Genotoxicity of [1H]benz[de]isoquinoline-1,3[2H]dione,5 amino-2-,[2-dimethylamino) ethyl] (BIDA) in human lymphocytes

We have investigated the genotoxicity of BIDA in cultured human lymphocytes. Lymphocytes were cultured and stimulated with phytohemagglutinin (PHA) for 72 h. Doses of 0.1, 0.25, 0.5, 0.75, and 1 microgram/ml of BIDA were added to the culture at 1 h (G2 phase), and 6 h (S/G2 phase) before harvesting. Cells were harvested at the end of the 72-h culture period with 1-h colcemid treatment to accumulate mitosis, and further prepared by standard cytogenetic technique. BIDA induced chromatic type breakages and chromatid exchanges at both 1 h and 6 h. The mean number of breakages per cell was 0, 0.1, 1.0, and 1.7 after treatment with 0.1, 0.25, and 0.75 micrograms/ml, respectively. Ai 1 microgram/ml, BIDA severely inhibited cell progression and very few mitoses were observed. At 6 h the mean number of breakages per cell was 0.3 at 0.25 microgram/ml and 1.2 at 0.5 microgram/ml. Very few cells entered mitosis at 0.75 and 1 microgram/ml. To study the effect of BIDA on cells in G0 and G1, BIDA (0.75 microgram/ml) was added for 1 h to the cultures at the beginning of culture (G0), or 24 h after (G1) culture initiation. Afterward, cells were washed and reincubated in the conditioned medium for 71 or 47 h. No chromosomal aberrations were seen in these experiments. The number of chromatid breaks was minimal (0.1 to 0.4/cell). Our study suggests that BIDA induces chromatid type aberrations during G2 and S phases. The absence of chromosome type aberrations in cells treated during G0 and G1 suggests that either BIDA has no effect on these cells or that damaged cells fail to progress through S and G2 to reach mitosis.     

Dose- and schedule-dependent activation and drug synergism between thymidine and 5-aza-2'-deoxycytidine in a human promyelocytic leukemia cell line

The ability of thymidine (dThd) to enhance the metabolism and cytotoxicity of subsequent administered 5-aza-2'-deoxycytidine (5-aza-dCyd) was studied in L1210 cells and in the human promyelocytic leukemic cell line, HL-60. Exposure of L1210 cells to 0.1 mM dThd for 5 h resulted in an increase in the total intracellular and acid-precipitable accumulation of 5-aza-dCyd. Higher dThd concentrations and longer exposure intervals resulted in smaller increments in 5-aza-dCyd accumulation. In contrast, in HL-60 cells, a 24-hr exposure in 1 mM dThd resulted in the greatest intracellular accumulation of 5-aza-dCyd, 3.3 times more accumulation than in control cells. There was also a 4-fold increase in the acid-precipitable accumulation and nearly a 3-fold increase in DNA incorporation of 5-aza-dCyd in HL-60 cells exposed to the same dThd schedule. High-pressure liquid chromatographic analysis demonstrated a greater than 3-fold increase in the intracellular amounts of 5-aza-dCyd metabolites eluting in the triphosphate region in these human cells under identical conditions. Shorter dThd incubation exposure intervals (6 hr) and lower dThd concentration (0.1 mM) produced smaller increments in these studies. Both growth and clonogenic assays of HL-60 cells demonstrated a dose- and schedule sequence-dependent synergism between dThd and 5-aza-dCyd.     

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.     

Cell cycle synchronization of human lymphoid cells in vitro by 2,3-dihydro-1h-imidazo[1,2-b]pyrazole.

2,3-Dihydro-1H-imidazo[1,2-b]pyrazole (IMPY), a DNA synthesis-inhibitory drug, reversibly arrests growth of human lymphoblasts in vitro. DNA distribution histograms of cultures exposed to 0.5 to 2.0 mM IMPY show accumulation of cells with G1-early S DNA content. On reincubation in fresh medium, cell cycle traverse is resumed by the blocked cells in a synchronized manner. Maximum incorporation of [H3]thymidine into DNA (174 to 220% of control) and labeling indexes (72 to 86%) are seen after 4 hr of incubation, and a major increase in cell number is seen between the 9th and 13th hr. DNA distribution histograms of cells reincubated in fresh medium (after double block), show an initial increase in the number od cells with S-G2-M DNA content and a corresponding decrease in the number of G1-early S cells. After 4 hr of reincubation, a gradual increase in the number of G1-early S cells was seen as the earlier blocked cells completed cell cycle traverse and mitosis. Cells exposed to 2.0 mM IMPY took approximately 2 hr longer to traverse than did cells exposed to 0.5 mM IMPY.     

