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.     

Effects of a prospective antitumor agent, 1,4-bis(2''-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate, on cultured mammalian cells

The effects of 1,4-bis(2'-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate (CBH; NSC 57198) on cell viability, growth, progression through the cell cycle, survival, and differentiation were investigated in suspension cultures of murine lymphocytic leukemia (L1210) and erythroleukemic (FL) cells and normal human lymphocytes stimulated with phytohemagglutinin (PHA) and in adherent cultures of Chinese hamster ovary (CHO) cells. CBH was equally cytotoxic toward stationary and exponentially growing CHO cells. Cell viability was diminished by 50% following 24 hr exposure to approximately 50 μg CBH per ml. Treatment of quiescent human lymphocytes for 24 hr with up to 100 μg CBH per ml did not appreciably diminish cell viability though the subsequent stimulation of such lymphocytes with PHA was inhibited in a dose dependent fashion. L1210, FL cells, and PHA stimulated human lymphocytes were equally sensitive to CBH, 50% inhibition of growth was obtained following 24 hr treatment with 25 μg CBH per ml. Incubation for up to 48 hr with CBH did not result in differentiation of FL cells to mature hemoglobin containing cells. Constant exposure of L1210 cells and PHA-stimulated human lymphocytes to 10-50 μg CBH per ml resulted in accumulation of cells in G₂ + M phase; higher drug concentrations resulted in cell arrest in mid to late S phase and G₂ phase. A short 1-hr pulse of the drug resulted in a transient accumulation of L1210 cells in S and G₂ phases. However, cells recovered from a short pulse of drug and by 48 hr, both cell proliferation and the cell cycle distribution appeared normal. A detailed analysis of cell cycle progression of L1210 cells in the presence of the drug indicated that the duration of G₂ phase was extended at low concentrations (10 μg/ml) while the transit of cells through S was retarded with subsequent accumulation in late S and G₂ phase at higher (50 μg/ml) concentrations. Concomitant with cell arrest in S and G₂ phase an increase in cellular RNA content indicating unbalanced growth was observed. This state of unbalanced growth was reversible in cultures exposed to a 1-hr pulse of up to 100 μg CBH per ml; cellular RNA content returned to control values by 48 hr. No effect on nuclear chromatin as assayed by acid denaturation was observed. Though the exact mechanism of drug action is not known, the data are not incompatible with the drug acting as an alkylating agent.     

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.     

Effects of 5-aza-2'-deoxycytidine on survival and cell cycle progression of L1210 leukemia cells

The in-vitro effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-dCyd), on DNA synthesis, growth, cloning in agar, and cell cycle traverse of L1210 leukemia cells were studied. 5-Aza-dCyd at 0.1 microgram/ml for 10 hr (cytotoxic concentration) did not inhibit DNA synthesis but produced a very potent growth inhibition, and changed markedly the DNA flow cytometric histograms. A 5-h continuous exposure to the drug at concentrations ranging from 0.1 to 10 micrograms/ml caused an accumulation of cells in the S portion of the DNA histograms indicating a slowing of the progression of cells in the S phase. A longer exposure time (10 h) at the same concentrations led to a bimodal DNA distribution (peaks at G1 and G2-M) and a depletion of the S phase. When the exposure time to 5-aza-dCyd (0.1 microgram/ml) was extended to 15 and 20 h, there was a decrease in the G2-M peak and an augmentation of the G1 peak. To determine if 5-aza-dCyd produced a block in cell cycle progression, L1210 cells were treated for 10 h with colcemid and 5-aza-dCyd simultaneously for 10 h. Colcemid alone, or colcemid in combination with 5-aza-dCyd produced an accumulation of cells under a single G2-M peak. This indicates that 5-aza-dCyd did not block the progression of L1210 cells through S phase, but only produced a slowing down of this event. These results, indicating that 5-aza-dCyd does not block cell cycle progression and that its cytotoxic action is not self-limiting, are of importance for designing future clinical trials.     

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.     

Cytotoxic thresholds of vincristine in a murine and a human leukemia cell line in vitro.

