Combined Antimicrotubule Activity of Estramustine and Taxol in Human Prostatic Carcinoma Cell Lines1

(CANCER RESEARCH 52, 4433-4440, August 15, 1992] Combined Antimicrotubule Activity of Estramustine and Taxol in Human Prostatic Carcinoma Cell Lines1

Lisa A. Speicher, Linda Barone, and Kenneth D. Tew Department of Pharmacology; Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

ABSTRACT pie round selection of EMR clones required more than 1 year and resulted in cells with resistance levels not greater than Estramustine (EM) and taxol, two antimicrotubule agents with dis 5-fold. The EMR cells have a phenotype distinct from that of tinct and apparently opposing mechanisms of action, were found to be effective in combination in the preclinical treatment of EM-resistant and the MDR and also have no elevation in either glutathione levels sensitive, wild-type human prostata- carcinoma cell lines. Estramustine or glutathione-5-transferase activity. In addition, the EMR combined with 1 n\t taxol (concentration 100-fold less than that mea clones exhibit nearly one-half the chromosome number (50-60 sured in plasma of patients treated with taxol) produced greater than chromosomes) and one-half the DNA content of the parental additive effects on the inhibition of cell survival of both wild-type and line. Because EM is independent of the MDR phenotype, there EM-resistant cells. When taxol was used with another microtubule- is a sound rationale for including the drug in clinical regimens destabilizing drug, vinblastine, no significantly increased cytotoxicity in combination with antimitotic agents, which are part of the was observed. Other effects on wild-type and EM-resistant cells pro MDR phenotype. duced by the combination of EM and taxol included (a) an increased Taxol, isolated from the bark of the western yew Taxus brev- proportion of the cells in the S phase of the cell cycle; (b) no mitotic ifolia (14), is a complex diterpene which is distinct from other block; and (c) an increase in the percentage of micronucleated cells from a control value of 20Â°/oafter drug treatment. Immunofluores- antimicrotubule agents in that it binds directly to polymerized tubulin to promote microtubule assembly and stabilizes (rather cent microscopic analysis of the effect of this drug combination on the than destabilizes) microtubules from depolymerization (15- mitotic spindle apparatus revealed specific examples of aberrant mitotic figures, including multiple asters, cells with two distinct spindles, and 17). These excessively stable microtubules are no longer able to tripolar spindles able to traverse mitosis and complete cytokinesis. maintain their normal dynamic reorganization properties, re These data provide supportive preclinical evidence for the potential sulting in loss of function. The drug has been shown to affect development of an EM/taxol combination clinical regimen either for numerous microtubule-regulated functions resulting in inhibi prostate or other cancers. tion of cell proliferation and mitotic arrest (16,18); induction of microtubule bundling (19); formation of abnormal mitotic spin INTRODUCTION dle asters (20, 21); and inhibition of transmission of transmem brane mitogenic signals (22). Clinically, taxol is proving to be a The appreciation for a multifaceted approach to cancer treat potentially exciting agent in the treatment of numerous tumor ment came with the discovery of tumor cell heterogeneity in the types (for review see Ref. 23) as well as several refractory tu early 1970s (1). The failure of single therapies in cancer man mors including ovarian carcinoma (24), malignant melanoma agement as well as the ability of tumor cells to develop resis (25), and myeoblastic leukemias (26). Although taxol demon tance to drugs used for treatment, provided a strong rationale strates great clinical promise, patient toxicity at standardly used for combining anticancer agents. In fact, the majority of the doses may serve to limit its utility. curable cancers today require combination chemotherapy (2). The present study investigates the hypothesis that taxol used The present study describes in vitro cellular effects of the com in combination with EM would be a logical approach to en bination of two antimicrotubule drugs: EM,2 a microtubule hance cytotoxicity through combined, though distinct, antimi destabilizing agent, and taxol, a microtubule stabilizer, on crotubule activity. Toxicity assays combining clinically relevant EMR and WT DU 145 human prostatic carcinoma cell lines. concentrations of EM with a taxol level (l HM) significantly Estramustine (Fig. 1), a chemotherapeutic agent used to treat lower than that seen in the serum of patients treated with taxol advanced hormone-refractory prostate carcinoma (3,4) exhibits were performed on EMR and WT DU 145 cells. The effects of unique pharmacological properties, with a mechanism of action this combination on cell cycle traverse and the mitotic spindle distinct from the predicted alkylating or estrogen activities of microtubules are also described. its constituent moieties (5,6). The carbamate ester bond linking nor-nitrogen mustard to estradiol is responsible for the unusual properties of the drug as well as its long clinical half-life (7, 8). MATERIALS AND METHODS Estramustine can be classified as an antimicrotubule agent, Cell Culture. Human prostatic carcinoma cell lines (DU 145) were since its noncovalent binding to MAPs causes microtubule dis cultured and EMR were selected as previously described (13). Briefly, assembly, stathmokinesis, and eventual cell death (9-12). following an initial mutagenesis with ethylmethane sulfonate, colonies Development of EM-resistant human prostatic carcinoma surviving twice the EM IC9u value were harvested and passaged sequen cell lines revealed a drug-resistance profile quite dissimilar from tially in increasing EM concentrations. This selection process resulted those typically seen for antimicrotubule drugs (13). The multi- in clones exhibiting approximately 5-fold levels of increased EM resis tance compared to WT drug-sensitive cells. Cytotoxicity Assays. The effect of taxol plus EM on cell survival was Received 3/3/92; accepted 6/9/92. The costs of publication of this article were defrayed in part by the payment of determined by using colony forming assays. Cell monolayers were page charges. This article must therefore be hereby marked advertisement in accord trypsinized from flasks, counted, and plated directly into drug-contain ance with 18 U.S.C. Section 1734 solely to indicate this fact. ing media at a density of 250 cells/10-cm2 tissue culture well. Cells were 1Supported by Grant 5R01 CA 43783-07 and NIH Grant CA-09035-16. Partial maintained at 37Â°Cin5% CO2 for 12-14 days. Surviving colonies (>32 support also provided by a Bristol-Squibb drug resistance grant. 2 The abbreviations used are: EM, estramustine; EMR, estramustine-resistant; cells) were fixed with 95% ethanol, stained with 1% crystal violet, and WT, wild type; MAP, microtubule-associated protein; MDR, multidrug resistance; counted with the use of a Biotran III automatic totalizer (New Bruns IC50. ICco, 50 and 90% inhibitory concentration, respectively. wick Scientific, Edison, NJ). The percentage of surviving colonies was 4433

