Lethality, DNA alkylation, and cell cycle effects of adozelesin (U-73975) on rodent and human cells.

Adozelesin (U-73975) is an extremely potent cytotoxic agent which causes 90% lethality, after 2 h exposure in vitro, of Chinese hamster ovary and lung (CHO and V79), mouse melanoma (B16), and human ovarian carcinoma (A2780) cells at 0.33, 0.19, 0.2, and 0.025 ng/ml, respectively. Under similar conditions, Adriamycin and cisplatin had 90% lethality values in CHO cells of 150 ng/ml (= 249 nM) and 6800 ng/ml (= 2266 nM), respectively. The relative drug sensitivity of the cell lines (A2780 > V79, B16, CHO) was correlated to the relative amounts of [3H]adozelesin alkylated to DNA. The greater sensitivity of A2780 was due to (a) greater DNA alkylation at different drug doses and (b) greater intrinsic sensitivity of A2780 which resulted in greater cell kill at comparable DNA alkylation. Phase specific toxicity studies show that adozelesin was least lethal to CHO cells in mitosis and very early G1. Lethality increased as cells progressed through G1 and was maximal in late G1 and early S. Mitotic cells had lower drug uptake and correspondingly less drug binding to DNA than G1 or S-phase cells. However, based on the amount of drug alkylated per micrograms of DNA, cells in M, G1, and S were equally sensitive. Therefore, the lower sensitivity of M-phase cells was due to lower drug uptake. Adozelesin had three different effects on progression of CHO, V79, B16, and A2780 through the cell cycle: (a) slowed progression through S which resulted in significantly increasing the percentage of S-phase cells. This effect was transient; (b) cell progression was blocked in G2 for a long time period; (c) the response of the cell lines to the G2 block differed. CHO and V79 cells escaped G2 block by dividing and entered the diploid DNA cycle or did not undergo cytokinesis and became tetraploid. On the contrary, B16 and A2780 cells remained blocked in G2 and did not become tetraploid. Cell progression was inhibited in a similar manner when a synchronized population of M, G1, or S-phase cells were exposed to adozelesin.     

Perturbation by insulin of human breast cancer cell cycle kinetics

The growth of cultured human breast cancer cells is sensitive to physiological concentrations of insulin suggesting that it may regulate breast cancer growth in vivo. The mechanisms for the growth effects of insulin are poorly defined. In the present study, we examine the effects of insulin on the cell cycle kinetics of asynchronous MCF-7 human breast cancer cells growing in serum-free medium. When the [3H]thymidine labeling index is used to estimate the S-phase fraction, insulin added to asynchronously growing cells results in a time-dependent increase in the proportion of cells engaged in DNA synthesis. Computer analysis of DNA histograms obtained by flow cytometry of mithramycin-stained cells also shows a time-dependent progression of cells into and through the S-phase compartment. Sixteen hr after adding insulin to asynchronous cells, 66% of cells are in S-phase compared to 37% in controls. The effect of insulin on the cell cycle progression of MCF-7 cells is also dose dependent. Stimulation is observed with physiological insulin concentrations of 0.1 to 1.0 nM; maximal effects are observed with 1.0 to 10 nM insulin. Various insulin analogues enhance the progression of cells into S phase in proportion to their ability to bind to the insulin receptor in MCF-7 cells (porcine greater than or equal to chicken greater than guinea pig greater than deoctapeptide insulin), while unrelated peptide hormones have no effect on the cell cycle kinetics. Cell cycle analysis after the addition of colchicine to prevent mitosis and the reentry of cells into G1 demonstrates a shortened G1 in response to insulin. Continuous [3H]thymidine-labeling studies after the addition of colchicine suggest that the growth fraction is about 88% with or without insulin. In summary, insulin causes a marked perturbation of the cell cycle kinetics of MCF-7 human breast cancer cells by facilitating the transit of cells through G1. The data also suggest that this effect is mediated via the insulin receptor.     