Synergistic action of 5-aza-2'-deoxycytidine and 3-deazauridine on L1210 leukemic cells and EMT6 tumor cells.

The biochemical and biological effects of the combination of 5-aza-2'-deoxycytidine (5-aza-dCyd) and 3-deazauridine (3-DU) on L1210 leukemic cells and EMT6 tumor cells were investigated. The cytotoxic action of 5-aza-dCyd and 3-DU on both L1210 and EMT6 cells in vitro was synergistic when these agents were used in combination. The combination of 5-aza-dCyd and 3-DU produced a greater inhibition of in vitro growth of L1210 and EMT6 cells than did either agent alone. The in vivo antineoplastic activity of this combination was synergistic with respect to the increased survival time of BALB/c x DBA/2 F1 mice with L1210 leukemia. 3-DU, an agent that reduces the intracellular pool size of cytosine nucleotides, stimulated the incorporation of [3H]-5-aza-dCyd into DNA of both L1210 and EMT6 cells, suggesting that the synergistic action of this combination is related to the increased incorporation of 5-aza-dCyd in the presence of 3-DU.     

介导肿瘤细胞G2期周期阻滞的分子机制

肿瘤的形成与进展离不开肿瘤细胞中细胞周期调控蛋白的改变,利用肿瘤细胞的G2-M期周期阻滞效应在减缓肿瘤的生长、提高治疗疗效方面尤为重要.DNA损伤的感受器、信号传导因子和效应器是介导肿瘤细胞G2期周期阻滞的经典途径,且有作为新型肿瘤治疗靶点的可能.     

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.     

Combination chemotherapy with aclarubicin and cisplatinum against malignant intracranial tumor--an in vitro study

Augmentation of cytotoxicity against cultured human tumor cell lines (5 gliomas, 2 neuroblastomas, 2 sarcomas) using a combination of Aclarubicin (ACR) and Cisplatinum (CDDP) was analysed in vitro from the viewpoints of cell growth inhibition and alteration of the DNA histogram. Synergistic effects of the combination of the two agents were observed in 7 of the 9 cell lines. In sarcoma cell lines, effects were demonstrated even by a low dose of 0.1 microgram/ml CDDP and 0.01 microgram/ml ACR. Flow cytometry studies of the DNA histogram showed increase of accumulation in the S phase following the G2-M phase and reduction of G1 phase cells in response to the combination. From the present experimental studies in vitro, it is concluded that combination chemotherapy with ACR and CDDP may be effective for sarcoma and ACNU-resistant glioma. The mechanism of the synergistic effect produced by the combination is suggested to be impairment of RNA synthesis by ACR which prevents the repair of DNA damage induced by CDDP.     

Effects of dibutyryl cyclic adenosine 3':5'-monophosphate on the growth of cultured human small-cell lung carcinoma and the specific cellular activity of L-dopa decarboxylase

High activity of L-dopa decarboxylase separates small (oat)-cell from non-small-cell lung cancer in cell culture. The present study investigates relationships between the specific cellular activity of this enzyme and: (a) cell growth kinetics of an established line (O-H-1) of human small cell lung carcinoma, and (b) responses of these cells to treatment with cyclic adenosine 3':5'-monophosphate and sodium butyrate. The O-H-1 cells, as for most other established small-cell lines, grow as suspended cell aggregates. During growth, the specific cellular activity of L-dopa decarboxylase parallels levels for [3H]thymidine labeling index and the ratio of cells in G2-M to those in G1-G0 phases of the cell cycle. Each of these parameters is 2- to 3-fold higher during exponential versus stationary growth. Continuous treatment with dibutyryl cyclic adenosine 3':5'-monophosphate (dcAMP; 0.1 or 1 mM) and 1 mM theophylline produces simultaneous cessation of cell growth and an increase in cellular L-dopa decarboxylase activity. During this period, analyses of DNA histograms reveal an increase in the number of cells in the G2-M phase; the rate of increase in the ratio of G2-M to G1-Go cells paralleled the rate of increase in specific activity of the enzyme. The effects of the dcAMP were promptly reversible; release of the apparent G2-M block preceded regrowth of the cells and was accompanied by a return of L-dopa decarboxylase activity to base-line levels. The changes in enzyme activity were specific for cyclic adenosine 3':5'-monophosphate; another cyclic adenosine 3':5'-monophosphate analogue, 8-bromo adenosine cyclic 3':5'-monophosphate yielded similar increases in L-dopa decarboxylase to those seen with dcAMP, while 0.01 to 1 mM butyrate alone produced the inhibition of cell growth but no changes in specific activity of L-dopa decarboxylase or percentage of cells in the different phases of the cell cycle. We conclude that the specific activity of L-dopa decarboxylase, a key neuroendocrine marker for cultured small-cell lung carcinoma, is highest during proliferative growth and/or when these cells are in the G2M phase of the cell cycle. The differential effects of dcAMP and sodium butyrate offer potential for exploring neuroendocrine differentiation in this important lung cancer and related endocrine neoplasms.     

