Cytotoxic, Cell Cycle, and Chromosomal Effects of Methylxanthines in Human Tumor Cells Treated with Alkylating Agents1

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[CANCER RESEARCH 46, 2463-2467. May 1986] Cytotoxic, Cell Cycle, and Chromosomal Effects of Methylxanthines in Human Tumor Cells Treated with Alkylating Agents1

Howard J. Fingert,2 James D. Chang, and Arthur B. Pardee

Surgical Oncology Unit, Massachusetts General Hospital Cancer Center, Boston, Massachusetts 02114 [H. J. F.], and Department of Pharmacology, Harvard Medical School and Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [H. J. F., J. D. C, A. B. P.]

ABSTRACT cells to undergo mitosis before the completion of DNA repair (13, 14). The G2 phase of the cell cycle has been generally Human tumor cells, like rodent cells, are sensitive to effects of meth- observed to lengthen in many normal or malignant cells exposed ylxanthines (MEX) on lethality, cell cycle delays, and chromosome aberrations after DNA damage by anticancer drugs. Enhanced cytotox- to radiation, alkylating agents, or other antineoplastic drugs (15-17). According to this model, MEX do not necessarily icity of ;ilk\luting agents was observed when T24 human bladder tumor cells in culture were exposed to nontoxic concentrations of MEX such as inhibit biochemical DNA repair processes per se; instead, they caffeine or pentoxifylline. Tumor cell lethality was increased up to 10- act by reducing time available for repair, possibly through a fold by either caffeine or pentoxifylline (1 HIM) present during the first protein which is altered directly or indirectly by DNA damage cell cycle (16-24 h) after exposure to nitrogen mustard (HN2) or thiotepa. and which controls the transit from G2 to mitosis (14, 15). In Cycloheximide, a protein synthesis inhibitor, abolished the enhanced BHK cells, MEX allowed G2-delayed cells to reach mitosis lethality produced by MEX. In these synchronized human tumor cells without finishing the repair process and consequently caused further kinetic studies revealed that HN2 (0.5 jiM x 1 h) delayed transit shattered chromosomes, nuclear fragmentation, and cell death through S phase by about 1-2 h, and this delay was prevented by MEX. (13). This model is consistent with studies using other cell lines, After completion of S phase, HN2-treated cells were delayed 3-6 h in which demonstrated: (a) prevention by MEX of G2 delay after (,:, and MEX also prevented this delay, leading to mitoses at the rate of damage caused by radiation or alkylating agents (18); (b) en controls. Chromosome analysis of these mitotic cells revealed dramatic hanced radiation-induced cytotoxicity following MEX treat increases in aberrations induced by alkylator + MEX combinations. The greatest number of aberrations was seen in HN2-treated cells exposed ment in G2 (19, 20); (c) selective increases in alkylator-induced briefly to MEX in late S-G2. In contrast, no increased chromosome chromosomal aberrations by MEX in G2 (21); and (d) minimal damage was seen in cells exposed to MEX in mid-S phase. Taken or no change in DNA repair when MEX were added for brief together, our results are consistent with the model that MEX enhance periods after DNA damage (22). lethality of alkylator-treated human tumor cells by preventing delays in Human tumor cells, in contrast to rodent cells, were proposed cell cycle transit through G2, leading to chromosome aberrations which not to exhibit enhanced lethality by MEX, based on early are lethal. (â€¢_â€¢delaysin human tumor cells may provide time for repair experiments with HeLa and other cell lines (8). However, we processes that are critical for survival after sublethal DNA damage by found that human bladder tumor cells also showed enhanced HN2 or other anticancer alkylating agents. lethality by MEX-alkylator treatments. MEX increased by 10- fold the cytotoxicity produced by thiotepa, an alkylating agent INTRODUCTION used frequently for topical therapy of superficial bladder cancers (4). Other studies reported enhanced cytotoxicity by MEX- Enhancement of cytotoxicity by alkylating agents or other alkylator combinations in human bladder and colon tumor cells, anticancer drugs has been demonstrated by post-treatment with MEX,3 such as theobromine, caffeine, and related compounds using either established cell lines (2, 4) or primary bladder cancers in a soft-agar colony forming assay (23). (1-5). However, its potential application to clinical cancer We report here experiments that were aimed to provide better therapy is poorly understood, and few studies have been done understanding of the mechanism whereby MEX enhance the relating to human tumor cells in culture or in animal models. lethality of alkylating agents in human tumor cells. Our present Identification of a single molecular or cellular mechanism studies demonstrate: (a) that CAF and pentoxifylline similarly has been difficult, since MEX exhibit a variety of modifying enhance lethality of thiotepa or HN2 in human tumor cells; (b) effects in mammalian cells which have been treated with DNA- that CAF prevented both minor delays in S and major delays damaging agents (5-7). One common hypothesis is that MEX in G2, produced by alkylator treatment of synchronized tumor inhibit repair processes such as (a) postreplication-repair syn cells; (c) that dramatic increases in the number of chromosome thesis of DNA (8), a phenomenon which itself is poorly under aberrations were produced by the same MEX + alkylator treat stood in mammalian cells (9) or (b) inhibition of poly (ADP- ments; and (d) that the most dramatic increase of chromosome ribose) polymerase (10). Other studies suggest modifications in damage was observed when CAF was added during late S-G2 S phase events of DNA replication such as (c) increased number phase, while no increase was observed when CAF treatment of sites for DNA synthesis in damaged replication units or "replicons" (3, 11) or (d) antagonism of the DNA-synthesis was restricted to mid-S phase. In human tumor cells, these studies support the model that enhanced lethality by MEX is inhibition that is induced by DNA damage (12). How these due to prevention of G2 delays, leading to chromosome aber effects relate to lethality enhancement by MEX is unclear. rations which are lethal. An alternative mechanism is that MEX act in G2 to induce