Kinetic response to cultured human lymphoid cells to rubidazone

Analysis of rubidazone, the benzoylhydrazone derivative of daunorubicin, for its effects on cell cycle progression of a human lymphoid cell line showed a kinetic response pattern similar to that of adriamycin. Thus rubidazone induced a G2-block, the magnitude and duration of which were dependent on concentration and incubation time. However, in contrast to adriamycin, a marked phase-dependent sensitivity for the induction of G2-accumulation was observed; cells treated in early and mid-S-phase were most sensitive. This age-dependent kinetic response may account for the smaller G2-accumulation in asynchronous cultures and the closer correlation of the magnitude of this kinetic effect with concentration and duration of rubidazone treatment. Prolonged exposure to high concentrations of rubidazone also delayed the traverse through G1 and/or the G1-S transition, whereas the S-phase transit was not impaired. Interference with cell cycle progression through G1 into S-phase caused a stepwise accumulation of cells in G2-phase.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: III. Effect of antiestrogen

Estrogen (E) stimulatory effects on mammary carcinoma are well documented; however, little is known of the precise mechanism regulating E-induced cell proliferation. This study attempts to investigate the in vitro effects of E and antiestrogen (AE) on promoting cell proliferation and the cell cycle kinetics of an experimental mammary carcinoma model, CAMA-1. Sublines IR and IN, which have been shown to respond to E somewhat differently in 3H-thymidine (3H-dThd) uptake and in the induction of progesterone receptor (Cancer Res 41: 5004-5009, 1981), were evaluated concurrently. The present report showed that E stimulated and AE, as shown by tamoxifen (TAM) and nafoxidine hydrochloride (NAF), inhibited cell growth and 3H-dThd uptake in a dose-related manner for both sublines supplemented with serum. In a chemically defined medium with 1% steroid-stripped serum, the E-stimulatory effect was nullified, and cells were more sensitive to AE. These results suggest that serum component(s) is (are) involved in AE and E action. The TAM inhibition could be partially reversed by E. A partially synchronized G1 population, arrested by serum deprivation for 48 hrs, responded promptly to E stimulation. While E promoted a faster G1 exit, faster for IN cells than for IR subline, AE inhibited cell progression at the G1 phase. Furthermore, E stimulated a higher proportion of S-phase formation during the first cycle of hormone treatment while AE inhibited this process. These results are consistent with the notion that TAM and E act on some common events at the G1 phase in cell proliferation. The net result of E stimulation was the promotion of new cell to traverse the cell cycle, the acceleration of G1 exit, and an increase in the proportion of S-phase and dividing cells per cycle. In contrast, AE inhibited the progression of cells at the G1 phase, resulting in a marked decrease of S-phase and dividing cells per cycle. Our results also demonstrate the importance of careful kinetic studies in evaluating the E and AE responses of mammary carcinoma cells in culture, and this is best conducted with synchronized populations.     

Effects of aclacinomycin on cell survival and cell cycle progression of cultured mammalian cells

The effects of aclacinomycin (ACM; NSC 208734) on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent (Chinese hamster ovary) cell systems. Cell cycle progression and the effect of the drug on various transition points in the cell cycle (i.e. G1 to S phase, through a window in early S phase and G2 phase to mitosis) were monitored by flow cytometry. Formation of Chinese hamster ovary cell colonies was inhibited by 50% following 24 hr of exposure to 0.05 micrograms ACM per ml whereas 1 hr of exposure to 1.0 micrograms ACM per ml reduced colony formation by only 30%. Stationary cultures required a drug concentration more than 5 times higher to reduce colony formation by an equivalent amount when present for 24 hr. Short-term (1-hr) exposure to drug concentrations up to 1.0 micrograms/ml had no effect on colony formation of stationary-phase Chinese hamster ovary cells. Cell growth was inhibited by 50% in suspension cultures of Friend leukemia and L1210 cells when exposed for 24 hr to 0.024 and 0.053 micrograms ACM per ml, respectively. Continuous drug exposure of Friend leukemia and L1210 cells to ACM concentrations of 0.05 to 0.1 micrograms/ml led to a slow down in cell progression manifested as an accumulation of cells in G2 + M phase by 24-hr and then in G1 phase by 48-hr culture. However, brief (1-hr) exposure of L1210 cells to 0.5 micrograms/ml resulted in an irreversible accumulation of cells in G2 + M phase. A more detailed examination of drug effects on the cell cycle determined that 0.1 micrograms ACM per ml resulted in a slow down in L1210 cells leaving G1 phase and entering mitosis and an accumulation of cells in G2 phase, although early S-phase cells appeared unaffected. At a 5 times higher drug concentration, exit of cells from G1 was almost completely halted, passage of cells through early S was slowed, and the entrance of cells into mitosis plateaued 3.5 hr after addition of the drug; G2-phase cells were only mildly affected. The RNA content of all cells examined was reduced by 35 to 50% depending upon dose and time of exposure. These findings are discussed in terms of the known biochemical effects of ACM on RNA and protein synthesis.     

