Adozelesin,a potent new alkylating agent: cell-killing kinetics and cell-cycle effects

Summary Adozelesin (U-73975) was highly cytotoxic to V79 cells in culture and was more cytotoxic than several clinically active antitumor drugs as determined in a human tumor-cloning assay. Phase-specificity studies showed that cells in the M + early G1 phase were most resistant to adozelesin and those in the late G1 + early S phase were most sensitive. Adozelesin transiently slowed cell progression through the S phase and then blocked cells in G2. Some cells escaped the G2 block and either divided or commenced a second round of DNA synthesis (without undergoing cytokinesis) to become tetraploid. Adozelesin inhibited DNA synthesis more than it did RNA or protein synthesis. However, the dose needed for inhibition of DNA synthesis was 10-fold that required for inhibition of L1210 cell growth. The observation that cell growth was inhibited at doses that did not cause significant inhibition of DNA synthesis and that cells were ultimately capable of completing two rounds of DNA synthesis in the presence of the drug suggests that adozelesin did not exert its cytotoxicity by significant inhibition of DNA synthesis. It is likely that adozelesin alkylates DNA at specific sites, which leads to transient inhibition of DNA synthesis and subsequent G2 blockade followed by a succession of events (polyploidy and unbalanced growth) that result in cell death.     

Cell cycle effects of CC-1065

CC-1065 is the most potent antitumor agent tested in our laboratory. It is lethal to B16 and CHO cells and to a variety of human tumors in the clonogenic assay at 1 ng/ml and is effective against L1210 leukemia and B16 melanoma in vivo at 1 to 50 micrograms/kg. CC-1065 inhibits DNA synthesis and binds to DNA in a nonintercalative manner in the minor groove. We report here the kinetics of inhibition of DNA synthesis and of cell progression and the phase-specific toxicity of the drug. To determine phase-specific toxicity, we started synchronous CHO cultures from mitotic cells harvested after Colcemid pretreatment. These cultures showed that mitotic cells were the most sensitive, and sensitivity decreased as the cells progressed through G1 to S and G2. Experiments with B16 and CHO mitotic cells harvested without Colcemid pretreatment also showed that mitotic cells were more sensitive than G1/S-phase cells. Cell progression studies showed that CC-1065 did not affect progression from mitosis to G1 or from G1 to S. Cells progressed slowly through S at low levels (1 ng/ml) of the drug but were blocked in S at 5 ng/ml. Cell progression from G2 to M was blocked by CC-1065. DNA synthesis in B16 cells was measured at different times after 2-hr exposure to CC-1065. The percentage of inhibition of DNA synthesis was minimum at 4 hr and maximum at 19 hr after drug exposure. Since B16 cell progression studies showed a marked change in percentage of S-phase cells during this time, the DNA synthesis rate was recalculated as cpm/S-phase cell. After this correction (i.e., expressing DNA synthesis as cpm/S-phase cell), the percentage of inhibition of DNA synthesis was minimum at 0 hr and gradually increased to maximum inhibition at 19 hr without the decrease seen previously at 4 hr.     

Synergistic and additive combinations of several antitumor drugs and other agents with the potent alkylating agent adozelesin

Adozelesin is a highly potent alkylating agent that undergoes binding in the minor groove of double-stranded DNA (ds-DNA) at A-T-rich sequences followed by covalent bonding with N-3 of adenine in preferred sequences. On the basis of its highpotency, broad-spectrum in vivo antitumor activity and its unique mechanism of action, adozelesin has entered clinical trial. We report herein the cytotoxicity for Chinese hamster ovary (CHO) cells of several agents, including antitumor drugs, combined with adozelesin. The additive, synergistic, or antagonistic nature of the combined drug effect was determined for most combinations using the median-effect principle. The results show that in experiments using DNA- and RNA-synthesis inhibitors, prior treatment with the DNA inhibitor aphidicolin did not affect the lethality of adozelesin. Therefore, ongoing DNA synthesis is not needed for adozelesin cytotoxicity. Combination with the RNA inhibitor cordycepin also did not affect adozelesin cytotoxicity. In experiments with alkylating agents, combinations of adozelesin with melphalan or cisplatin were usually additive or slightly synergistic. Adozelesin-tetraplatin combinations were synergistic at several different ratios of the two drugs, and depending on the schedule of exposure to drug. In experiments using methylxanthines, adozelesin combined synergistically with noncytotoxic doses of caffeine or pentoxifylline and resulted in several logs of increase in adozelesin cytotoxicity. In experiments with hypomethylating agents, adozelesin combined synergistically with 5-azacytidine (5-aza-CR) and 5-aza-2-deoxycytidine (5-aza-2-CdR). Combinations of adozelesin with tetraplatin or 5-ara-2-CdR were also tested against B16 melanoma cells in vitro and were found to be additive and synergistic, respectively. The synergistic cytotoxicity to CHO cells of adozelesin combinations with tetraplatin, 5-aza-CR, or pentoxifylline was not due to increased adozelesin uptake or increased alkylation of DNA by adozelesin.     

Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants

The antitumor drug adozelesin is a potent cytotoxic DNA-damaging agent. Here we determined how adozelesin affects chromosomal DNA replication at a molecular level in a yeast model system and examined the influence of checkpoint kinase genes, the human homologues of which are mutated in cancer. Analysis of replication intermediates using two-dimensional gel electrophoresis showed that adozelesin inhibited the activity of a replication origin and stalled replication fork progression through chromosomal DNA at the origin. RAD53 and MEC1 protein kinase genes, homologues of human CHK2 and ATM, respectively, regulate an intra-S-phase DNA damage checkpoint and, when mutated, permit unchecked replication of damaged DNA in S-phase. Mutations in these genes did not abrogate adozelesin-induced inhibition of origin activity and fork progression at the replication origin. However, novel replication intermediates indicative of DNA breaks were detected only in the rad53 mutant, suggesting a role for the wild-type gene in maintaining chromosome integrity in the presence of the drug. In contrast to the inhibition of the active replication origin by adozelesin, normally silent origins present in the same chromosome were activated by adozelesin in rad53 and mec1 mutant cells. Thus, an antitumor drug that damages DNA can induce an abnormal replication pattern in a chromosome by activating silent origins, depending upon defects in yeast checkpoint kinase genes, the homologues of which are mutated in cancer. Implications of an abnormal replication pattern for the epigenetic regulation of gene expression are discussed.     

Induction of p53-dependent and p53-independent cellular responses by topoisomerase 1 inhibitors.

We have previously shown that loss of p53 function in A2780 human ovarian adenocarcinoma cells confers increased clonogenic resistance to several DNA-damaging agents, but not to taxol or camptothecin. We have now extended these studies, comparing wild-type p53-expressing A2780 cells with isogenic derivatives transfected with a dominant negative mutant (143; val to ala) p53. We show that, as well as retaining equivalent clonogenic sensitivity to camptothecin, mutant p53 transfectants of A2780 cells do not acquire significantly increased resistance to the camptothecin analogues topotecan and SN-38, the active metabolite of CPT-11. Compared with vector-alone transfectants they are, however, relatively (2.2-fold) resistant to GI 147211, a further camptothecin analogue undergoing clinical trial. Treatment of A2780 with camptothecin and each analogue produces an increase, maximal at 24-48 h after drug exposure, of cells in the G2/M phase of the cell cycle and a decrease in both G1 and S-phase cells. The G2 arrest is independent of p53 function for camptothecin and the three analogues. All four compounds can induce apoptosis in A2780, which is reduced in mutant p53 transfectants, as measured using the terminal DNA transferase-mediated b-d UTP nick end labelling (TUNEL) assay. Thus, although p53-dependent apoptosis is induced by camptothecin, topotecan and SN-38 in this human ovarian carcinoma cell line, these drugs induce p53-independent death, as measured by clonogenic assay.     