Received 9/9/85; revised 1/28/86; accepted 1/30/86. MATERIALS AND METHODS The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Cell Cultures and Synchronization. The T-24 cell line, originally 1Supported by USPHS Grant CA 01157 to H. J. F., Grant CA 22427 to A. established from a human transitional cell carcinoma of the bladder B. P.. and a grant from the Donaldson Charitable Trust. (24), was grown as a monolayer in Dulbecco's modification of Eagle's 2To whom requests for reprints should be addressed, at MGH Cancer Center, medium supplemented with 10% fetal calf serum (Flow Laboratories), Fruit St., Boston. MA 02114. 'The abbreviations used are: MEX, methylxanthines; CAP, caffeine; HN2 penicillin (100 units/ml), and streptomycin (100 Mg/ml) at 37Â°Cin a (nitrogen mustard), methyl (fi-chloroethyl)amine. humidified, 10% CO2 atmosphere. The generation time under these 2463

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. EFFECTS OF METHYLXANTHINES IN HUMAN TUMOR CELLS conditions was 17 h. New cultures were started every 6 weeks from Flow Cytometry. Cells were prepared for flow cytometry as described frozen aliquots. The human karyotype was reconfirmed in our labora by Yen and Pardee (28). Cells grown in 60-mm culture plates were tory, and the non-HeLa isoenzyme pattern was confirmed by Dr. JÃ¶rgen washed three times with a hypotonie staining solution of propidium Fogh (Sloan Kettering Institute for Cancer Research. Rye, NY). Each iodide (50 Mg/ml in 0.1% sodium citrate). Cells were stained for 15 min batch of cells was determined to be free of mycoplasma by the method with propidium iodide at 4Â°Cand then dislodged gently by rubber of Schneider et al. (25). scraper to suspend the nuclei in the staining solution. Flow cytometry To synchronize cells at the G,/S border, exponential cultures were was performed with a Biophysics System Cytofluorograf model 4800A. first plated at high density (3-4 x 10" cells per 75-cm2 flask). They The data presented were repeated in at least 5 independent experiments. were allowed to grow in complete media for 2 days, at which time they Chromosome Analysis. Quantification of chromatid breaks, isochro- were densely confluent and nonproliferating, as demonstrated by flow matid breaks, and gaps followed procedures described by Hansson (21). cytometry. Since overgrowth led to cell death and detachment, a prac Colcemid (0.2 Mg/ml) was added to cultures 2 h before harvest, and tical method to obtain optimal confluence was achieved by initial plating mitotic cells were identified with Giemsa stain. In the data presented, of multiple cell densities (e.g., 3.6, 3.8, and 4.0 x IO6 cells) and the frequency of gaps was omitted from the total aberration yield. subsequent selection of dense flasks without cell detachment or debris. One hundred to 200 metaphase cells were examined per treatment Cells were then replated in fresh medium at 4 x IO5 cells per 60-mm condition, using slides which were coded. Aberrations scored as chro Petri dish. Densities less than 1-2 x IO5 cells/60-mm dish led to matid breaks showed dislocations greater than the chromatid diameter inconsistent synchrony. Eight h after this replating. hydroxyurea (0.5 with distinct misalignment. Isochromatid breaks showed dislocation IHM)was added. Prior studies determined that 8-h exposure to this and misalignment of both sister chromatids (Fig. 6). Chromosome hydroxyurea concentration was adequate to block thymidine incorpo analyses were repeated in 3 independent experiments. ration without cytotoxicity. Eight h later the cells became synchronized at the Gi/S border. Hydroxyurea was then removed, cells were rinsed twice, and fresh medium without hydroxyurea was added back to release RESULTS cells from the d/S border. Synchrony at the Gi/S border and subse quent cell cycle progression were monitored by flow cytometry and MEX Potentiation of Alkylator-induced Cytotoxicity. In prior tritiated-thymidine incorporation for autoradiography. About 85% of experiments reported from our laboratory, synergism was ob the total cell population progressed into the cell cycle within 2 h after served