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.     

Tracking the cell cycle origins for escape from topotecan action by breast cancer cells

The anticancer agent topotecan is considered to be S-phase specific. This implies that cancer cells that are not actively replicating DNA could resist the effects of the drug. The cycle specificity of topotecan action was investigated in MCF-7 cells, using time-lapse microscopy to link the initial cell cycle position during acute exposures to topotecan with the antiproliferative consequences for individual cells. The bioactive dose range (0.5-10 microM) for 1-h topotecan exposures was defined by rapid drug delivery and topoisomerase I trapping. Topotecan caused pan-cycle induction and activation of p53. Lineage analysis of the time-lapse sequences identified cells initially in S-phase and G2, and defined the time to mitosis for cells originating from G2, S-phase and G1. Topotecan prevented all mitoses from S-phase cells and G1 cells (half-maximal effects at 0.14 microM and 0.96 microM, respectively). No dose of topotecan completely prevented mitosis among G2 cells, and at saturating doses of topotecan about half the cells of G2 origin continued dividing (the half-maximal effects was at 0.31 microM). Overall, topotecan differentially targeted G1-, S- and G2-phase cells, but many G2 cells were resistant to topotecan, presenting a clear route for cell cycle-mediated drug resistance.     

S-Phase arrest by nucleoside analogues and abrogation of survival without cell cycle progression by 7-hydroxystaurosporine

The mechanisms of resistance to nucleoside analogues established in preclinical models are rarely found in primary tumors resistant to therapy with these agents. We tested the hypothesis that cells sense sublethal incorporation of analogues into DNA during replication and react by arresting further DNA synthesis and cell cycle progression. After removal of drug, cells may be able to repair damaged DNA and continue proliferation, thus escaping nucleoside analogue toxicity. As a corollary, we evaluated whether dysregulation of this mechanism causes cell death. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, we found that DNA synthesis decreased, cells arrested in S-phase transit, and 60-70% of the population accumulated in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium. S-phase-arrested cells were then treated with otherwise nontoxic concentrations of UCN-01, which caused rapid onset of apoptosis without cell cycle progression specifically in cells with an S-phase DNA content. Thus, S-phase arrest by nucleoside analogues sensitizes cells to UCN-01, which appears to activate signaling for death mechanisms and/or inhibit survival pathways. These results differ from those in cells arrested at the G2 checkpoint, in which UCN-01 abrogates cell cycle arrest, permitting cells to progress in the cell cycle before apoptosis.     

Bimodal effects of 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) on cell cycle checkpoints.

