• Abstract and Figures
• Public Full-text

[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. (•_•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 37°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. Jörgen 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 4°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.

Load more

  6

Copy Link