Additive interaction of gefitinib ('Iressa', ZD1839) and ionising radiation in human tumour cells in vitro

Cultures of human carcinoma A-431, A-549 and HeLa cells were challenged with gamma-rays without or with concomitant exposure to gefitinib, a potent inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR). The outcome of treatment was determined from cell and colony count, cell cycle progression and DNA double-strand break formation and rejoining. Apoptosis was measured in parallel from hypodiploid DNA and using an annexin V assay. Gefitinib developed a cytostatic effect in all cell lines, with drug sensitivity correlating the level of EGFR expression. A weak cytotoxicity of gefitinib was observed in HeLa cells only, although the drug was unable to induce significant cell cycle redistribution in this cell line. In contrast, substantial G1 block and S-phase depletion was observed in A-431 and A-549 cells exposed to gefitinib. The drug brought about additive to subadditive interaction with radiation with regard to growth inhibition, clonogenic death and induction of apoptosis. Consistently, gefitinib did not hinder the rejoining of radiation-induced DNA double-strand breaks in any cell line. The results demonstrate that gefitinib may elicit cytotoxicity at high concentration, but does not act as a radiosensitiser in vitro in concomitant association with radiation.     

Etoposide-induced cell cycle delay and arrest-dependent modulation of DNA topoisomerase II in small-cell lung cancer cells.

As an approach to the rational design of combination chemotherapy involving the anti-cancer DNA topoisomerase II poison etoposide (VP-16), we have studied the dynamic changes occurring in small-cell lung cancer (SCLC) cell populations during protracted VP-16 exposure. Cytometric methods were used to analyse changes in target enzyme availability and cell cycle progression in a SCLC cell line, mutant for the tumour-suppressor gene p53 and defective in the ability to arrest at the G1/S phase boundary. At concentrations up to 0.25 microM VP-16, cells became arrested in G2 by 24 h exposure, whereas at concentrations 0.25-2 microM G2 arrest was preceded by a dose-dependent early S-phase delay, confirmed by bromodeoxyuridine incorporation. Recovery potential was determined by stathmokinetic analysis and was studied further in aphidicolin-synchronised cultures released from G1/S and subsequently exposed to VP-16 in early S-phase. Cells not experiencing a VP-16-induced S-phase delay entered G2 delay dependent upon the continued presence of VP-16. These cells could progress to mitosis during a 6-24 h period after drug removal. Cells experiencing an early S-phase delay remained in long-term G2 arrest with greatly reducing ability to enter mitosis up to 24 h after removal of VP-16. Irreversible G2 arrest was delimited by the induction of significant levels of DNA cleavage or fragmentation, not associated with overt apoptosis, in the majority of cells. Western blotting of whole-cell preparations showed increases in topoisomerase II levels (up to 4-fold) attributable to cell cycle redistribution, while nuclei from cells recovering from S-phase delay showed enhanced immunoreactivity with an anti-topoisomerase II alpha antibody. The results imply that traverse of G1/S and early S-phase in the presence of a specific topoisomerase II poison gives rise to progressive low-level trapping of topoisomerase II alpha, enhanced topoisomerase II alpha availability and the subsequent irreversible arrest in G2 of cells showing limited DNA fragmentation. We suggest that protracted, low-dose chemotherapeutic regimens incorporating VP-16 are preferentially active towards cells attempting G1/S transition and have the potential for increasing the subsequent action of other topoisomerase II-targeted agents through target enzyme modulation. Combination modalities which prevent such dynamic changes occurring would act to reduce the effectiveness of the VP-16 component.     

Cell cycle perturbations in cisplatin-sensitive and resistant human ovarian carcinoma cells following treatment with cisplatin and low dose rate irradiation