1R,2R-Diaminocyclohexane(trans-diacetato)(dichloro)-platinum(IV) (DACH-acetato-Pt) is a novel platinum-based agent that is highly effective against cisplatin-resistant ovarian tumor cells. To probe its cellular mechanism, the effects of DACH-acetato-Pt (0-6.4 microM) on cell cycle checkpoints were examined using the ovarian cancer A2780 cell line as the model system. We found that DACH-acetato-Pt at > or =0.2 microM dramatically inhibited cell growth and induced cell death. At concentrations < or =0.6 microM (low effective concentrations), DACH-acetato-Pt specifically induced G(1) phase arrest by selectively inhibiting cyclin-dependent kinase 4 (Cdk4) and Cdk2 activities. The Cdc2 activity, which regulates G(2)-M phase progression, was unaffected by the drug at these concentrations. At concentrations >0.6 microM (high effective concentrations), DACH-acetato-Pt first transiently inhibited S-phase progression and then blocked cell cycle progression at both G(1) and G(2) phases. These cell cycle effects were associated with sequential inhibitions of Cdk2/cyclin A activity, Cdk4 and Cdk2 activities, and Cdc2 kinase activity. Following the cell cycle effects, both the low and high effective concentrations of DACH-acetato-Pt induced cell death through apoptosis. These results indicate that DACH-acetato-Pt activates multiple cell cycle checkpoints in a bimodal manner and suggest that the cell cycle effects demonstrated in these studies may be linked to its ability to induce apoptosis.     

Cell cycle phase-specific cytotoxicity of the antitumor agent maytansine.

The objective of this investigation was to study the effects of maytansine on the cell cycle kinetics of HeLa cells. The results of this study indicate that maytansine is a very potent mitotic inhibitor and that it has no effect on macromolecular synthesis. Maytansine-induced cytotoxicity was dependent upon the position of the cell in the cell cycle. Mitotic and G2 cells are most sensitive to this agent, while G1 phase cells are the most resistant, with S-phase cells being intermediate. Small (0.82 X 10(-8) M) fractionated doses given at an interval of 8 hr have been found to be more cytotoxic than was a large (1.63 X 10(-8) M) single dose. In evaluating the drug combinations, we observed that the schedule in which 1-beta-D-arabinofuranosylcytosine treatment was followed by maytansine treatment exhibited greater cell kill than the reverse sequence. No schedule-dependent effects were observed when maytansine was tried in combination with Adriamycin.     

Flow cytometric analysis by bromodeoxyuridine/DNA assay of cell cycle perturbation of methotrexate-treated mouse L1210 leukemia cells

The in vitro effects of methotrexate (MTX) on cell cycle progression and DNA synthesis of L1210 leukemia cells were studied by the bromodeoxyuridine (BrdUrd)/DNA analysis technique. Low dose (10(-8) M) MTX, which slightly inhibits clonal replication of the cells, delays progress across the S phase, and treatment for 24 h results in a slight increase of the S-phase population. Much higher doses (10(-7) M and 10(-6) M) of MTX, which strongly reduce the clonogenicity, prevented the progression of cells at the G1-S boundary and across the S phase, but not in the other phases. The cells arrested at the G1-S boundary were able to incorporate BrdUrd in the medium for 6-12 h after the start of treatment and then lost the ability to incorporate BrdUrd. By determining the colony inhibitory activity of MTX, it could be shown that not only S-phase cells but non-S-phase cells are susceptible to cytotoxicity of MTX. MTX-induced S-phase arrest is closely associated with an alteration in the distribution of BrdUrd-labeled cells, and MTX apparently inhibits BrdUrd incorporation into L1210 cells as the dose and duration of treatment increase. These results suggest that MTX-induced cell cycle perturbation is related to inhibition of DNA synthesis.     

Effects of genistein on the growth and cell cycle progression of normal human lymphocytes and human leukemic MOLT-4 and HL-60 cells.