with theobromine or caffeine in bladder tumor cells release, as determined by autoradiography. HN2 was added to appro treated with thiotepa, a drug commonly used for clinical treat priate cultures l h before release from hydroxyurea. ment of bladder cancer (4). When T-24 bladder tumor cells Drug Treatment. Thiotepa (Lederle) was added to cultures from stock solutions dissolved in 0.9% NaCI and frozen at â€”70Â°C.HN2was added were exposed to thiotepa for l h in independent experiments, a dose-dependent decrease in cell survival was observed. Fifty from frozen aliquots from a stock solution of 100 JJMHN2 dissolved in sterile phosphate-buffered saline. At the end of alkylator treatment, % lethality (LD50) was produced by thiotepa at about 20 pg/ medium was removed, and cultures were rinsed twice before fresh ml. Subsequent exposure to pentoxifylline, as with caffeine, for medium was added back. 24 h markedly reduced cell survival (LD50 about 5 Mg/ml Caffeine (Sigma) and pentoxifylline (Hoechst) were dissolved in 0.9% thiotepa; Fig. 1). These MEX produced no significant cell NaCI. All MEX were made fresh as 100 mM stock solutions, and drug lethality in control plates without thiotepa. These MEX also additions did not alter pH of the media. enhanced lethality produced by the bifunctional alkylator HN2 Colony Formation. Following exposure to thiotepa or HN2 during (Fig. 2). Addition of MEX after 24 h had no significant effect exponential growth, the culture medium was changed, and T-24 cells on cell survival in alkylator-treated cultures (4). Using 2-way were trypsinized and plated for cloning efficiency in the presence or analysis of variance (26), both MEX produced a statistical absence of MEX. The cloning efficiency of untreated controls was 60 decrease in cell survival (P< 0.01) compared to alkylator alone. to 70% on standard 60-mm dishes (Falcon). Plates were fixed after 9 However, the degree of enhancement (approximately 3-fold days, and colonies greater than 200 cells were identified with crystal increased initial slope) was consistently less than previous stud- violet stain. Percentage of survival was computed as the number of colonies of treated cells relative to controls, so that all data points were corrected for the cloning efficiency of untreated controls. All points 100 represent the average of 2 plates from a typical experiment, and all cloning experiments were repeated 3-5 times. Cloning experiments ""5 were also done by plating cells 4 to 6 h before MEX additions to avoid nonspecific effects on cell attachment, and both methods gave identical results. Statistical differences were computed by 2-way analysis of variance with replicate plates from a single experiment (26), using multiple concentrations of alkylator in each treated group. The data presented were reconfirmed at least 3 times with various batches of cells, drugs, 10 and media. Autoradiography. Autoradiography was performed by using a modi fication of Hamlin's procedure (27). Cells were grown in 35-mm plates and pulsed at times indicated for 60 min with tritiated thymidine (0.5 o No Methylxonthine \ Â¿iCi/ml;specific activity, approximately 80 Ci/mmol; New England Nuclear). At the end of labeling, cells were rinsed twice with cold â€¢Â»Caffeine phosphate-buffered saline and then fixed in methanol/acetic acid, 2:1 (vol/vol), for 15 min at room temperature. After air drying, emulsion (Kodak NTB-2) was applied gently, and the plates were developed after 4 days with Kodak D19 developer. The cells were lightly stained with 10 20 30 Giemsa and percentage of labeled nuclei was determined by microscopic THIOTEPA CONCENTRATION counting. A minimum of 300 cells/plate was counted for each deter (Â¿ig/ml x 1hour) mination, and the data points represent the average value of two plates Fig. 1. MEX sensitivity of alkylator-treated human tumor cells. Drug treat from a typical experiment. Autoradiography experiments were repeated ment and percentage of survival were described in "Materials and Methods." at least 3 times per treatment condition. MEX (1 mM) were present for 24 h after removal of thiotepa. Bars, SE. 2464