Effect of derivatives of chrysophanol, a new type of potential antitumor agents of anthraquinone family, on growth and cell cycle of L1210 leukemic cells

The new C-methyl modified derivatives of the anthraquinones chrysophanol and emodin, recently synthesized by us, are potentially bifunctional agents having the ability to intercalate to nucleic acids and also having alkylating properties. Two of these compounds, namely 3-(N,N-bis(2-chloroethyl)-amino)methyl-1,8-dihydroxy-9,10-anthraquinone (Compound 31.662) and its 1,8-di-O-methylated analog (Compound 31.655) have been presently tested on murine leukemic L1210 cells in vitro with respect to their cell cycle specificity. During the initial 24 h of treatment the cytostatic effects of the drugs predominated, manifesting as suppression of cell progression through S (especially through the early portion of S phase) and G2. After 24 h, the cytotoxic effects became apparent, and there was also the appearance of cells with doubled DNA content suggestive of either endoreduplication or impairment of cytokinesis; these cells at higher ploidy level were progressing through S and G2. The observed effects were time- and dose-dependent, occurring at 0.1-0.4 micrograms/ml concentration of 31.662 and 2.0-10.0 micrograms/ml of its methylated analog, either during continuous- or after a 4-h pulse-treatment. Modulation of the cell cycle by the studied drugs is similar to that generally caused by intercalators as well as alkylating agents. However, because no positive evidence of intercalation of the studied drugs to nucleic acids was found, it is possible that alkylation of DNA or other cell constituents may be the primary lesion(s) leading to perturbation of the cell cycle.     

Low concentrations of misonidazole counteract effects of extreme hypoxia on cells in S.

Populations of NHIK 3025 cells synchronized by mitotic selection were exposed at 37 degrees C to extreme hypoxia in absence and presence of misonidazole (MISO). Cells in G1, S or G2 and mitosis were treated for 3 h. Inhibition of cell-cycle progression by this treatment was measured by flow cytometry of DNA histograms and cell inactivation was measured by colony formation. The exposure to hypoxia alone of cells in G1 or in G2 and mitosis led to only minor cell-cycle inhibition, and hardly reduced cell survival. However, the exposure of cells in S to hypoxia alone had a strong inhibitory effect on cell-cycle progression, and cell survival was only 40% of untreated cells. Low concentrations of MISO (0.05-0.4 mM) during exposure of cells in S to hypoxia, produced less cell-cycle inhibition than after hypoxia alone, and cell survival was restored to 100%. The presence of MISO during the 3h exposure to hypoxia of cells in G1 or in G2 and mitosis only increased the effects of hypoxia alone. MISO at concentrations greater than 0.8 mM during hypoxia produced cell inactivation, for all phases of the cell cycle, comparable to that already known from the literature.     

Diverse effects of camptothecin, an inhibitor of topoisomerase I, on the cell cycle of lymphocytic (L1210, MOLT-4) and myelogenous (HL-60, KG1) leukemic cells