Genistein (GEN) is an isoflavone known to inhibit both tyrosine protein kinases and DNA topoisomerase II. The effects of GEN on cell proliferation and cell cycle kinetics of human myelogenous leukemia HL-60 and lymphocytic leukemia MOLT-4 cell cultures were studied, and the data were compared to results obtained with normal human lymphocytes stimulated to proliferate with phytohemagglutinin. GEN concentrations greater than 50 micrograms/ml (185 microM) were cytotoxic to HL-60 and MOLT-4 cells following exposure for 24 h; in HL-60 cell cultures, a population of cells with decreased DNA content and nuclear fragmentation characteristic of apoptosis was observed within 8 h. The 50% inhibition concentration after 24 h of exposure for HL-60 and MOLT-4 cells was 8.5 and 13.0 micrograms/ml, respectively. Normal proliferating lymphocytes survived a 24-h exposure of up to 200 micrograms/ml GEN. Short-term (4-8 h) exposures of MOLT-4 or HL-60 cells to 5-20 micrograms/ml GEN resulted in a suppression of cell progression through S or through both S and G2 phases, respectively, while equivalent treatment had no effect on proliferating lymphocytes. A stathmokinetic experiment using MOLT-4 cells revealed that as little as 5 micrograms/ml GEN suppressed cell exit from S to G2 phase by 40%, with a terminal point of action at or near the S-G2 border. Cell progression through the very early portion of G1 phase (G1A, characterized by postmitotic chromatin decondensation) was also suppressed by approximately 40%, whereas cell advancement through the remainder of the G1 phase was not markedly affected. Longer (24 h) exposure of proliferating lymphocytes to 20 micrograms/ml GEN led to an S-phase arrest, while similar treatment of leukemic cells caused cell arrest in G2 phase and an increase in the number of cells entering the cycle at higher DNA ploidy. The mitogen-induced transition of lymphocytes from G0 to G1 phase was extremely sensitive to inhibition by GEN; the 50% inhibition concentration was 1.6 micrograms/ml. The chemotherapeutic value of GEN may be due to the fact that, in terms of cytotoxicity, this agent is more active against proliferating leukemic cells than against normal proliferating lymphocytes. The sensitivity of the G0 to G1 transition in normal lymphocyte cultures and the suppressive effect of GEN on the G1A exit in MOLT-4 cells both suggest that protein kinases involved in chromatin decondensation may be a target of this drug. In light of the observation that lymphocyte stimulation is sensitive to the presence of GEN, the drug is expected to be a strong immunosuppressant.     

Radiosensitivity of thymidylate synthase-deficient human tumor cells is affected by progression through the G1 restriction point into S-phase: implications for fluoropyrimidine radiosensitization

Recent studies of fluoropyrimidine (FP)-mediated radiosensitization (RS) have focused on the molecular mechanisms underlying regulation of the cell cycle, particularly at the G1-S transition. Although thymidylate synthase (TS) inhibition by FP is necessary, we hypothesize that FP-RS is temporally dependent on progression of cells into S-phase under conditions of altered deoxynucleotide triphosphate pools, particularly an increased dATP:dTTP ratio, which subsequently results in enhanced DNA fragmentation and cell death. To better understand the mechanism of FP-RS, we characterized the cellular and biochemical responses to ionizing radiation (IR) alone, using different synchronization techniques in two isogenic, TS-deficient mutant cell lines, JH-1 (TS-) and JH-2 (Thy4), derived previously from a human colon cancer cell line. After G0 synchronization by leucine deprivation, these clones differ under subsequent growth conditions and dThd withdrawal: JH-2 cells have an intact G1 arrest (>72 h) and delayed cell death (>96 h), whereas JH-1 cells progress rapidly into early S-phase and undergo acute cell death (<24 h). No difference in the late S-phase and G2-M cell populations were noted between these growth-stimulated, G0-synchronized TS-deficient cell lines with dThd withdrawal. Biochemically, the intracellular ratio of dATP:dTTP increased substantially in JH-1 cells as cells progressed into early S-phase compared with JH-2 cells, which remained in G1 phase. Synchronized JH-1 cells showed significantly decreased clonogenic survival and an increase in DNA fragmentation after IR when compared with JH-2 cells. RS was demonstrated by an increase in alpha and decrease in beta, using linear quadratic analyses. An alternative synchronization technique used mimosine to induce a block in late G1, close to G1-S border. Both JH-1 and JH-2 cells, synchronized in late G1 and following growth stimulation, now progressed into S-phase identically (<24 h), with similarly increased dATP:dTTP ratios under dThd withdrawal conditions. These late G1-synchronized JH-1 and JH-2 cells also showed a comparable reduction in clonogenic survival and similar patterns of increased DNA fragmentation following IR. We suggest, based on the cellular and biochemical differences in response to IR between G0- and late G1-synchronized cells, that S-phase progression through the G1 restriction point under an altered (increased) dATP:dTTP ratio is a major determinant of FP-RS.     