CICH2CM2 >IM-C-0-^ io CICHjCHj Fig. 1. Chemical structure of estramustine. Nor-nitrogen mustard is linked to i.i estradici through a carbamate-ester bond. O calculated by comparison to control wells and plotted as a function of 12345 drug concentration. IC50 values were calculated from linear regression ESTRAMUSTINE CONCENTRATION (uM) analysis of plotted values. Flow Cytometry. Flow cytometry was used to study the effect of EM and taxol on the cell cycle. Cells were exposed to EM (WT, 0-5 J

ESTRAMUSTINE CONCENTRATION (uM) RESULTS Fig. 2. Combinational effects of EM and taxol on cell survival of wild type (A), EMR 4 (B), and EMR 9 (C) cell lines. Data are presented as percentage of cell Taxol Significantly Enhances Effect of Estramustine on Cell survival as a function of estramustine concentration in either the absence or Survival. Colony-forming assays were used to examine the cy- presence of taxol (WT, 0.5 HM;EMR, 1.0 nm). Combined EM/taxol data are normalized to account for the cell survival inhibitor)' effect of taxol alone (ap totoxic consequences of EM in combination with taxol in WT proximately 20% inhibition of cell survival). Values represent results from at least and EMR cell lines. The taxol concentrations (WT, 0.5 n\i; two experiments done in triplicate Â±SEM. Taxol significantly (* represent P values <0.05) enhances the cytotoxicity of EM in both WT and EMR cell lines. EMR, 1.0 n\i) used for the experiments were 2 to 3 logs lower (O) EM alone; (â€¢)EM plus taxol. than levels achieved in the serum of patients treated with taxol. A lower taxol concentration was used for WT cells because of a small increase in sensitivity (versus EMR) to the drug. As a 1.8 and 14.0 MM,respectively. In the presence of taxol, these single agent, the concentrations of taxol used resulted in an ICjo values decreased to 0.6 and 6.0 MMfor WT and EMR cells, approximate 20% inhibition of cell survival. In plotting the respectively. The ability of taxol to augment cytotoxic effects of combination data, this single agent effect was compensated by the antimicrotubule drug, vinblastine, was also tested. As seen making this equal to 100%. As illustrated in Fig. 2, when taxol in Fig. 3, no additive cytotoxicity occurred with this drug com was combined with increasing concentrations of EM, a greater bination. than additive toxic effect occurred in both sensitive and resis Effect of Estramustine and Taxol on Cell Cycle. Both EM tant cells. The EM IC50 values for the WT and EMR cells were and taxol have been reported to block cells in metaphase. Using 4434

