Earl G. Adams

Cell kill kinetics and cell cycle effects of taxol on human and hamster ovarian cell lines

Taxol is a clinically active anticancer drug, which exerts its cytotoxicity by the unique mechanism of polymerizing tubulin monomers into microtubules and stabilizing microtubules. Our studies with ovarian (hamster CHO and human A2780) cells showed that taxol is a phase-specific agent that is much more cytotoxic to mitotic cells than interphase cells. First, the dose-survival pattern of taxol resembled that of other phase-specific agents, in which cell-kill reached a plateau at a certain concentration. This suggests that the asynchronous cell population consists of a taxol-sensitive (presumably mitotic) fraction and a taxol-resistant fraction. Second, the cells were more responsive to increased exposure time than to increased dose above the plateau concentration. Third, in both asynchrounous and synchronous cultures taxol was much more cytotoxic to mitotic than interphase (G1, S and G2) cells. Fourth, the taxol concentration needed to kill cells corresponded to the dose needed to block cells in mitosis. Although taxol blocked cells in mitosis, the mitotic block was of short duration. Cells escaped the mitotic block, without cytokinesis, and entered the next round of DNA synthesis to form multinucleated polyploid cells. Taxol was 15-to 25-fold more toxic to A2780 (human ovarian carcinoma) cells compared to CHO cells. This difference in sensitivity correlated with a higher intracellular taxol concentration in A2780 as compared to CHO as determined by either an ELISA assay or by [H3]-taxol uptake.

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

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

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

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

Effects of treatment with immunomodulatory drugs on thymus and spleen lymphocyte subpopulations and serum corticosterone levels.

Immunofluorescence was used to characterize the lymphocyte subpopulations of mice treated with six immunomodulatory drugs: hydrocortisone acetate (HCA), corticosterone acetate (corticosterone), cyclophosphamide, cytosine arabinoside (Ara-C), 15(S)-methyl prostaglandin E1 (15(S)-methyl PGE1), and 2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone (ABPP). The number of thymus and spleen cells bearing Thy-1, Ig, Lyt-1 and Lyt-2 antigens and the density of the antigens on each cell (IF profiles) were determined. Microscopic examination of cells stained with rhodamine-labeled anti-Lyt-2 and fluorescein-labeled anti-Lyt-1 was used to measure the proportion of Lyt-1+2-, Lyt-1+2+, and Lyt-1-2+ cells in the spleen and thymus of drug-treated animals. The changes in lymphocyte subpopulations were compared with the varied effects of these drugs on antibody formation and graft vs host (GVH) reaction. Three immunosuppressive drugs, HCA, cyclophosphamide, and Ara-C, depleted the thymus of cells expressing a large quantity of Thy-1. The drug-resistant cells were larger and had more Lyt-1 than cells from control animals. HCA treatment depleted the thymus of Lyt-1+2+ cells; the cortisone resistant cells were primarily Lyt-1+2-. Cyclophosphamide and the antiviral immunostimulant, ABPP, caused similar, but less marked, alterations. The proportion of Lyt-1-2+ cells in the thymus was reduced by treatment with all the drugs, but the density of Lyt-2 on the drug-resistant cells was not altered. Treatment with Ara-C or 15(S)-methyl PGE1 produced a very modest evaluation in Lyt-1+2- cells. 15(S)-Methyl PGE1, which suppresses some immuno-inflammatory reactions, had no discernible effect on thymocyte size or the IF profile of Thy-1, Lyt-1, or Lyt-2. In the spleen, the amount of Thy-1 and of immunoglobulin on cells bearing these markers was changed very little by drug treatment. The proportion of splenic B cells was diminished by treatment with cyclophosphamide and, to a lesser extent, by HCA, while the proportion of spleen cells bearing detectable Thy-1 and Lyt-1 increased correspondingly. The proportion of cells bearing Lyt-2 was altered by only two drugs; cyclophosphamide increased both Lyt-1+2+ and Lyt-1-2+ spleen cells and ABPP (an interferon inducer which stimulates antibody formation) decreased both Lyt-2+ subpopulations. Treatment with two drugs caused the serum corticosterone concentration to rise: ABPP increased serum corticosterone substantially while the prostaglandin induced a smaller and more transitory increase. An indirect mechanism, via corticosteroid release, might explain the thymic depletion observed in mice treated with 15(S)-methyl PGE1 and ABPP, but neither the suppression of the GVH reaction by these drugs nor polyclonal activation of B cells by ABPP can be attributed to endogenous corticosteroids. Our data show that enumeration of splenic lymphocyte subpopulations by immunofluorescence techniques may aid in elucidating the mode of action of immunomodulatory drugs.

