Formation of Cadaverine As an Effect of A-Difluoromethylornithine on Chick Embryo Fibroblasts Transformed by Rous Sarcoma Virus
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(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 Gìphase 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 °Cor in cellular cadaverine levels was also demonstrated during the 42°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 (42°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 °C and spermine are aliphatic bases essential for cell growth (3, 8, (permissive condition) or at 42°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, «-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 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1985 American Association for Cancer Research. REPLACEMENT OF PUTRESCINE BY CADAVERINE IN FIBROBLASTS 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 °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 °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 £ 2.0 40 I.O •lisa X EOEEE IEI X o^ftJWÕO« — eme E o 30 OWMWO'0 CE Lu o.