combined EM/taxol treatment appeared to be less than control value concomitant with the proportional increase in S. Microscopic analysis of fixed (drug-treated) cells was also performed to investigate the potential mitotic block resulting from an EM/taxol treatment. Over 2000 cells were scored for ce :=> io- each condition listed in Table 2.2 As expected, EM treatment resulted in an increased percentage of mitotic WT cells (the resistant phenotype of the EMR cells would prevent a mitotic Ãœ block at the EM concentration used). In agreement with the aÂ«; flow cytometric data, no mitotic buildup was observed for EMR cells when the drugs were combined. The percentage of WT 0.1 0.2 0.^ 0.4 05 cells in mitosis after EM/taxol treatment was not significantly VINBLASTINE CONCENTRATION (nM) different (P > 0.05) from EM treatment alone (11.3 versus 11.5%)). Induction of Micronucleation by Estramustine and Taxol Treatment. Light microscopy was used to examine the effects B of EM and taxol on the induction of micronucleation. Results

Table 1 Effect of EM and taxol on cell cycle of wild-type and EM-resistant DU 2 145 cell lines i Fluorescence activated flow cytometry was used to analyze the effect of estra- => ICH mustine and taxol on the cell cycle of wild type and EM-resistant cell lines. Cells were treated with drug for 28 h prior to fluorescence-activated cell sorter analysis. Data are presented as percentage of cells in S or Gj-M after treatment with EM O alone or EM plus l n Mtaxol. Ã Â« % of cells

0.0 0.5 1.0 1.5 WildtypeNo drug1 VINBLASTINE CONCENTRATION (nM) nMtaxolSUM EM5 taxolEMR4NoMMEM + 1 HM

drug1 1001 l nMtaxol20 MMEM20 taxolEMR9NoMMEM + 1 HM

drug1 nMtaxol20 MMEM20 MMEM + 1 nM taxolG,50.044.238.117.345.745.242.329.1j44.542.438.430.7S39.546.548.675.841.843.545.763.544.046.150.063.2G2-M10.59.313.36.912.511.312.07.411.511.511.66.1

CJ Table 2 Effect of EM and taxol on mitolic index of WT and EMR cells Cells were treated with EM (WT, 2.5 MM;EMR, 20 MM);taxol (1 nM) or their Ã›.O 0.5 1.0 1.5 combination for approximately 40 h. At least 2000 cells were scored for each mii

Table 3 Effect of EM and taxol on micronucleation flow cytometry, the effect of an EM/taxol combination (28-h Cells were treated as described in Table 2 legend. At least 1000 cells were exposure) on the cell cycle of WT and EMR cells was com scored. Data are expressed as percentage of total number of cells with more than one nucleus. The combination of EM and taxol resulted in a significant increase pared. In contrast to an expected mitotic block, the majority of in the percentage of micronucleated cells (P < 0.05). the cells were observed in S phase. Results are listed in Table 1. micronucleationWild % of Taxol alone yielded a slight increase in the percentage of WT cells in S (46.5 versus 39.5%) but caused no such increase in the +taxol20.0 percentage of EMR cells in S (43.5 versus 41.8%). In contrast, type EMR 4 1.5 8.6 2.1 23.1 when combined with EM, taxol treatment resulted in a signif EMR 9Control0.5 0.9EM"3.2 8.3TaxolÂ»3.53.3EM 23.3 icant increase in the number of both WT and EMR cells in the " Concentration of EM; G, 2.5 MM;EMR, 20 MM. S phase (P < 0.05). The percentage of cells in G2-M after * Concentration of taxol, 1 nM. 4435