V79 Chinese hamster lung cells resistant to the bis-alkylator bizelesin are multidrug-resistant

Bizelesin (U-77779) is a highly potent bis-alkylating antitumor agent that is effective against several tumor systems in vitro and in vivo. V79 cells that were 125-to 250-fold resistant to bizelesin developed after constant exposure to gradually increasing concentrations of the drug. Resistant cells exhibited a multidrug-resistant phenotype and genotype as indicated by cross-resistance to several structurally and functionally unrelated drugs, e. g., colchicine, actinomycin D, and Adriamycin, and overexpression of mdr mRNA. Very low levels of cross-resistance to the alkylating, agents cisplatin and melphalan were seen. Multidrug-resistant mouse leukemia (P388/Adriamycin-resistant) and human (KB/vinblastine-resistant) cells were also resistant to bizelesin. Bizelesin resistance was unstable and decreased when cells were grown in the absence of the drug. Resistant and sensitive cell lines had similar levels of glutathione, and bizelesin cytotoxicity for resistant cells was not markedly affected by treatment with buthionine sulfoximine. Cross-resistance between bizelesin and several of its analogs is reported.

Synergistic combination of menogarol and melphalan and other two drug combinations.

SummaryMenogarol is a new anthracycline undergoing phase I clinical trial. We report here the lethality after 2 hr exposure to 2 drug combinations of menogarol and several antitumor agents. A new statistical procedure was used to identify synergistic combinations. Most of these combinations were additive, except for menogarol plus melphalan, which was synergistic. Adriamycin plus melphalan was also synergistic. The menogarol-melphalan combination was studied in detail with regard to the effect of dose and drug-schedule, lethality for exponential and plateau phase cells and effect on cell cycle progression. Although the combination was synergistic for exponential cells it was additive for plateau phase cells. The combination exerted a synergistic effect in inhibiting progression of cells through the cell cycle. After 2 hr menogarol exposure cells were blocked in G2 for about 12 hr following which the block was reversed. This reversal was inhibited when menogarol was combined with melphalan. The uptake of menogarol or melphalan was not changed in the presence of the other drug.

Cytotoxicity of combinations of prostaglandin D2 (PGD2) and antitumor drugs for B16 melanoma cells in culture

Prostaglandin D2 (PGD2) is lethal to murine and human melanoma cells at high doses, but synchronizes cells at G1 at non-toxic doses (2.5 or 5 μg/ml). We tested the lethality to B16 mouse melanoma cells of combinations of PGD2 with anticancer drugs. The drugs selected were mostly those used in treating human melanoma: actinomycin D, Bleomycin, BCNU, cis-platin, melphalan, 5-fluorouracil, and 1-β-D-arabinofuranosylcytosine (ara-C). PGD2 was combined with the drugs according to 3 different protocols:Protocol 1An asynchronous culture was given a long term (24 hr) exposure simultaneously to PGD2 + drug. Combinations with Bleomycin, ara-C or melphalan were additive or slightly antagonistic whereas PGD2 plus actinomycin D was significantly antagonistic.Protocol 2Cells synchronized in G1 by 24 hr PGD2 exposure were then given a short-term (2 hr) treatment with PGD2 + drug. Combinations with cis-platin, Bleomycin, BCNU or 5-fluorouracil were additive or slightly antagonistic, whereas melphalan and actinomycin D combinations were significantly antagonistic.Protocol 3Cells were released from a PGD2-induced G1 block and were exposed to drug at different times during cell progression. Actinomycin D was antagonistic when added immediately after release from the G1 block, but was significantly synergistic when added 10 to 12 hr later.The effect of the combinations cannot be explained by available cell cycle or biochemical information. The antagonism between PGD2 and several of the drugs resembles the “cytoprotective” effect of PGD2 towards various noxious agents.