Effects of 9-OH-ellipticine on cell survival, macromolecular syntheses, and cell cycle progression in sensitive and resistant Chinese hamster lung cells

In an effort to understand the mechanism of action of the DNA-intercalating antitumor agent 9-hydroxyellipticine (9-OH-E), we have examined the effects of this drug on the cell survival, macromolecular syntheses, and cell cycle progression in sensitive and resistant cells. Our results show that 9-OH-E toxicity on sensitive and resistant cells involves different mechanisms of action: the drug toxicity in the sensitive cells appears to result from lethal lesions mediated through the interaction of the drug with an intracellular protein, independently of any effect of the drug on the macromolecular syntheses; in the resistant cells, the cell death occurs concomitantly with the inhibition of these syntheses. Cell cycle progression analysis after 9-OH-E treatment showed that, in the sensitive cells, the drug is inducing a G1 and a G2 block, which are both released in the presence of 1 mM caffeine, without any effect on the 9-OH-E toxicity. In the resistant cells, a G2 block was also observed but only when the cells were resuming their growth after about a 30- to 40-h growth arrest. Caffeine release of this block, which again had no effect on 9-OH-E toxicity, was only observed when it was added from 40 to 60 h after 9-OH-E treatment, when the cells resumed their growth. Finally in the sensitive cells, cycloheximide exerted an inhibitory effect on 9-OH-E toxicity when it was added before and during the cell exposure to the drug. This effect was interpreted as indicating that 9-OH-E toxicity in the sensitive cells relies on a protein which is not induced by the drug but has to be present in the cells when the drug is added. The possible implication of DNA topoisomerases in 9-OH-E toxicity mechanism is discussed.     

Type I interferon prolongs cell cycle progression via p21WAF1/CIP1 induction in human colon cancer cells

Type I interferon (IFN) was originally identified as an immunomodulatory cytokine because of its antiviral activity. Further characterization of its biological effects revealed a prominent role in the direct control of cell growth and potent immunomodulatory and antiangiogenic actions. IFN-alpha and IFN-beta had both been classified as type I IFN, but differences in their antitumor activities were reported. We confirmed the difference in the antiproliferative activities of IFN-alpha2b and IFN-beta toward HT29 and SW480 cells. IFN treatment was observed to prolong cell cycle progression; in particular, the accumulation of S-phase population was one of the most characteristic changes. The prolongation of S-phase progression and transition into G2/M-phase was suggested to be a crucial action of type I IFN on colon cancer. Additionally, IFN activated the p21 promoter gene and induced p21WAF1/CIP1 expression. Furthermore, the cell cycle prolongation effect of IFN was suppressed when p21 expression was downregulated. Therefore, we confirmed that p21WAF1/CIP1 was a crucial target molecule for the effects of IFN on the cell cycle. Additionally, the ability of p21 induction differed between IFN-alpha2b and IFN-beta and correlated with their inhibitory activities toward cell growth. We conclude that type I IFN prolongs cell cycle progression by p21WAF1/CIP1 induction in human colon cancer cells.     

Effects of chartreusin on cell survival and cell cycle progression

Chartreusin was lethal to both L1210 and P388 cells in culture with 90% of the cells being killed after a 24-hr exposure to 1.1 and 2.6 microgram/ml, respectively. The lethality of the drug increased in direct proportion to dose and exposure time. Both L1210 and Chinese hamster ovary cells in S phase were more sensitive to the lethality of the drug than were their corresponding non-S-phase cells. L1210 cells were partially synchronized by exposing an asynchronous culture to [methyl-3H]thymidine (20 Ci/mmol) and Colcemid for 3 hr. Synchronous culture of Chinese hamster ovary cells was established by planting mitotic cells. The progression of cells through the cell cycle was studied with flow microfluorometry both in the presence of the drug and after the drug had been washed off. In the presence of chartreusin the progression of mitotic cells into G1 was not affected. The movement of G1 cells into S was slower, and the movement of G2 cells into mitosis was blocked. When the drug was removed, the G2 to M block persisted for at least 4 hr but the progression of G1 cells to S was no longer inhibited.     