Purpose: To investigate cell cycle pertubations in plateau-phase human ovarian carcinoma cells following treatment with cisplatin, low dose-rate irradiation (LDRI), or combined cisplatin and LDRI, in order to understand cell cycle mechanisms by which these two treatment modalities interact. Methods: Human ovarian carcinoma cells sensitive (A2780) and resistant (2780^CP) to cisplatin were grown to plateau phase and given protracted cisplatin treatments (A2780 0.7 and 2 μg/ml; 2780^CP 5 and 15 μg/ml) and/or LDRI (0.41 cGy/min). Cell cycle distribution following treatment was determined by two-parameter flow cytometry, based on bromodeoxyuridine (BrdU) uptake and DNA content using propidium iodide staining.Results: The cisplatin-sensitive A2780 cells exposed to cisplatin alone for up to 28 h showed depletion of the G1 fraction and accumulation in S-phase, although the percentage of S-phase cells actively incorporating BrdU dropped to almost zero. The cisplatin-resistant 2780^CP cells exposed to cisplatin alone showed a G1 arrest when exposed to 15 μg/ml, but not when exposed to 5 μg/ml. LDRI alone caused little cell cycle redistribution different from controls in either cell line. When LDRI was combined with cisplatin, no significant cell cycle redistribution was observed, apart from a decline in the actively incorporating S-phase fraction. Conclusions: Cisplatin caused A2780 cells to accumulate in nonincorporating S-phase, with no evidence of G1 arrest. Cisplatin-resistant 2780^CP cells showed a G1 block when exposed to a high enough cisplatin concentration. This could indicate a mechanism of cisplatin resistance in these cells. LDRI alone or in combination with cisplatin did not result in significant cell cycle redistribution. Received: 22 December 1995 / Accepted: 28 September 1996     

Radiosensitizing potential of gemcitabine (2′,2′-difluoro-2′-deoxycytidine) within the cell cycle in vitro

Purpose: Gemcitabine (2′,2′-difluorodeoxycytidine; dFdCyd) is a new deoxycitidine analog which exhibits substantial activity against solid tumors and radiosensitizing properties in vitro. To examine cell cycle–specific effects of a combined treatment with gemcitabine and radiation, the in vitro clonogenic survival of two different cell lines was measured for cells from log-phase culture, G1 and S-phase cells.Methods and Materials: Chinese hamster (V79) and human colon carcinoma (Widr) cells were exposed to different radiation doses and for different points of time relative to gemcitabine treatment (2 h). Experiments were also carried out with different cell-cycle populations obtained after mitotic selection (V79) or after serum stimulation of plateau-phase cells (Widr). The resulting survival curves were analyzed according to the LQ model, and mean inactivation doses (MID) and the cell cycle–specific enhancement ratios (ER) were calculated from the survival curve parameters.Results: Effectiveness of combined treatment of log-phase cells was greatest when cells were irradiated at the end of the gemcitabine exposure [ER: 1.28 (V79), 1.24 (Widr)]. For later times after the removal of the drug, radiosensitization declined, approaching independent toxicity. From the time course of interactive-type damage decay half-life values of 75 min (V79) and 92 min (Widr) were derived. Gemcitabine did not radiosensitize G1 Widr cells or V79 cells from the G1/S border, but substantial radiosensitization was observed for the S-phase cell preparations [ER: 1.45 (V79-lateS), 1.57 (Widr)].Conclusions: Treatment of cells with gemcitabine immediately before irradiation eliminates, or at least greatly reduces, the variation in radiosensitivity during the cell cycle that is manifested by radioresistance during S phase. This reversal of S-phase radioresistance could imply that gemcitabine interferes with the potentially lethal damage repair/fixation pathway. Other approaches have been taken to overcome S-phase radioresistance, such as hyperthermia or densely ionizing radiation, and combined treatments with dFdCyd could prove of value to complement such efforts.     

Detection of S-phase overreplication following chronic hypoxia using a monoclonal anti-BrdUrd

We have examined cytokinetic perturbations induced in Chinese hamster V-79 cells in vitro during and following exposure to chronic hypoxia employing simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content. These data indicate hypoxia inhibited G1 progression into S-phase, but did not significantly delay G2M division and progression into G1. Also, upon reaeration after 20 hr in hypoxia, cells originally in G1 exhibited significant kinetic delay. BrdUrd pulse/chase and pulse/fix data indicated DNA replication was reduced, but not completely inhibited during hypoxia. Also, between 6 and 20 hr of chronic hypoxia and following reaeration, a subset of the original S-phase cells overreplicated their DNA, such that these cells had greater than 2C DNA content. This subpopulation was estimated on the average to comprise approximately 20% of the total population (30% of the treated S-phase subpopulation) by 24 hr following reaeration after 20 hr hypoxia. These results are discussed in light of the similarities between overreplication and gene amplification observed under certain conditions with other agents, which like chronic hypoxia, are used to transiently disrupt DNA synthesis.     