100

80 30 JL

20 Control 40 0.5/xM HN2 HN2 10 20

8 12 16 HN, HOURS AFTER RELEASE FROM HYDROXYUREA Fig. 3. Fraction of cells in S phase after release from hydroxyurea. Cells were treated with HN2 for l h before release from hydroxyurea and followed by autoradiography, as described in "Materials and Methods." One mM caffeine was Fig. 2. Sensitivity of HN2-treated tumor cells to post-treatment with CAP, CAF plus cycloheximide (CHM), or 8-bromo-cAMP (Br-cAMP). Drug treatment added to cells with closed symbols immediately after release. and percentage of survival were described in "Materials and Methods." HN2 (0.5 MM)was present for 1 h, and CAF (1 mM), CAF plus CHM (0.18 MM),or 8- bromo-cAMP (0.1 mM) were present 0 to 24 h after removal of HN2. Bars, SE. 13 HOURS Â«P< 0.01 compared to HN2 or HN2 + CAF + CHM. 100 r Control ies from our laboratory with BHK rodent cells, where a 10-fold 0.5/iM HN2 increased slope was observed by MEX-alkylator combinations (13). Low concentrations (0.18 MM)of cycloheximide, which inhib its protein synthesis (13, 14), completely abolished the en hanced lethality by MEX (Fig. 2). Taken together, these results suggest that activity of MEX to enhance lethality in human tumor cells, as proposed for rodent cells (14), depends on synthesis of macromolecules such as proteins during the first cell cycle after DNA damage. In contrast, 8-bromo-cyclic AMP (0.01-0.5 mM) produced no enhanced lethality after alky hitor damage (Fig. 2). Coupled with other negative studies using the phosphodiesterase-inhibitor 3-isobutyl-l-methylxanthine (2, 29) or dibutyryl cAMP (30, 31), these results suggest that enhanced cytotoxicity by MEX does not result from effects on HOURS AFTER RELEASE cAM P metabolism. FROM HYDROXYUREA Effect of MEX on S-Phase Delay Produced by HN2. Prior Fig. 4. Fraction of cells in G2 + M phase after release from hydroxyurea. Cells were treated with 0.5 MMHN2 for l h before release from hydroxyurea and studies from our laboratory (13, 14) used HN2 to investigate followed by flow cytometry as described in "Materials and Methods." 1 mM cell cycle alterations produced by MEX-alkylator treatments in caffeine was added to cells with closed symbols immediately after release from rodent cells. In the present studies we similarly analyzed cell hydroxyurea. Inset shows typical histogram of DNA content at 13 h: left peak, G, cells; right peak. G2 + M cells. cycle progression in human tumor cells. HN2 was added to the T-24 cells while they were arrested at the d-S boundary (pro cedure described in "Materials and Methods"). One h later, demonstrated by flow cytometry and mitotic index, this is considerably greater than the S-phase delay (1 h) induced by cells were released from G,/S by rinsing two times and adding 0.5 /UMHN2. Fig. 4 shows the percentage of cells in G2 + M at fresh medium without HN2 or hydroxyurea. Drug effects on various times after treatment with HN2 (0.5 MM),which was subsequent cell cycle progression were monitored by flow cy- added for 1 h before release from the G,/S border. MEX thus tometry, pulse-labeled autoradiography, or total tritiated-thy- produced a more dramatic decrease in G2-phase delay compared midine incorporation. As shown in Fig. 3, HN2 treatment for to effects on S-phase. Control cultures reached maximum G2 l h produced a dose-dependent delay in progression through S- about 9-10 h after release from hydroxyurea; the maximum phase. Addition of caffeine (1 mM) after removal of HN2 mitoses were observed by 11 h. In contrast, 0.5 MMHN2-treated prevented this delay, allowing cells to proceed through S phase cells reached maximum G2 only after 13 h and demonstrated a at the rate of controls. Caffeine had no significant effect on S- 3-6-h delay in progression through G2. Since 0.5 MMHN2 phase progression of control (undamaged) cells. produced only about a 1-2-h delay in S phase, this delay in G2 Similar results (compared to autoradiography) were obtained transit represents about 100% increase compared to controls. in 2 separate experiments by measuring total tritiated-thymi- Post-treatment with caffeine (1 HIM)allowed HN2-treated cells dine incorporation for 5 h after HN2 treatment of synchronized to traverse G2 at the control rate. Thus, MEX induced the cells. This was decreased by 25% (+/-2.6%) in HN2-treated damaged cells to traverse both S and G2 at the rate of controls, cells, and caffeine restored uptake to control levels. as demonstrated by the appearance of new cells with d DNA Effect of MEX on G2 Delay Produced by HN2. After comple content (Fig. 4, inset). tion of S phase, HN2-treated cells showed a 3-6 h delay during Effect of MEX on Chromosome Damage. HN2 treatment (0.5 G2, which was prevented by post-treatment with MEX. As MMx 1 h) at the Gi/S border produced a small increase in the 2465