Exposure of mouse lymphocytic L1210 cells to 0.02-0.5 micrograms/ml of camptothecin (CAM) causes a slowdown in the rate of cell progression through S and G2 phases of the cell cycle; the "terminal" point of CAM action is about 1 h prior to mitosis. Some cells also enter higher DNA ploidy and progress through the cycle at that ploidy. CAM exerts similar effects (S- and G2-phase arrest, entrance to higher DNA ploidy, low initial cytotoxicity) on human lymphocytic MOLT-4 leukemia cells. In contrast, treatment of human promyelocytic HL-60 cells with CAM results in the immediate (occurring as early as 2 h after treatment) death of S- and G2+M-phase cells; the dead cells exhibit decreased DNA stainability with intercalating dyes, suggestive of DNA degradation. Although CAM is less cytotoxic to another human myelogenous leukemic cell line, KG1, the latter cells also respond like HL-60, namely by selective death in S and G2. The data indicate that there may be a tissue (leukemia type) specificity in the response of cells to camptothecin and suggest that myelogenous leukemias, especially those characterized by high proliferation rates, may be especially sensitive to the cytotoxic action of this and perhaps other topoisomerase I inhibitors.     

Cytostatic effect of deoxyspergualin on a murine leukemia cell line L1210

The mode of antiproliferative action of deoxyspergualin (NKT-01) was examined. The growth-inhibitory effect on a murine leukemia cell line L1210 following treatment with NKT-01 was time-dependent, and there was little or no effect on the syntheses of DNA and RNA. Thus, the inhibitory activity of NKT-01 was not attributable to the inhibition of DNA and RNA syntheses. The influence of NKT-01 on cell cycle progression was studied by flow cytometric analysis. Bromodeoxyuridine/DNA distribution patterns in cells that were treated for 72 h, showed that the growth inhibition is due to the delay of cell cycle progression but not to cytotoxicity. This finding was also supported by evidence that the treated cells were re-proliferative in fresh medium. In addition, a majority of drug-treated cells was prevented from traversing from the G0/G1 phase to the S phase by 144 h or longer exposure to NKT-01. The results suggest that NKT-01 is cytostatic, preventing G0/G1-S progression.     

Response of 9L tumor cells in vitro to spirohydantoin mustard.

The effects of spirohydantoin mustard (SHM), a potential antitumor agent for central nervous system tumors, in the in vitro 9L rat brain tumor model were studied. In cell culture medium at 37 degrees, the drug was totally detoxified within 30 min. Dose-response curves for exponentially growing and plateau-phase cells were similar and indicated that a small fraction of cells were resistant to SHM. When exponentially growing cells were treated with SHM (5 microgram/ml for 1 hr), recovery from potentially lethal damage occurred within 28 hr. When cells were perturbed by SHM, the S, G2, and M phases were prolonged, there was a G2 block, some cells entered mitosis, but few divided, and cells tended to accumulate in mid-S, then moved synchronously to G2-M. The rate at which cells moved was concentration dependent and was much slower at high concentrations. The ability of SHM to both synchronize cells and block DNA synthesis may be useful in multiagent therapy regimens.     

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.     

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.     

Caffeine modulates the effects of DNA-intercalating drugs in vitro: a flow cytometric and spectrophotometric analysis of caffeine interaction with novantrone, doxorubicin, ellipticine, and the doxorubicin analogue AD198

Exposure of L1210 cells to DNA-intercalating antitumor drugs Novantrone (mitoxantrone; 20 ng/ml), doxorubicin (0.5 micrograms/ml), ellipticine (5 micrograms/ml), or the doxorubicin analogue AD198 (0.4 micrograms/ml), for 1 h, results in inhibition of cell proliferation, arrest of cells in the G2 phase of the cell cycle, and an increase in the number of cells entering higher DNA ploidy. These effects are significantly reduced when 5 mM concentrations of the methylxanthines caffeine or pentoxifylline are present either simultaneous with, or, in some cases, when added for 1 h immediately following pulse exposure to the drug. Both caffeine and pentoxifylline alone (5 mM) have little effect on cell growth or cell cycle progression. The possible mechanism of cell protection against intercalating drugs provided by caffeine was studied spectrophotometrically by measuring the interaction between Novantrone and the caffeine chromophore and in a model system using permeabilized L1210 cells and measuring the effect of caffeine in reducing binding of the intercalating dye acridine orange to cellular DNA and RNA. The data indicate that the observed protection of cells against intercalating drugs by caffeine or pentoxifylline is most likely a consequence of the direct interaction between the methylxanthines and the planar aromatic molecules of the intercalating drugs: formation of caffeine-drug complexes in solution effectively lowers the concentration of the free drug and thereby reduces its pharmacological activity. The principle of selective entrapment of the intercalator by compounds like caffeine may be considered in designing strategies to modulate the activity of intercalating drugs in vivo, e.g., in lowering drug toxicity when inadvertently applied at too high doses.     