L1210 murine leukemia and CEM human lymphoblastoid leukemia cells were exposed to vincristine sulfate in vitro. The response of these cell lines to this agent was measured by the colony-forming ability of L1210 cells in soft agar and inhibition of growth of CEM in suspension culture. Incremental increases of vincristine concentrations in excess of 2 x 10(-9) M produced a progressive reduction of survival of L1210 cells and suppression of CEM growth under the condition of constant drug exposure. A maximum cytotoxic effect was reached with drug concentrations between 10(-8) and 10(-7) M. When L1210 cells were exposed to vincristine for a variable length of time ranging from 0.5 to 24 hr, 10(-7) M produced a noticeable cytotoxic effect following an incubation of only 30 min. A 50% cell kill of L1210 cells and a 50% reduction of CEM cell growth were produced by 10(-7) M following a 1- to 3-hr period of exposure; 6 to 12 hr were required to produce a similar effect at a vincristine concentration of 10(-8) M. Therefore, the antitumor effect of vincristine is critically dependent on both concentration and duration of exposure. These data suggest the possibility that the effectiveness of vincristine as an antitumor agent could be enhanced if methods are developed to prolong exposure of neoplastic tissues for longer periods of time than currently produced by conventional methods of administration.     

Mechanisms of action of a novel macromolecular antitumor antibiotic, SN-07, on mouse leukemia L1210 cells in culture

The mechanisms of action of a novel macromolecular antitumor antibiotic (SN-07) were examined using cultured mouse lymphoid leukemia L1210 cells. A shoulder exponential-type cytotoxicity was observed when the cells were treated with 3.13 to 100 ng/ml SN-07 for 1 hr and surviving colonies were counted after a 14-day incubation. It was found that 500 ng/ml SN-07 inhibited both RNA and DNA syntheses significantly at 40 and 80 min, respectively, while 8,000 ng/ml did not affect protein synthesis at 80 min. Treatment with a low concentration (80 ng/ml) of SN-07 for 1 hr inhibited both RNA and DNA syntheses after a 24-hr post-incubation. The alkaline elution technique revealed that 8,000 ng/ml SN-07 induced DNA interstrand cross-links time-dependently for 1 to 4 hr, and a 1-hr treatment with 80 to 8,000 ng/ml SN-07 induced DNA breaks after a 24-hr post-incubation. According to flow cytometric analysis, most L1210 cells progressed to the G2 phase in the cell cycle at a cytostatic concentration (25 ng/ml) of SN-07, and typical inhibition of the cell cycle progression was observed at a cytocidal concentration (200 ng/ml).     

Effect of dimethyldioctadecylammonium bromide induced macrophages on malignant cell proliferation

Murine peritoneal macrophages elicited by dimethyldioctadecylammonium bromide (DDA), which is a potent immunologic adjuvant, were examined for cytotoxic and growth inhibiting activity for malignant cells. DDA macrophages had no cytolytic activity for murine B16BL-6 melanoma or human SMS-SB pre-B leukemia cells even in the presence of up to 1 microgram bacterial endotoxin (lipopolysaccharide, LPS)/ml. However, they exhibited a variable inhibitory effect on the growth of several lines of leukemia cells. The number of SMS-SB and human NALL cells remained essentially static in the presence of DDA macrophages while they increased significantly when cultured with resident macrophages. In contrast, L1210 cells increased 5-8-fold in the presence of macrophages elicited either by DDA or the inflammatory agent proteose peptone (PP). Although DDA macrophages retarded L1210 growth relative to PP macrophages, both populations responded to LPS in a comparable dose dependent manner to become essentially cytostatic at 1 microgram LPS/ml.     

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.     

Effect of adriamycin on the reproductive integrity of cultured leukemia L1210 and P388 cells

Proliferating cultured P388 cells exhibited a greater degree of sensitivity to adriamycin than did proliferating cultured L1210 cells, although both leukemia cell populations had approximately the same doubling time. The rate of reduction in viability when cultured L1210 cell populations were exposed to adriamycin (0.0625-2.0 microgram/ml, concentrations that are comparable to tissue drug levels during therapy) was concentration-dependent. Therefore, the results indicated a possible therapeutic advantage to be gained by an increase in drug concentrations (within the limits of acceptable host toxicity) at the target cell site.     

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.     

Inhibitory effects of phytohemagglutinin isolectins L4 and E4 on L1210 cells

Phytohemagglutinin isolectins L4 and E4 inhibit the growth and proliferation of cultured L1210 murine leukemia cells. L1210 cells were incubated with L4 or E4, and the metabolic and morphological characteristics of the cells were assessed. Dose-dependent inhibition of up to 90% occurs for [3H]thymidine and [14C]uridine incorporation. L4 is 30 to 50 times more potent an inhibitor than is E4. Inhibition begins 2 to 3 hr after exposure of L1210 cells to L4 and persists for as long as the cells are exposed to this isoleuctin. Total DNA and oxygen consumption in L4-treated cultures is also decreased. Whereas protein synthesis assessed by [14C]valine incorporation is less affected, glucose utilization remains unchanged. The binding of L4 and E4 to L1210 cells and human lymphocytes is similar and is reversible by porcine thyroglobulin. Porcine thyroglobulin also reverses L4-induced inhibition of nucleotide incorporation. Cell aggregation is the major morphological consequence of isoleuctin treatment observed by light or electron microscopy. L1210 cells are agglutinated at lower doses of isoleuctins than are normal murine lymphocytes. No evidence of cell death as estimated by 51Cr release or trypan blue uptake has been noted. Our data indicate that L4 and E4 have cytostatic properties and demonstrate that the reversible binding of a macromolecule to the surface of a malignant cell can modulate synthetic pathways and the rate of proliferation.     