percentage of cells with chromosome aberrations, which were were HN2-treated at G,/S, followed by brief caffeine treatments analyzed in the subsequent mitoses. In order to use equal which corresponded to mid-S phase (4-8 h after HN2) or preparative techniques and ensure similar representation of mainly G? phase (8-12 h after HN2). Fig. 5 demonstrates mitotic cells in various test groups, colcemid was added to each minimal effect in mid-S phase, and the most profound enhance group at the beginning of M phase as determined by flow ment was after many cells had entered G2 phase. Equivalent cytometry and mitotic index. results were obtained by computation of total aberrations (chro Post-treatment by CAP produced a marked increase in chro matid breaks plus isochromatid breaks) per 100 chromosomes. mosome damage (Fig. 5). Fig. 6 illustrates several types of While no enhancement was observed when CAF was present in damaged chromosomes, observed after combined treatments mid-S phase, our results do not exclude effects in late S phase, with alkylators plus MEX. This CAF concentration (1 ITIM) due to partial overlap with 62 cells in the period 8-12 h after alone produced no increase in chromosome aberrations over HN2. baseline. DISCUSSION Since CAF prevented delays in cell cycle progression both in S and Gj, chromosome damage was quantified in cells that In human tumor cells, the studies reported here demonstrate that MEX have potent effects on cytotoxicity, chromosome aberrations, and cell cycle delays induced by anticancer alkylating agents. Our results are consistent with the G2-related S+G2 S mechanism proposed by Painter and Young in studies on ataxia (32), Busse et al. (19) in studies with X-rays, and Lau and I 60 2 Pardee in studies with alkylator damage in rodent cells (13). Ã¯ Taken together, these studies suggest that MEX enhance le io thality of some DNA-damaging agents by prevention of G2 g 40 a delays, leading to chromosome aberrations and cell death. This model supports the concept that G2 delays are essential for complete DNA repair, which requires a critical time for com

