[CANCERRESEARCH40,2695-2700,August1980] 0008-5472/80/0040.0000$02.00 ComparativeEffects of Vindesine,Vinblastine,and Vincristineon Mitotic Arrest and HormonalResponseof Li 210 LeukemiaCell& Sandra M. H. Howard, Athanasios Theologides, and J. R. Sheppard2 Department of Medicine, University of Minnesota Medical School (S. M. H. H., A. T.J,and the Dight Institute for Human Genetics (J. R. S.], University of Minnesota, Minneapolis, Minnesota 55455 ABSTRACT Although it is structurally related to VBL and to VGA, it has been reported to differ pharmacokinetically from the latter 2 The Vinca alkaloids vinblastine, vincristine, and vindesine compounds (20, 21, 35, 37). We undertook a study of these 3 were compared for their capacity to arrest Li 2i 0 cells in compounds to compare their effects on cellular proliferation, mitosis and for their effect on the response of Li 210 cells to Ml, cellular DNA content, and hormonal responsiveness. In this fI-adrenergic and prostaglandin E, hormones. Both micro study, we observed that OVA was more potent (as determined scopic and flow cytofluorimetric studies showed that, of the by molar ratio) than were VBL and VCR on these physiological three drugs, vindesine was the most potent for inhibiting growth effects. Moreover, evidence is presented indicating that OVA and arresting Li 2i 0 cells in mitosis. has a positive effect on the capacity of Li 210 cells to synthe In the hormone response studies, vindesine was also found size cyclic AMP in response to isoproterenol and PGE1. to be the most potent of the three drugs. Cells pretreated for 30 mm with 1o-@M concentrations of the drugs increased the MATERIALS AND METHODS initial hormone response after 5 mm of Li 2i 0 cells to either hormone by 100%. After a i -hr exposure to either isoproterenol A suspension culture of Li 210 cells was maintained in a or prostaglandin E1, however, the vindesine-treated cells had humidified 5% CO2@air atmosphere at a density of approxi cyclic adenosine 3':S'-monophosphate levels several times mately 3.5 x i O@cells/mI by thrice-weekly splitting of the higher than did controls. Vinblastine- and vincristine-treated cells. Culture media consisted of Roswell Park Memorial Insti cells were not different from the controls after i hr of hormone tute Medium 1640 (Grand Island Biological Co., Grand Island, exposure. Dose-response studies show that the vindesine ef N. V.) supplemented with 10% heat-inactivated fetal calf serum fect on enhanced cyclic adenosine 3':S'-monophosphate ac (Lot 1065; Pacific Laboratories, Inc., Richmond, Calif.), 2 mM cumulation increases substantially with drug concentrations L-glutamine (Grand Island Biological Co.), and i 00 units of from 10 to 1o@ M. These and other studies indicate that the penicillin-streptomycin (Grand Island Biological). Cells main effect of the Vinca alkaloids on cyclic adenosine 3':5'-mono tamed as described grew exponentially with a doubling time of phosphate metabolism occurs at the transduction mechanism 24 to 26 hr. OVA, VBL, and VCR were kindly supplied in pure which couples the hormone receptor to the catalytic component form without preservatives by Eli Lilly and Co., Indianapolis, of the adenylate cyclase complex. Ind. After sterilization by Millipore filtration, each drug was serially diluted in media to the concentration used in the assays. INTRODUCTION Four different concentrations, 10_6 to i O@M, were evaluated for each drug. The molecular mechanisms responsible for cytotoxic and The drugs were added to the cell suspensions worth a antitumor activity of Vinca alkaloids have been a controversial density of 3.5 x 1O@cells/mI. At zero time, a total cell count topic (6, 8, 18, 23, 4i ). The interaction between these drugs was done, and metabolic death was assessed as percentage and microtubules has been particularly well characterized (38), of viability by trypan blue dye exclusion (26). An aliquot of cells and a well-accepted mechanism of Vinca alkaloid-induced cell was also concentrated on a cytospin (Shandon Elliot Corp., death has been mitotic arrest caused by microtubule dysfunc Camberly, Surrey, England), fixed, and stained with Wright tion (5, 10, 34, 36). Several studies suggest that microtubule Giesma stain for Ml (percentage of cells in mitosis; 500 cells organization and cyclic nucleotide metabolism are closely re counted) and for evaluation of morphology. lated (9, 13, 27, 33). Since cyclic nucleotides are known to One additional aliquot was stained with propidium iodide for participate in the control of cell division (22, 30), it is possible DNA histogram analysis by FMF (17). An Ortho Model 4801 that Vinca alkaloids could inhibit proliferation through a cyclic fluorimeter was used for these studies. Cells prepared for FMF nucleotide-dependent process. To understand their cytotoxic were centrifuged gently (200 x g), resuspended in propidium action more