Sensitivity of leukemia cell lines to cytotoxic alkyl-lysophospholipids in relation to O-alkyl cleavage enzyme activities

The human leukemia cell lines K562, HL60, and Raji and the mouse leukemia cell line L1210 showed a differential susceptibility to the action of the alkyl-lysophospholipid (ALP) 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). After 48 hours, the 50% growth-inhibition doses (ID50) of ET-18-OCH3 were found to be 0.78 microgram/ml (HL60), 1.53 microgram/ml (Raji), 4.41 micrograms/ml (K562), and 5.05 micrograms/ml (L1210), as determined by [3H]thymidine incorporation. At the same time, cell viability was determined by trypan blue exclusion and revealed median lethal doses (LD50) of 3.5 micrograms/ml (HL60), 15 micrograms/ml (Raji), 24 micrograms/ml (L1210), and 38 micrograms/ml (K562). Since O-alkyl cleavage enzyme previously was suggested as being important in the detoxification of cytotoxic ALPs, the enzyme activity was compared with the susceptibility to ET-18-OCH3 in the distinct cell lines. In comparison to an approximate sevenfold to elevenfold (ID50 and LD50, respectively) difference in the susceptibility of the above leukemia cell lines to ET-18-OCH3, no significant difference in the specific activities (0.13-0.21 nmol/min/mg) of the O-alkyl cleavage enzyme was found in the above leukemia cell lines. Therefore, the differential sensitivity of the above lines investigated cannot be explained by differences in O-alkyl cleavage enzyme activity. Experiments with radiolabeled ET-18-OCH3 in Raji cells suggest, rather, a critical role for phospholipases C and/or D in ALP metabolism.     

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.     

Response of transplantable tumors in mice and of macromolecular synthesis to 17 beta-acetamido-3-aza-A-homo-4 alpha-androsten-4-one

17 beta-acetamido-3-aza-homo-4 alpha-androsten-4-one has cytostatic activity against Ehrlich ascites tumor, L1210, and P388 leukemias in mice when administered intraperitoneally. The effect of the homo-aza-steroid on the incorporation of radioactive precursors to DNA and RNA of L1210 leukemia cells was investigated. It was found that treatment of cells with 12.5 micrograms/ml of the drug for 1 hour inhibited DNA synthesis by 81%. This was partly because the drug affected the radioactive thymidine pool in the cell. The inhibitory effect was found to be reversable. The incorporation of [3H]thymidine into DNA was lower when cells were incubated in the presence of S9 mix. It was also found that the same compound inhibited RNA synthesis by 67%.     

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.     

In vitro antitumor mechanism of a new platinum complex, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato+ ++) platinum(II)

The antitumor mechanism of (-)-(R)-2- aminomethylpyrrolidine (1.1-cyclobutanedicarboxylato)platinum(II) (DWA2114R) was examined using cultured murine L1210 leukemia cells by estimating its effects on parameters such as proliferation, macromolecular synthesis, morphology and cell cycle progression. Each parameter was estimated in cells concomitantly exposed to the drug for 24-48 hr. More than 0.1 microM of DWA2114R markedly inhibited cell proliferation as well as DNA synthesis, and it decreased in mitotic index in a concentration-dependent manner. One microM of DWA2114R decreased DNA synthesis by 80% in the cells treated for 24 hr, while the inhibition of RNA synthesis was less than 40%. A significant inhibition of protein synthesis was caused only by treatment with a high concentration (100 microM) of the drug. Under complete cytostatic conditions (10 microM of DWA2114R), cell volume markedly increased and about 40% of the total cells were polynucleate. In addition, flow cytometrical analysis revealed that most of these cells were accumulated in the G2/M phase of the cell cycle, and a new peak located in the G2/M phase of tetraploid cells emerged. On the other hand, the cells treated with 100 microM of the drug did not increase in volume and their progress in the cell cycle was almost completely blocked.     