Ecteinascidin-743 (ET-743), a natural marine compound, with a unique mechanism of action

The mode of action of Ecteinascidin-743 (ET-743), a marine tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, which has shown very potent antitumour activity in preclinical systems and encouraging results in Phase I clinical trials was investigated at a cellular level. Both SW620 and LoVo human intestinal carcinoma cell lines exposed for 1 h to ET-743 progress through S phase more slowly than control cells and then accumulate in the G2M phase. The sensitivity to ET-743 of G1 synchronised cells was much higher than that of cells synchronised in S phase and even higher than that of cells synchronised in G2M. ET-743 concentrations up to four times higher than the IC(50) value caused no detectable DNA breaks or DNA-protein cross-links as assessed by alkaline elution techniques. ET-743 induced a significant increase in p53 levels in cell lines expressing wild-type (wt) (p53). However, the p53 status does not appear to be related to the ET-743 cytotoxic activity as demonstrated by comparing the drug sensitivity in p53 (-/-) or (+/+) mouse embryo fibroblasts and in A2780 ovarian cancer cells or the A2780/CX3 sub-line transfected with a dominant-negative mutant TP53. The cytotoxic potency of ET-743 was comparatively evaluated in CHO cell lines proficient or deficient in nucleotide excision repair (NER), and it was found that ET-743 was approximately 7-8 times less active in ERCC3/XPB and ERCC1-deficient cells than control cells. The findings that G1 phase cells are hypersensitive and that NER-deficient cells are resistant to ET-743 indicate that the mode of action of ET-743 is unique and different from that of other DNA-interacting drugs.     

Cellular mechanisms associated with the lack of chronic thermotolerance expression in HeLa S3 cells.

Chronic thermotolerance is an operational definition for that resistance to cell killing by heat which develops during a protracted exposure at temperatures generally in the range of 41.5-42.5 degrees C which is usually observed as a reduction in the slope of the survival curve. While Chinese hamster ovary (CHO) cells are generally more sensitive to high-temperature heat shock than HeLa cells, studies of cells maintained in suspension culture at 41.5 degrees C demonstrated CHO cells to be more resistant to cell killing at this temperature than HeLa cells, due to the expression of chronic thermotolerance in the hamster cell line and the corresponding lack of chronic thermotolerance expression in the HeLa cell line. Experiments were conducted in the two cell lines while heating under identical conditions, in order to detect any cell line-specific changes in heat-induced perturbation of cell cycle progression and the expression of chronic thermotolerance. Our results showed that CHO cells exhibited a G1 block which lasted throughout the course of the 32-h heating period. HeLa cells, however, failed to accumulate in G1, progressing instead into S phase where spontaneous premature chromosome condensation and nuclear fragmentation were observed. This accumulation of cells with condensed chromatin possessing S-phase DNA content exhibited a linear, one-to-one functional relationship with the fraction of dead cells. Previous studies (M.A. Mackey and W.C. Dewey, Int. J. Hyperthermia, 5:405-415, 1989) demonstrated that synchronized S-phase CHO cells heated at 41.5 degrees C and 42 degrees C were unable to express chronic thermotolerance. Therefore, we hypothesize that progression of cells out of G1 phase into S and G2-M phases leads to lethal processes that prevent the expression of chronic thermotolerance in the HeLa cell line. This hypothesis is strengthened by the observed correlation between the accumulation of "mitotic-like" cells and decreased survival, suggesting that the G1 block observed in CHO cells is causally connected with the expression of chronic thermotolerance.     

Cytotoxicity of tetraplatin and cisplatin for human and rodent cell lines cultured as monolayers and multicellular spheroids

The cytotoxicity of tetraplatin (dl-trans), its d- and l-isomers, and cisplatin for four human tumor cell lines (myeloma 8226, ovarian 2008, A2780, and OVCAR-3), their cisplatin-resistant variants, and three rodent cell lines (V79, EMT6/Ro, and L1210) were compared. Tetraplatin was more, or equally as, potent as cisplatin for the human cell lines and for L1210 but was clearly less potent for V79 and EMT6/Ro. The d-trans tetraplatin was more potent than the l-trans. Cisplatin resistant human tumor cells were less resistant to tetraplatin. On comparing sensitivity of V79 and EMT6/Ro cells in two growth models, we observed that all of the platinum compounds were more cytotoxic to cells in multicellular spheroids than in exponentially growing monolayers. Uptake studies, however, showed that tetraplatin was more cytotoxic to spheroids because spheroids accumulated more drug than monolayers.     