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein.

We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G1 phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p16(INK4a) was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclin-dependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p16(INK4a) would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of progression through S-phase under such conditions.     

Flow cytometric analysis of adenosine analogue lymphocytotoxicity

The induction of G1-phase arrest in T-lymphoblasts by cytostatic concentrations of 2'-deoxyadenosine (R. M. Fox, R. F. Kefford, E. H. Tripp, and I. W. Taylor, Cancer Res., 41: 5141-5150, 1981) prompted a flow cytometric analysis of the cell cycle effects of three other adenosine analogues with known effects on polyadenylated RNA metabolism in an attempt to further explore the nature of 2'-deoxyadenosine 5'-triphosphate-mediated lymphotoxicity. Cytostatic concentrations of 9-beta-D-arabinofuranosyladenine induced an S-phase block, while 3'-deoxyadenosine (cordycepin) and tubercidin (7-deazaadenosine) induced a cycle-nonspecific block. Furthermore, total cellular RNA content was unaltered by 2'-deoxyadenosine or 9-beta-D-arabinofuranosyladenine, but 3'-deoxyadenosine and tubercidin caused a marked reduction in total cellular RNA at minimally cytostatic concentrations. At concentrations of 0.3 to 20 microM, all of these nucleosides were toxic to nondividing peripheral blood lymphocytes, suggesting that in these cells their mechanism of action does not involve reactions associated with DNA replication. Inhibition of polyadenylated RNA metabolism by triphosphate derivatives of adenosine analogues may account for lymphocytotoxicity in nondividing cells, but the demonstrated diverse effects of these nucleosides on nucleic acid metabolism in dividing cells preclude elucidation of the mechanism of the unique induction of G1-phase arrest by 2'-deoxyadenosine.     

Cytokinetic evaluation of the four-drug combination of bleomycin, vincristine, mitomycin C, and methotrexate (BOMM) in cultured Burkitt's lymphoma cells and human bone marrow

The four-drug combination of bleomycin, vincristine, mitomycin C and methotrexate produces a high response rate in patients with squamous cell carcinoma. In this study we have examined the cytokinetic effects of this drug combination in vitro and in human bone marrow in vivo. The in vitro analysis revealed that mitomycin C produces a concentration dependent slowing of S-phase transit with partial G2/M and G1/S blocks in cell cycle progression. A partially synchronized S-wave occurs 4-8 and 16-20 hours following a two-hour drug exposure. Bleomycin produces a dose dependent G2/M block during a 14-hour drug exposure. Drug removal did not result in appreciable cell cycle synchrony. In vitro exposure to the two drug combination resulted in loss of both the marked G2/M accumulation seen with bleomycin, and the partially synchronized S-phase waves seen following mitomycin C exposure. The sequential changes in bone marrow cytokinetics were determined in eight patients during and following administration of vincristine, mitomycin C and a continuous four-day infusion of bleomycin. The major cytokinetic changes observed were an increase in G2/M phase cells 18 hours following vincristine, and an increase in thymidine incorporation and S-phase cells 24 and 48 hours following the end of the bleomycin infusion. The clinical course of 24 patients was reviewed. Eleven patients who received methotrexate 36 to 42 hours following bleomycin had a subsequent median leukocyte nadir of 1.8 x 10(3)/mm3. Thirteen patients receiving methotrexate 60 to 72 hours after bleomycin had median leukocyte nadir of 3.9 x 10(3)/mm3. The objective response rates in the two groups was 75% and 80%, respectively. This study demonstrates that bone marrow cytokinetic analysis may allow schedule modification to avoid myelosuppression without loss of therapeutic activity.