Cellular pharmacology of DUP-785, a new anticancer agent

DUP-785, a new inhibitor of dihydroorotate dehydrogenase, is currently undergoing clinical evaluation for anticancer activity. We developed a GC/MS method to quantitate dihydroorotate that accumulates in cultures of L1210 cells exposed to growth inhibitory concentrations of DUP-785. This method was used to follow the onset, extent, and duration of inhibition of de novo pyrimidine synthesis in intact L1210 cells and to compare this inhibition with cell proliferation and cellular concentrations of pyrimidine nucleotides. There were direct relations between inhibition of de novo pyrimidine synthesis, changes in pyrimidine nucleotide concentrations, and cell proliferation following short (less than 24 hr) drug exposures; with prolonged exposures (greater than 24 hr), however, there was a departure from these relationships in that restoration of pyrimidine nucleotide pools and de novo pyrimidine pathway activity did not restore cell proliferation. Exposure of L1210 cells to 15 microM DUP-785 produced a maximum cell kill (99.9% as determined by cloning efficiency) at 24 hr, and no increase in cell kill was observed with drug exposure up to 96 hr.     

Potentiation of the anti-tumor effect of actinomycin D by tumor necrosis factor α in mice: Correlation between in vitro and in vivo results

The anti-tumor effects of actinomycin D (Act D) and recombinant human tumor necrosis factor (TNF)-α have been studied on 4 established murine tumor cell lines: MmB16 melanoma, Lewis lung (LL/2) carcinoma, L1 sarcoma and L1210 leukemia. During short-term incubation (24 hr) Act D produced dose-dependent cytostatic/cytotoxic effects against MmB16, LL/2 and L1 tumor cells but did not reduce the viability of these cells even at high concentration (10 μg/ml), below a threshold of 30–60%. However, L1210 leukemic cells were highly susceptible to Act D, and no viable cells were detected in cultures incubated with 1 μg/ml of Act D. TNF-α alone, when used under the same culture conditions, had only a negligible effect on all cell lines tested. However, the combination of this cytokine with Act D produced synergistic cytotoxic effects against MmB16, LL/2 and L1 cells but not against L1210 leukemia cells. In an in vivo model of regional therapy in which tumor-bearing mice were treated with Act D and TNF-α, a correlation with in vitro results was observed. In mice bearing MmB16 melanoma, LL/2 carcinoma and L1 sarcoma, the most potent anti-tumor effects were observed in mice treated with Act D and TNF-α together. This treatment led to a delay of tumor growth and induced complete tumor regression in some cases. On the contrary, TNF-α did not enhance the effect of Act D in mice injected with L1210 leukemia cells. Our results show that TNF-α can potentiate the anti-tumor effects of Act D against tumors weakly susceptible to Act D and may be a useful adjuvant to chemotherapy in the local treatment of neoplasia. © 1996 Wiley-Liss, Inc.     

Camptothecin, teniposide, or 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide, but not mitoxantrone or doxorubicin, induces degradation of nuclear DNA in the S phase of HL-60 cells

Short-term (2-6 h) exposure of human promyelocytic HL-60 cell cultures to the DNA topoisomerase I inhibitor camptothecin (0.05-0.5 microgram/ml) or to the topoisomerase II inhibitor, teniposide (VM-26; 0.3-3.0 micrograms/ml) or 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine; 0.8 microgram/ml) triggered rapid degradation of DNA specifically in S-phase cells. As a result of the selective death of S-phase cells, only G1 cells remained in these cultures. On the other hand, mitoxantrone (0.02-0.4 microgram/ml) or doxorubicin (adriamycin; 0.4-10.0 micrograms/ml) did not induce DNA degradation in S phase but arrested HL-60 cells in S and G2 phases. In contrast to HL-60 cells, human lymphocytic leukemic MOLT-4 cells responded to all of these drugs (camptothecin, teniposide, amsacrine, mitoxantrone, and adriamycin) at all concentrations tested, invariably by being arrested in G2 and S phases and also by entering a higher DNA ploidy cycle. The data illustrate the differences in the sensitivity of S-phase cells in myelogenous versus lymphocytic leukemic lines to both DNA topoisomerase I and II inhibitors and emphasize the tissue (leukemia type)-specific factors that modulate the cytostatic and cytotoxic effects of these inhibitors. The qualitatively different response of HL-60 cells to camptothecin, teniposide, or amsacrine (by rapidly triggered DNA degradation in S phase) as compared to mitoxantrone or adriamycin (by cell arrest in G2 and S) suggests that, despite the generally assumed common mode of action attributed to these drugs (i.e., via stabilization of the cleavable DNA-topoisomerase complexes), there are significant differences in the mechanisms by which they exert cytostatic/cytotoxic effects.