Cell killing and kinetic effects of diglycolaldehyde on dividing and nondividing mammalian cells in vitro.

The effects of the anticancer drug diglycolaldehyde (NSC-118994) were studied on Chinese hamster ovary cells growing in vitro. Dividing cells, specifically those in S-phase, were more sensitive to the drug than were nondividing cells, although a large fraction of nondividing cells was also killed by doses up to 800 microgram/ml. Dose-dependent effects on cell progression kinetics were observed in all phases of the cell cycle except in mitosis, during which treated cells progressed at control rates into G1-phase. The inhibition of cell progression from G1- into S-phase (most sensitive phase of the cell cycle) put self-limiting restrictions on the cell killing effects of the drug.     

Induction of apoptosis by taxol and cisplatin and effect on cell cycle-related proteins in cisplatin-sensitive and -resistant human ovarian cells.

The effect of taxol (TX) and cisplatin (CDDP), singly or in association, was assessed on two human ovarian cancer cell lines, one sensitive (A2780) and one resistant (A2780 cp8) to CDDP. Cell lines showed a similar sensitivity to TX, whereas different cytotoxicity results were obtained in the two cell lines as a function of TX and CDDP sequence. Specifically, TX followed by CDDP induced simply additive effects in both cell lines, whereas the opposite sequence produced antagonistic effects in A2780 cells and synergistic effects in A2780 cp8 cells. TX, with or without CDDP, induced oligonucleosomal DNA fragmentation typical of the apoptotic process, but the biochemical mechanisms undergoing apoptosis were different in the two cell lines. In fact, in A2780 cells, TX (with or without CDDP) treatment markedly increased p53 as well as p21waf1 protein expression. In A2780 cp8 cells, drug treatment enhanced p53 levels, whereas the expression of p21waf1 was always undetectable at mRNA and protein levels. In the latter cell line, a premature activation of p34cdc2 kinase was observed in correspondence with the drug-induced increase in the S-phase cell fraction. Such an activation was not ascribable to an increase in the overall expression of p34cdc2 or cyclin B1 proteins, but to a dephosphorylation of p34cdc2 kinase. Overall, our results indicate that TX-induced apoptosis in human ovarian cancer cells may be sustained by different events at the cell cycle-control level.     

The Effect of the MDM2-p53 Loop on the Sensitivity of Ovarian Cancer Cells to Cisplatin

OBJECTIVE To explore whether MDM2 transfection can alter the MDM2-p53 autoregulatory feedback loop so as to change the sensitivity of ovarian cancer cell lines to cisplatin. METHODS The ovarian cancer cell line A2780 expressing wild-type P53 and the ovarian cancer cell line SKOV-3 with the p53 null type were stably transfected with pCMV-MDM2 or pCMV as a control. The blocked expression of P53 was determined by Western blots. Cytotoxicity was assessed using the MTT assay and the trypan blue exclusion assay. Flow cytometry was used to detect changes in the cell cycle and removal of platinum -DNA adducts was measured by atomic absorption spec-troscopy. RESULTS (1) Parental A2780 and A2780-V cells (IC50= 15.14±1.39 μmol) have similar cisplatin sensitivities, whereas sensitivity to cisplatin in A2780-M cells (IC50=7.98±1.32 μmol) was 2 to 3 fold greater (P=0.001). The trypan blue exclusion assay demonstrated that cisplatin killed a higher percentage of A2780-M cells compared to A2780-V cells. There was no significant change following MDM2 transfection in SKOV-3 cells. (2) After cisplatin treatment, A2780-M cells showed a pronounced S-phase arrest, however, A2780 cells with the intact wild-type P53, arrested primarily at the G2/M transition. (3) Platinum uptake was similar for all of the A2780 cell lines after ciaplatin treatment, but the removal of plat-inum-DNA adducts was reduced in the A2780-M cells compared with A2780-V cells. CONCLUSION MDM2 increases cisplatin cytotoxicity in ovarian cancer cells by blocking the expression of p53 through the MDM2-p53 autoregulatory feedback loop.     