o 20 pletion before mitosis. MEX increase DNA damage by pre IL venting sufficient time for repair in G2; they do not inhibit biochemical repair reactions per se. D. The results presented here do not preclude additional mech - 0-12 0-12 4-8 4-8 8-12 8-12 CAF(HRS) anisms, such as direct or indirect inhibition of a poorly defined DNA repair pathway in late S-G2, leading to an increase in Fig. S. Effects of caffeine on the frequency of mitotic cells with chromosome aberrations (illustrated in Fig. 6). Cells were given 0.5 ti\t HN2 treatment l h lethal chromosome aberrations (and, possibly, a coordinate before release from hydroxyurea. and 1 mM caffeine was added during indicated inhibition of the delay in cell-cycle traverse). However, evidence Â¡menais. Colcemid was added during the last 2 h before harvest as cells entered against such inhibition of repair reactions was provided by M phase. studies demonstrating synergistic cytotoxicity but no observable change of DNA repair assays in cells treated with MEX + alkylating agents (2, 22). The effects of MEX in human tumor cells are similar to previous investigations from our laboratory with BHK rodent cells (13, 14).4 These studies are consistent with the working hypothesis that this action of MEX requires production of a protein. Puromycin and cycloheximide, two drugs which inhibit synthesis of proteins, blocked the actions of caffeine, using concentrations with minimal effect on cell cycle progression (14). Experiments with synchronized BHK cells demonstrated that cycloheximide is not delaying growth, so much that dam aged cells are prevented from entering into the caffeine-sensitive period. While the results presented here with human tumor cells are consistent with this model, we did not study multiple inhibitors or their specific actions on cell cycle progression. The present studies with human tumor cells did demonstrate several consistent differences from prior work with rodent cells (13, 14): (a) a reproducible effect on S-phase delay was shown in T-24 human tumor cells, while no such change was observed in BHK rodent cells; (b) profound fragmentation of the cell nucleus was observed in rodent cells after MEX-alkylator treat ments and cell division. In contrast, no such gross morpholog ical changes were observed in the human cells, although en hanced chromosome damage was demonstrated; and (c) survival of rodent cells exhibited greater sensitivity to MEX-alkylator treatments compared to human cells, especially after treatment Fig. 6. Typical chromosome spread of HN2-treated tumor cells with caffeine with alkylating agents at minimal toxicity. post-treatment. Cells were treated with 0.5 >iMHN2 for l h before release from Our investigations of MEX activity in rodent (13,14), human hydroxyurea, and I m.Mcaffeine was added 8-12 h later. Arrows show chromatid and isochromatid breaks. 4 S. K. Das. C. C. Lau, and A. B. Pardee, unpublished observations. 2466

