. Omithine decar mines in mycoplasma and in mycoplasma-infected tumor cells. Biochem. J., boxylase: essential in proliferation but not in differentiation of human promye- 202.267-270,1982. locytic leukemia cells. Science (Wash. DC), 276: 75-77,1982. 3. Bachrach, U. Function of Naturally Occurring Polyamines, pp. 1-211. New 21. Marlon, L. J., Levin, V. A., Hervatin, S. J., Koch-Weser, J., McCann, P. P., York: Academic Press, Inc., 1976. and Sjoerdsma, A. Potentiation of the antitumor therapeutic effects of 1,3- 4. Bachrach, U., Don, S., and Wiener, H. Polyamines in normal and in transformed bis(2-chloroethyl)-1-nitrosourea by a-difluoromethylomithine, an omithine de chick embryo fibroblasts. Cancer Res., 34: 1577-1580, 1974. carboxylase inhibitor. Cancer Res., 41: 4426-4431,1981. 5. Bachrach, U., and Shtorch, A. Are polyamines essential for the transformation 22. Medrano, E. E., Goldemberg, S. H., and Algranati, I. D. Differential effect of a- of fibroblasts by Rous sarcoma virus? In: C. M. Caldarera and U. Bachrach difluoromethyl-omithine on the proliferation of Balb 3T3 and chemically trans (eds.), Advances in Polyamines in BiomÃ©dicalResearch, pp. 3-19. Bologna, formed 3T3 cells. J. Cell. Physiol., 777: 141-147, 1983. Italy: Editrice, Bologna, 1983. 23. Metcalf, B. W., Bey, P., Danzin, C., Jung, M. J., Casara, P., and Vevert, J. P. 6. Boutwell, R. K. Evidence that an elevated level of omithine decarboxylase Catalytic irreversible inhibition of mammalian omithine decarboxylase by sub activity is an essential component of tumor promotion. Adv. Polyamine Res., strate and product analogues. J. Am. Chem. Soc.. 700: 2551-2552, 1978. 4:127-134,1982. 24. Oredsson, S. M., Deen, D. F., and Marton, L. J. Decreased cytotoxicity of c/s- 7. Caldarera, C. M., and Moruzzi, G. Polyamines and nucleic acid metabolism in diamminedichloroplatinum(ll) by Â«-difluoromethylomithine depletion of polya the chick embryo. Ann. NY Acad. Sci., 171: 709-722,1970. mines in 9L rat brain tumor cells in vitro. Cancer Res., 42: 1296-1299,1982. 8. Cohen, S. S. Introduction to the Polyamines, pp. 1-179. New York: Prentice 25. Oredsson, S. M., Deen, F. D., and Marton, L. J. Influence of polyamine depletion Hall, Inc.. 1971. caused by a-difluoromethylornithine, an enzyme-activated irreversible inhibitor 9. Dion, A. S., and Cohen, S. S. Polyamine stimulation of nucleic acid synthesis of omithine decarboxylase, on alkylation and carbamoylation-induced cytotox in an uninfected and phage-infected polyamine auxotroph of Escherichia coli icity in 9L rat brain tumor cells in vitro. Cancer Res., 43: 4606-4609,1983. KÂ«.Proc. Nati. Acad. Sci. USA, 69: 213-217, 1972. 26. Oredsson, S. M., Gray, J. W., Deen, D. F., and Marton, L. J. Decreased 10. Don, S., and Bachrach, U. Polyamine metabolism in normal and virus-trans cytotoxicity of 1-/i-o-arabinofuranosylcytosine in 9L rat brain tumor cells pre- formed chick embryo fibroblasts. Cancer Res., 35: 3618-3622, 1975. treated with Â«-difluoromethylornithine in vitro. Cancer Res., 43: 2541-2544, 11. Don, S., Wiener, H., and Bachrach, U. Specific increase in polyamine levels in 1983. chick embryo cells transformed by Rous sarcoma virus. Cancer Res., 35:194- 27. Pegg, A. E., and McCann, P. P. Polyamine metabolism and function: a brief 198,1975. review. Am. J. Physiol., 243: C212-C221,1982. 