Effects of N-hydroxy-N'-aminoguanidine derivatives on ribonucleotide reductase activity, nucleic acid synthesis, clonogenicity, and cell cycle of L1210 cells

Derivatives of N-hydroxy-N'-aminoguanidine were recently shown to be efficient inhibitors of mammalian ribonucleotide reductase and cancer cell growth. We investigated the effects of the 1-isoquinolylmethylene and the 2-quinolylmethylene derivatives of N-hydroxy-N'-aminoguanidine on intracellular targets, cell viability, and cell cycle of L1210 mouse leukemia cells. A 2-h exposure of L1210 cells to either drug in the low micromolar concentration range led to inhibition of intracellular ribonucleotide reductase activity and DNA synthesis. After a 24-h incubation in the presence of these drugs, RNA synthesis was also markedly diminished. The clonogenicity of L1210 cells was inhibited after treatment with the drugs for 24 and 48 h, the I50 values being comparable to the drug concentrations required for 50% inhibition of DNA synthesis and cell proliferation. The isoquinoline compound was always more inhibitory to reductase activity, nucleic acid synthesis, and clonogenicity than the quinoline compound. As shown by flow cytometry, the N-hydroxy-N'-aminoguanidine isoquinoline derivative at 0.5-10 microM led to an elevation of G0/G1 cells and a decrease of G2/M and S cells. At 10 microM of the drug this shift remained unchanged over 48 h. L1210 cells treated with 0.5, 1, and 2 microM of the drug overcame the block after 4 to 12 h of exposure and progressed through S- and G2/M-phase in a synchronized manner.     

Levels of thymidylate synthetase during normal culture growth of L1210 cells

The binding of 5-fluoro-2'-deoxyuridylate generated from 5-fluoro-2'-deoxyuridine in intact cells was used to measure changes in the level of thymidylate synthetase during the course of population growth of murine leukemia L 1210 cells. By the use of elutriation techniques and flow cytometric analysis, the amount and activity of thymidylate synthetase associated with the various phases of the cell cycle were determined for the L 1210 cells during unperturbed in vitro culture growth. Fluctuations of thymidylate synthetase levels were associated with the cell cycle; there was a positive correlation (P less than 0.001) between the percentage of the total cell population in S phase and the concentration of thymidylate synthetase, although there was an increase in the level of this enzyme in association with an increase in G2-M cells, this did not achieve statistical significance. A negative correlation between G1 cells and the concentration of thymidylate synthetase was also observed. The maximum amount of thymidylate synthetase was nearly 900 fmol/10(6) cells and occurred in cell populations during logarithmic growth when the percentage of the population in S phase and G2-M phase was greater than 50 and 20%, respectively. In late culture growth (plateau) when only 25% of the cell population was in S phase and nearly 75% of the population was in G1 phase, the level of enzyme was reduced to 200 fmol/10(6) cells.     

Cell cycle specific toxicity of the Hoechst 33342 stain in untreated or irradiated murine tumor cells

Hoechst 33342 stain has been used previously in conjunction with fluorescence activated cell sorting to separate cell populations into the various phases of the cell cycle. The present experiments were designed to evaluate the toxicity of this stain in cells derived from solid KHT sarcomas which were untreated or irradiated prior to dissociation. In both untreated and irradiated cells, stain toxicity increased with increasing exposure times but was always greater in treated cells; for example, after stain exposures of 1 h at 10 microM or 2 h at 5 microM, survival was reduced 25- to 45-fold in irradiated cells and 4- to 5-fold in untreated cells. To determine whether this toxicity occurred equally in all phases of the cell cycle or preferentially in a particular phase, cells derived from untreated or irradiated KHT tumors were separated into the various phases of the cell cycle using centrifugal elutriation. Cells in each phase then were divided in two, one-half being stained with 5 microM Hoechst 33342 for 2 h, the other half being handled in an identical manner but not stained. These particular stain exposure conditions were chosen for detailed evaluation because they provided the best DNA histograms. In both untreated and irradiated cells the cytotoxic effects of Hoechst 33342 were found to be significantly greater in cells in the S phase than in cells in G1 and G2-M phases of the cell cycle. This toxicity, particularly its cell cycle specificity, suggests a potentially severe limitation for the use of Hoechst 33342 in combination with fluorescence activated cell sorting in studies aimed at determining treatment efficacies in various phases of the cell cycle.