Detection of etoposide resistance by measuring DNA damage in individual Chinese hamster cells

The comet assay, which measures DNA strand breakage in individual cells, was used to examine the relation between DNA damage, cell survival, and resistance to the topoisomerase II inhibitor etoposide (VP-16). Chinese hamster V79-171b cells and a VP-16-resistant subline (VPr) were exposed to VP-16 as monolayers or spheroids. The comet assay was comparable in sensitivity to the DNA precipitation and alkali unwinding assays for detecting DNA strand breaks induced by VP-16. However, unlike conventional DNA damage assays, the comet assay also indicated heterogeneity in cell response. For V79 multicell spheroids exposed to VP-16, the external cycling cells were 50 times more sensitive to killing and DNA damage than the internal noncycling cells; the comet assay indicated the fraction of cells resistant to the drug. VPr cells, which were 10 times more resistant to killing and DNA damage by VP-16 than the parent cell line, could also be identified in mixed populations with the use of this method. These results suggest that the comet assay could be useful in predicting tumor cell response to DNA-damaging agents.     

Effects of ellipticine on cell survival and cell cycle progression in cultured mammalian cells

The effects of ellipticine [5,11-dimethyl-6H-pyrido(4,3-b)carbazole; NSC 71795] on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent [Chinese hamster ovary (CHO)]tumor cell systems as well as in mitogen-stimulated human peripheral blood lymphocyte cultures. Cell cycle progression and the terminal point of action of the drug were monitored by flow cytometry. Ellipticine was cytostatic for all cell lines tested, blocking cells in G2 phase following 24 hr constant exposure at concentrations in the range of 1.0 microgram/ml. A 10 times higher drug concentration was required to block cells in G2 if the cells were exposed for only 30 min to the drug followed by 23.5 hr culture in drug-free medium. Formation of CHO cell colonies was inhibited by 50% following exposure to ellipticine for 2 hr at 6.0 microgram/ml or for 24 hr at 0.3 microgram/ml. Fifty % cell kill in asynchronously growing Friend leukemia and L1210 cells was obtained following exposure to ellipticine for 24 hr at 2.0 microgram/ml and 1.15 microgram/ml, respectively, whereas human peripheral blood lymphocytes required 66 hr exposure to 1.0 microgram/ml to kill 50% of the cells. Phytohemagglutinin-stimulated lymphocytes were remarkably resistant to the cytotoxic effect of ellipticine but did display a dose-dependent inhibition of stimulation and accumulation in G2 whether the drug was added prior to our during active cell proliferation. Ellipticine, at cytostatic concentrations, had a marked effect on cellular RNA content. Friend leukemia cells, blocked in G2 by the drug, doubled their RNA content compared to control cells. L1210 and CHO cells, but not lymphocytes, also increased in RNA content following ellipticine treatment. Drug concentrations which blocked cells in G2 also led in the case of Friend leukemia and L1210 but not CHO cells to an increase in the proportion of cells with greater than 4C amounts of DNA.     

DNA double-strand breaks measured in individual cells subjected to gel electrophoresis

Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as low as 5 Gy. The radiation dose-response relationship for initial formation of double-strand breaks was identical for cell lines irradiated in G1, regardless of their sensitivity to killing by ionizing radiation. However, for cells irradiated in S phase, DNA migration was significantly reduced. For Chinese hamster V79 cells, Chinese hamster ovary cells, WiDr human colon carcinoma cells, and L5178Y-R mouse lymphoblastoid cells, S-phase DNA appeared to be about 3 times less sensitive to X-ray damage than DNA from other phases of the cell cycle. However, for the very radiosensitive L5178Y-S cells, the migration of replicating DNA was reduced only slightly. For Chinese hamster V79 and Chinese hamster ovary cells, damage was repaired at a similar rate in all cells of the population, and 85% of the breaks were rejoined within 2 h after irradiation. The radiosensitive L5178Y-S cells repaired damage more slowly than V79 or Chinese hamster ovary cells; 2 h after exposure to 50 Gy, approximately 50% of the damage was still present.