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. EM/TAXOL AND PROSTATE CANCER are shown in Table 3. Less than 1% of control WT and EMR DISCUSSION cells had greater than one nucleus. This value increased by 3-8% when EM and taxol were used as single agents. However, The unique pharmacological properties and target of EM afford sound reasoning for its use in combination with other when the two drugs were combined, greater than 20% of the cell population of both the WT and EMR cells were micronucle- antimicrotubule agents. Earlier studies which demonstrated that EM was independent from the multidrug-resistant ated. Effect of Estramustine and Taxol on Mitotic Spindle. The phenotype (13) further support this concept. Indeed, in vitro effects of taxol and EM on interphase as well as mitotic micro- studies demonstrated that EM enhanced the effect of vinblas- tubules were observed by indirect immunofluorescent micro tine on microtubule disassembly in transformed mouse ovarian scopy, using a ÃŸ-tubulinprimary and rhodamine-labeled sec and DU 145 cell lines (29). The present study suggests that EM may be used in combination with the microtubule-stabilizing ondary antibody. The drug concentrations used for these studies did not produce detectable alterations in interphase mi- agent, taxol, to produce significant enhancement of cell toxic- crotubule morphology (data not shown); however, many aber ity. Colony-forming assays used to study the cytotoxic conse rant mitotic figures were observed in both WT and EMR cells. quences of EM with taxol demonstrated that this drug combi Fig. 4 illustrates multiple aster formation in WT cells after nation had a greater than additive effect on inhibition of cell treatment with 2.5 MMEM and 1.0 HMtaxol for approximately survival. Of significance, the concentration of taxol necessary to 40 h. On rare occasions cells appeared to exhibit two distinct augment the toxic effects of EM was 100-fold less than those mitotic spindle apparati at metaphase (Fig. 4A). In addition, a levels found in the serum of patients treated with taxol (30). small proportion of the dividing cells appeared to exhibit func The toxicity caused by taxol at standard doses may serve to tional, tripolar mitotic spindles. Examples of cells with three limit its clinical utility; therefore, these data establish an en spindle poles and three sets of corresponding chromosomes couraging foundation for evaluation of this combination in hu were seen at various stages of mitosis, including metaphase man malignancies. (Fig. 4fi), anaphase (Fig. 5, Aâ€”D),and telophase (Fig. 5, E and The effect of taxol in combination with vinblastine was also F). It appears that these cells do in fact separate to form three examined in WT and resistant lines. Similar to EM, vinblastine distinct cells as evidenced by examples of three cells in both produces microtubule disassembly; however, this effect is me early (Fig. 5, G and //) and late (Fig. 6) stages of cytokinesis diated through a direct binding to tubulin rather than MAPs. which share one midbody. No significant increased cytotoxicity was observed with the

Fig. 4. Immunofluorescent localization of mitotic microtubules of wild type cells treated with 2.5 >IMEM and 1.0 nm taxol. (A) two mitotic spindles within a single cell; (B) multiple aster formation; (C and D) two mitotic spindles sharing three asters. Bar, 10 JIM. 4436

Fig. 5. Immunofluorescent and corresponding phase contrast photographs of WT cells treated with EM and taxol demonstrating tripolar spindles at individual stages of mitosis: (A and B) early anaphase; (C and D) late anaphase; (E and F) telophase; and (G and //) early cytokinesis. Bar, 10 MM.