clearly, we have examined the effect of Vinca iodide solution (5 mg propidium iodide in a 0.i 2% solution of alkaloids on the cyclic nucleotide metabolism of Li 210 cells. sodium citrate), and kept at 4°until FMF analysis was per A new Vinca alkaloid derivative, OVA,3 is currently under formed. The G1, 5, and G2 + M phases of the cell cycle were evaluation in clinical cancer chemotherapy (2, 7, 20, 21, 35). calculated using a modification of the estimation method of Baisch et a!. (i ). The histogram generated by the fluorimeter is I Supported by Grants CA-i 9527 from the NIH, the Minnesota Medical Foun datlon, and the Leukemia Task Force. divided into 5 sections (Chart 1): a, b, c, d, and e, where b is 2 To whom requests for reprints should be addressed. the modal channel of the G1 peak, and d is twice the distance 3 The abbreviations used are: DVA, desacetyl vinblastine amide sulfate (yin from the origin as b and represents the G2 + M peak. The G1, deems); VBL vinblastine; VCR, vincristine; MI, mitotic index; cyclic AMP, cyclic adenoelne 3':5'-monophosphate; PGE,, prostaglandin E,. 5, and G2 + M fractions are calculated as indicated in the Received January 21, 1980; accepted April 29, 1980. chart. Analysis of populations with large S fractions results in AUGUST1980 2695 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1980 American Association for Cancer Research. S. M. H. Howard et al. scintillation cocktail (90 g naphthalene and 9 g PPO in 1 liter dioxane), and the level of radioactivity was determined in a Beckman LS-i 00 scintillation counter. Recoveries ranged be tween 95 and i 00%. The method of determining the cyclic AMP content of the extracts was essentially that of Brown et a!. (3). The cyclic AMP-binding protein was prepared from whole bovine adrenal glands of freshly slaughtered animals according to the proce dures of Brown et a!. (3). Undiluted binding protein was stored ab c de frozen at —20°.Priorincubation of the extract with beef heart Phase Summations Percent Calculations phosphodiesterase (Boehringer Mannheim, Indianapolis, Ind.) G1@2a+b T@G1+S+G2.t'M eliminated 95 to i 00% of the competition between the extract and cyclic [3HJAMPfor the binding protein. s@c -(a+e) %G1:lOOx G2+M:2e+d O/S@10Qxi.. RESULTS %G2+M100 G@÷M Cell Growth and Mitotic Arrest. Untreatedcontrolcellsgrew exponentially throughout the time course of the experiment. A gradual slowing of cell growth with OVA and VBL occurred at DNA Histogram i 0-@M, and cell growth was completely suppressed at 1O@ Chart 1. Cell cycle phase distribution of a cellular population determined by M, the highest concentration tested. VCR affected cell growth flow cytometry using propidium iodide as a fluorescent probe. at the highest concentration only. A graph of relative cell counts (treated cell counts divided by control cell counts) is shown in underestimation of S and overestimation of G1. We emphasize Chart 2. OVA at 1O@and 1 8M was the most effective agent that percentages obtained from DNA histograms are estimates. in slowing cellular proliferation. Viability of the drug-treated The above studies were repeated after 6-, 24-, and 30-hr cells remained at approximately 95% over this 30-hr period. exposures of the cells to the drugs. A drug concentration-dependent increase in the mitotic index Hormonal Response. The physiologicalresponse to hor over time was found for all 3 drugs. At as early as 6 hr of mones was measured by analysis of cellular cyclic AMP levels. exposure to VBL and OVA at i O@ M, the cells exhibited The /1-adrenergic agonist, L-isoproterenol-D-bitartrate, was ob increased Ml's of 8.3 and i 2.6%, respectively, compared to a tamed from Sigma Chemical Co., St. Louis, Mo. Stock solutions Ml of 2.4% for controlcultures(Table i ). However, by 24 hr, were made fresh for each experiment using i mM ascorbate. these effects had disappeared. At concentrations of 10@8and PGE1,a gift of Or. John Pike (Upjohn Co., Kalamazoo, Mich.), i 0@ M, VBL and OVA caused an additional increase in Ml, was stored at —20°as stock solutions of 5 mg/mI 95% whereas VCR showed an increase in MI only at 1O'@M. The ethanol. The prostaglandin stock solution was diluted immedi ately before use in 50 [email protected],pH 7.4, at 300. All solutions were diluted at least i 0-fold upon addition to the cell systems. - OVA VBL VCR The solvents alone had no observed effects on cyclic AMP /c780 0 ,@ /c77• U A levels or hormonal response. All experimental procedures were carried out at 37°using 0 @ prewarmed solutions unless otherwise noted. The cells were 1.0 washed 3 times, each with 3 ml of 10 mM sodium phosphate buffer containing 0.i 5 M NaCI, 1 mM MgCI2, and i mM CaCI2, -S,, Q9 pH 7.4 (phosphate-buffered [email protected] removal @0.8 of the final wash, i ml of phosphate-buffered saline-Ca2―-Mg2― (with or without 10 @MR020,a phosphodiesterase inhibitor, 0.7 - which was the gift of H.