12. Fried, J., Perez, A. G., and Clarkson, B. D. Rapid hypotonie method for flow 28. Pegg, A. E., and McGill, S. Decarboxylation of omithine and lysine in rat cytofluorometry of monolayer cell cultures. J. Histochem. Cytochem., 26:921 - tissues. Biochim. Biophys. Acta, 568: 416-427,1974. 933, 1978. 29. Persson, L. Decarboxylation of omithine and lysine by omithine decarboxylase 13. Gamliel, H., Gurfel, D., Leiberowitz, R., and Polliack, A. Air-drying of human from kidneys of testosterone treated mice. Acta Chem. Scand. B, 35: 451- leucocytes by scanning electron microscopy, using the GTGO procedure. J. 459,1981. Microsc., 737:87-95, 1983. 30. Raina, A., JÃ¤nne,J., Hannonen, P., and Holtta, A. Synthesis and accumulation 14. Gazdar, A. F., Kilton, L. J., and Bachrach, U. Increased omithine decarboxylase of polyamines in regenerating rat liver. Ann. NY Acad. Sci., 777: 697-708, activity in murine sarcoma virus infected cells. Nature (Lond.), 262: 696-698, 1970. 1976. 31. Russell, D. H., and Dune, B. G. M. Polyamines as Biochemical Markers of 15. Heby, 0. Role of polyamines in the control of cell proliferation and differentia Normal and Malignant Growth, pp. 1-178. New York: Raven Press, 1978. tion. Differentiation, 79: 1-20, 1981. 32. Seiler, N. Use of the dansyl reaction in biochemical analysis. Methods Biochem. 16. Holtta, E., Vartio, T., JÃ¡nne,J., Vaheri, A., and Movi, T. Temperature-dependent Anal., 78: 259-337, 1970. and transformation associated changes in polyamine metabolism in normal 33. Sunkara, P. S., Fowler, S. K., Nishioka, K., and Rao. P. N. Inhibition of polamine and Rous sarcoma virus-infected chick embryo fibroblasts. Biochim. Biophys. biosynthesis by Â«-difluoromethylornithine potentiates the cytotoxic effects of Acta, 677: 1-6,1981. arabinosyl cytosine in HeLa cells. Biochem. Biophys. Res. Commun., 95:423- 17. JÃ¤nne,J., Alhonen-Hongisto, L., Seppanen, P., and SÃ»mes,M. Use of polya 430, 1980. mine antimetabolites in experimental tumors and in human leukemia. Med. 34. Williams-Ashman, H. G., and Canellakis, Z. N. Polyamines in mammalian Biol. (Helsinki), 59: 448-457,1981. biology and medicine. Perspect. Biol. Med., 22: 421-453, 1979.

CANCER RESEARCH VOL. 45 MAY 1985 2163

Fig. 1. Scanning electron microscopy of normal CEF. Primary cultures were Fig. 2. Scanning electron microscopy of transformed CEF. Primary cultures treated with 3 x 10~3M DFMO, and treatment was continued in the secondary were transformed by RSV and treated with 3 x 1(T3 M DFMO. Drugs were also cultures (a), b, untreated secondary normal cultures. Cells were examined by added to the secondary transformed cultures (a). Â£>,untreatedsecondary trans scanning electron microscopy. formed cultures. Cells were examined by scanning electron microscopy.

CANCER RESEARCH VOL. 45 MAY 1985 2164

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1985 American Association for Cancer Research. Formation of Cadaverine as an Effect of α -Difluoromethylornithine on Chick Embryo Fibroblasts Transformed by Rous Sarcoma Virus

Uriel Bachrach and Atalia Shtorch

Cancer Res 1985;45:2159-2164.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/45/5/2159

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/45/5/2159. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.