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Fig. 6. Immunofluorescent (A, C, and D) and phase contrast (I!) micrographs of WT cells treated with EM and taxol demonstrating three cells sharing one midbody in early (A and B) and late (C and />) cytokinesis. Data suggest that one cell completed division into three corresponding daughter cells. Bar, 10 UM. vinblastine/taxol combination. The prediction might be that a croscopic analysis also indicated the lack of a mitotic block with microtubule-stabilizing agent would counter the effects of a the drug combination. These data support the concept that microtubule-disrupting agent. However, one must consider the microtubules may have an important role in the regulation of S dynamic nature of the microtubule. Microtubule subunits phase traversal. The first studies to suggest such a role for undergo continual turnover (31); thus, an inhibition of this microtubules demonstrated that colchicine either prevented the subunit turnover through either microtubule disassembly or sta initiation of, or prolonged the period of, DNA synthesis (32, bilization would result in a nonfunctioning microtubule net 33). Combinations of low-dose antimicrotubule agents and work. The greater than additive cytotoxic effects of the EM/ growth stimulants suggested a role for microtubules in growth taxol combination are conceivably related to the different factor regulation of DNA synthesis (34). Since this time, nu microtubule protein targets of EM and taxol (MAPs and tubu- merous studies have suggested that the microtubule network lin, respectively). Such is not the case with vinblastine and can regulate DNA synthesis through enhancement or inhibition taxol, with both drugs interacting directly with tubulin. A pre of mitogenic signals (35-37). vious report demonstrated that EM-induced microtubule disas We propose an alternative mechanism of DNA synthesis sembly was reversed with the addition of taxol (12). In contrast, modulation by the microtubules. It is possible that the EM/ when microtubules were disassembled with vinblastine, addi tion of taxol did not reinitiate microtubule assembly.3 MAPs taxol combination causes a disruption in communication be tween the cytoskeleton and the nuclear matrix, with a subse may serve to stabilize and/or protect microtubules when agents quent effect on the regulation of DNA synthesis. Drug that poison these structures are present. Thus, in the presence treatment produced many cells with abnormally shaped nuclei of EM, a drug such as taxol could be more effective in disrupt exhibiting blebbing and multilobed and clover-shaped mem ing the microtubule network. In addition, EM could remove brane conformations (data not shown). These abnormal nuclei MAPs from the microtubule and subsequently sterically in could result from a disruption of the microtubule network with crease the number of target sites available for taxol binding. In contrast to an expected block in G2-M, flow cytometric concomitant nuclear scaffold changes. This idea is supported by analysis revealed that EM/taxol treatment produced a signifi the reported relationship between the cytomatrix and the nu cant increase in the percentage of cells in S phase. Light mi- clear matrix (38) and the fact that the nuclear matrix has a role in the process of DNA synthesis (39, 40). Such a debilitation of the nuclear matrix could result from direct EM binding (6) or as 3 B. Hartley-Asp, personal communication. 4438