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. EFFECTS OF METHYLXANTHINES IN HUMAN TUMOR CELLS tumor (4), and normal (33) cells have illustrated several diffi 11. Murnane, J. P.. Byfield, J. E., Ward, J. F., and Calabro-Jones, P. Effects of methylated xanthines on mammalian cells treated with bifunctional alkylat culties in such comparisons between various cell types. Sensi ing agents. Nature (Lond.), 285: 326-329, 1980. tivity can be altered dramatically by experimental conditions 12. Painter, R. Effects of caffeine on DNA synthesis in irradiated and unirra- (34), such as proliferative rate of cells at the time of drug diated mammalian cells. J. Mol. Biol., Â¡43:289-301, 1980. 13. Lau, C. C., and Pardee, A. B. Mechanism by which caffeine potentiates treatments (4). For example, the dramatic effects of MEX on lethality of nitrogen mustard. Proc. Nati. Acad. Sci. USA, 79: 2942-2946, chromosome aberrations, cytotoxicity, and cell cycle delays 1982. 14. Das, S. K., Lau, C. C., and Pardee, A. B. Abolition by cycloheximide of reported here could be abrogated by decreasing the growth caffeine-induced lethality of alkylating agents in hamster cells. Cancer Res., fraction (4). Comparisons of MEX sensitivity in various cell Â«.-4499-4504, 1982. types, therefore, must be controlled for proliferative rate and 15. Tobey, R. A. Different drugs arrest cells at a number of distinct states in G2. Nature (Lond.), 254: 245-247, 1975. cell cycle kinetics. A similar requirement for analysis of growth 16. Wennerberg, J., Aim, P., Biorklund, A.. Killander, D., Langstrom, E., and rates has been suggested by investigations using other drugs to Trope, C. Cell cycle perturbations in heterotransplanted squamous-cell car inhibit DNA repair (35). As discussed above, protein synthesis cinoma of the head and neck after mitomycin C and cisplatinum treatment. Int. J. Cancer, 33: 213-222, 1984. may be important for lethality enhancement by MEX. Differ 17. Vindelov, L. L., Hansen, H. H., Gersei, A., Hirsch, F. R., and Nissen, N. I. ences in MEX sensitivity, reported with various cell types (2, Treatment of small-cell carcinoma of the lung monitored by sequential flow 3, 8, 22), may be due in part to synthesis (or turnover) of a key cytometric DNA analysis. Cancer Res., 42: 2499-2505, 1982. 18. Bases, R., MÃ©ndez,F.,Liebeskind, D., Elequin, F., and Neubort, S. DNA of protein and/or other macromolecules under various experimen HeLa cells during caffeine-promoted recovery from X-ray induced G2 arrest. tal conditions (14). Int. J. RadiÃ¢t.Biol., 437-445, 1980. 19. Busse, P. M., Bose, S. K., Jones, R. W., and Tolmach, L. J. The action of Understanding the necessary conditions for enhanced lethal caffeine on X-irradiated. He La cells. III. Enhancement of X-ray-induced ity by MEX in tumor cells may provide a rational basis for the killing during G2 arrest. RadiÃ¢t.Res., 76: 292-307, 1978. 20. Lucke-Huhle, C. <>-Irradiatimi imliunl G2 delay: a period of cell recovery. future design of cancer treatment protocols in animal models RadiÃ¢t.Res., 89: 298-308, 1982. and in humans. For example, the data presented here suggest 21. Hansson, K., Kihlman, B. A., Tanzarella, C., and Palitti, F. Influence of that MEX: (a) should be maintained through (but not beyond) caffeine and 3-aminobenzamidine in G2 on the frequency of chromosomal aberrations induced by thiotepa, mitomycin C, and /V-methyl-A'-nitro-A"- the first cell cycle after alkylator therapy in vivo; (b) will be nitrosoguanidine in human lymphocytes. MutÃ¢t.Res., 126: 251-258, 1984. most effective in tumors (or tumor cell populations) with high 22. Sivak, A., Rudenko, L.. and Teague, L. G. Variations among species and cell rates of proliferation and protein synthesis; and (c) will be less types in the effects of caffeine on mutagen-induced cytotoxicity and post effective if combined with other treatments which inhibit mac- replication repair of DNA. Environ. Mutagen., 4: 143-162, 1982. 23. Fingert, H. J., Alley, M. C., Lieber, M. M., and Pardee, A. B. Enhanced romolecule synthesis and/or progression through G|. Selective lethality by methylxanthines in human bladder cancer cells treated with toxicity in tumor versus normal cells was indicated by prelimi thiotepa or mitomycin C (Abstract). Proc. Am. Assoc. Cancer Res., 25:354, 1984. nary studies from our laboratory, which showed less MEX 24. Fogh, J. Cultivation, characterization, and identification of human tumor effects in normal human cells (33), and other studies with cells with emphasis on kidney, testis, and bladder tumors. Nati. Cancer Inst. Monogr., 49:5-12, 1978. human or rodent tumors transplanted into mice (1,5, 36). 25. Schneider, E. L., Stanbridge, E. K.. and Epstein, C. J. Incorporation of 3H- In view of the known pharmacokinetic properties and clinical uridine and 3H-uracil into RNA: a simple technique for the detection of safety of pentoxifylline (37), this agent may have greater prom mycoplasma contamination of cultured cells. Exp. Cell Res., 84: 311-318, 1974. ise than other MEX for future animal or human trials. Using 26. Kleinbaum, D. G., and Kupper, L. L. Applied Regression Analysis and Other human tumors in cell culture or in animal models (4, 36, 38), Multivariate Methods. North Scituate, MA: Duxbury Press, 1978. further studies are testing the efficacy of pentoxifylline and 27. Hamlin, J. L., and Pardee, A. B. S-phase synchrony in monolayer CHO cultures. Exp. Cell Res., Â¡00:265-275, 1976. other MEX to improve the therapeutic index of clinically used 28. Yen, A., and Pardee, A. B. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. Cytotoxic, Cell Cycle, and Chromosomal Effects of Methylxanthines in Human Tumor Cells Treated with Alkylating Agents

Howard J. Fingert, James D. Chang and Arthur B. Pardee

Cancer Res 1986;46:2463-2467.

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