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. EM/TAXOL AND PROSTATE CANCER a consequence of drug-induced changes in the ordered micro- 6. Tew, K. D., Erickson, L. C., White, G., Wang, A. L., Schein, P. S., and Hartley-Asp, B. Cytotoxicity of estramustine, a steroid-nitrogen mustard tubule array of the cytoskeleton. The end result could be an derivative, through non-DNA targets. Mol. Pharmacol., 24: 324-328, 1983. interference with the normal communications between the two 7. Gunnarsson, P. O., Forshell, G. P., Fritjofsson, A., and Norlen, B. J. Plasma cell compartments and subsequent disturbance of S phase. In concentrations of estramustine phosphate and its major metabolites in pa tients with prostatic carcinoma treated with different doses of estramustine light of these studies, it is not unreasonable to speculate that phosphate (Estracyt). Scand. J. Urol. Nephrol., 15: 201-205, 1981. such a disturbance could prevent cells from entering mitosis, 8. Gunnarsson, P. ().. Anderson, S. B., Johansson, S. A., Nilsson, T., and Plym-Forshell, G. Pharmacokinetics of estramustine phosphate (Estracyt) in and this would account for the lack of a mitotic block observed prostatic cancer patients. Eur. J. Clin. Pharmacol., 26: 113-119, 1984. with combined EM/taxol treatment. 9. Hartley-Asp, B. Estramustine-induced mitotic arrest in two human prostatic The combination of EM and taxol also produced significant carcinoma cell lines, DU 145 and PC-3. Prostate, 5: 93-100, 1984. alterations in the mitotic spindles. The increased micronucle- 10. Stearns, M. E., and Tew, K. D. Anti-microtubule effects of estramustine, an antiprostatic tumor drug. Cancer Res., 45: 3891-3897, 1985. ation after drug treatment indicates altered spindle function. 11. Stearns, M. E., and Tew, K. D. Estramustine binds MAP-2 to inhibit micro- These results are similar to those from studies demonstrating tubule assembly in vitro. J. Cell Sci., 89: 331-342, 1988. 12. Kanje, M., Deinum, J., Wallin, M., Ekstrom, P., Edstrom, A., and Hartley- Vinca alkaloid induction of multinucleation (41). It was sug Asp, B. Effect of estramustine phosphate on the assembly of isolated bovine gested that disruption of the mitotic spindle could cause rever brain microtubules and fast axonal transport in the frog sciatic nerve. Cancer sion to an interphase state without completion of mitosis or Res., Â«.-2234-2239, 1985. 13. Speicher, L. A., Sheridan, V. R., Godwin, A. K., and Tew, K. D. Resistance cytokinesis. to the antimitotic drug estramustine is distinct from the multidrug resistant Immunofluorescent studies revealed multiple spindle asters phenotype. Br. J. Cancer, 64: 267-273, 1991. similar, in some cases, to those previously described to occur 14. Wani, M. C., Taylor, H. L., Wall, M. E., et al. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor with taxol treatment alone (20, 42). Cells with tripolar mitotic agent from Taxus brevifolia. J. Am. Chem. Soc., 93: 2325-2327, 1971. spindles and three corresponding sets of chromosomes were 15. Schiff, P. B., Fant, J., and Horwitz, S. B. Promotion of microtubule assembly also observed. Examples of these tripolar spindles were ob in vitro by taxol. Nature (Lond.), 22: 665-667, 1979. 16. Schiff, P. B., and Horwitz, S. B. Taxol stabilizes microtubules in mouse served at each phase of mitosis as well as cytokinesis. The data fibroblast cells. Proc. Nati. Acad. Sci. USA, 77: 1561-1565, 1980. suggest that in the presence of EM and taxol, an individual cell 17. Parness, J., and Horwitz, S. B. Taxol binds to polymerized tubulin in vitro. J. Cell Biol., 91: 479-487, 1981. is able to form a functional tripolar spindle such that the single 18. Fuchs, D. A., and Johnson, R. K. Cytologie evidence that taxol, an antine- cell completes division, producing three distinct daughter cells. oplastic agent from Taxus brevifolia, acts as a mitotic spindle poison. Cancer At this time, the viability of the progeny cells as well as their Treat. Rep., 62: 1219-1220, 1978. 19. Manfredi, J. J., Fant, J., and Horwitz, S. B. Taxol induces the formation of capacity to replicate is not known. To answer these questions, unusual arrays of cellular microtubules in colchicine-pretreated J774.2 cells. studies following a live cell through mitosis and cytokinesis will Eur. J. Cell Biol., 42: 126-134, 1986. be necessary. The concept that one cell can divide into three is 20. Manfredi, J. J., and Horwitz, S. B. Taxol: an antimitotic agent with a new mechanism of action. Pharmacol. Ther., 25: 83-125, 1984. not novel. In fact, in the early part of the century, Boveri (43) 21. DeBrabander, M., Geuens, G., Nuydens, R., et al. Taxol induces the assembly proposed this to be the mechanism through which tumor cells of free microtubules in living cells and blocks the organizing capacity of the arose. The aneuploid chromosome range in the WT cells was 80 centrosomes and kinetochores. Proc. Nati. Acad. Sci. USA, 78: 5608-5612, 1981. to 100; therefore, one cell could conceivably produce three 22. Crossin, K. L., and Carney, D. H. Microtubule stabilization by taxol inhibits progeny, each carrying a functional complement of chromo initiation of DNA synthesis by thrombin and by epidermal growth factor. somes. These results are supported by a recent study that dem Cell, 27: 341-350, 1981. 23. Rowinsky, E. K., Cazenave, L. A., and Donehower, R. C. Taxol: a novel onstrated induction of multipolar mitotic cells by the mitotic investigational antimicrotubule agent. J. Nati. Cancer Inst., 82: 1247-1259, inhibitor 2-(2-thenyl)sulfonyl-5-bromopyrimidine, and tripolar 1990. cells able to complete cytokinesis giving rise to three daughter 24. McGuire, W. P., Rowinsky, E. K., Rosenshein, N. B., et al. Taxol: a unique antineoplastic agent with significant activity in advanced ovarian epithelial cells (44). neoplasms. Ann. Intern. Med., /;/: 273-279, 1989. Results from a recent Phase II trial using escalating EM and 25. Einzig, A. L., Trump, D. L., Sasloff, J., et al. Phase II pilot study of taxol in constant, nontoxic vinblastine doses in advanced-hormone re patients (pts) with malignant melanoma (mm). Proc. Am. Soc. Clin. Oncol., 7:963, 1988. fractory prostatic carcinoma demonstrated responses in greater 26. Rowinsky, E. K., Burke, P. J., Karp, J. E., et al. Phase I and pharmacody- than 50% of the patients on study (45). This trial indicates that namic study of taxol in refractory acute leukemias. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. Combined Antimicrotubule Activity of Estramustine and Taxol in Human Prostatic Carcinoma Cell Lines

Lisa A. Speicher, Linda Barone and Kenneth D. Tew

Cancer Res 1992;52:4433-4440.

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