Synergistic action of taxol with tiazofurin and methotrexate in human breast cancer cells: Schedule-dependence

Taxol (paclitaxel, (NSC 125973)) is an active agent in the treatment of metastatic carcinoma of the breast; however, positive responses were observed in only about 60% of cases. Therefore, drug combinations which might improve the effectiveness are required. Since tiazofurin (2-β-D-ribofuranosylthiazole-4-carboxamide, NSC 286193), methotrexate (MTX) and taxol exert their anticancer action in different phases of the cell cycle and have different biochemical targets, we tested the hypothesis that tiazofurin and methotrexate might be synergistic with taxol. MDA-MB-435 human breast cancer cells were grown in monolayer in flasks. In the growth inhibition assay for tiazofurin, methotrexate and taxol the IC₅₀s were 12.5, 0.5 and 0.016 μM, respectively, and in the clonogenic assay 4.5, 0.065 and 0.004 μM. When taxol was given 6 hr before tiazofurin, antagonism was observed and summation was seen when drugs were given simultaneously. Synergism was obtained in both growth inhibitory and clonogenic assays when tiazofurin was followed 12 hr later by taxol. Methotrexate and taxol had an antagonistic effect when they were added simultaneously or when taxol was given 6 hr before methotrexate; summation was observed when taxol was followed 12 hr later by methotrexate. Synergistic action was obtained in the clonogenic assay when methotrexate was followed 12 hr later by taxol. The protocols yielding synergism should be of value in the design of taxol-based clinical trials for breast cancer.     

Experimental therapy of ovarian cancer with synthetic makaluvamine analog: in vitro and in vivo anticancer activity and molecular mechanisms of action

The present study was designed to determine the biological effects of novel marine alkaloid analog 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ) on human ovarian cancer cells for its anti-tumor potential and the underlying mechanisms as a novel chemotherapeutic agent. Human ovarian cancer cells (A2780 and OVCAR-3), and Immortalized non-tumorigenic human Ovarian Surface Epithelial cells (IOSE-144), were exposed to FBA-TPQ for initial cytotoxicity evaluation (via MTS assay kit, Promega). The detailed in-vitro (cell level) and in-vivo (animal model) studies on the antitumor effects and possible underlying mechanisms of action of the compounds were then performed. FBA-TPQ exerted potent cytotoxicity against human ovarian cancer A2780 and OVCAR-3 cells as an effective inhibitor of cell growth and proliferation, while exerting lesser effects on non-tumorigenic IOSE-144 cells. Further study in the more sensitive OVCAR-3 cell line showed that it could potently induce cell apoptosis (Annexin V-FITC assay), G2/M cell cycle arrest (PI staining analysis) and also dose-dependently inhibit OVCAR-3 xenograft tumors' growth on female athymic nude mice (BALB/c, nu/nu). Mechanistic studies (both in vitro and in vivo) revealed that FBA-TPQ might exert its activity through Reactive Oxygen Species (ROS)-associated activation of the death receptor, p53-MDM2, and PI3K-Akt pathways in OVCAR-3 cells, which is in accordance with in vitro microarray (Human genome microarrays, Agilent) data analysis (GEO accession number: GSE25317). In conclusion, FBA-TPQ exhibits significant anticancer activity against ovarian cancer cells, with minimal toxicity to non-tumorigenic human IOSE-144 cells, indicating that it may be a potential therapeutic agent for ovarian cancer.     

片仔癀对人卵巢癌OVCAR-3细胞增殖、凋亡及周期的影响

目的：研究片仔癀对人卵巢癌细胞株OVCAR-3增殖抑制作用,及其对细胞周期、细胞凋亡的影响。方法：采用MTT法观察片仔癀对人卵巢癌细胞株OVCAR-3细胞的增殖抑制率,流式细胞仪检测细胞凋亡及细胞周期,westem—blot检测相关蛋白的表达。结果：片仔癀以剂量依赖式抑制人卵巢癌细胞株OVCAR-3细胞增殖,片仔癀250、500、1000μg·mL-1作用于OVCAR-3细胞24h后,其早期凋亡率分别为6．6％、30．9％、43．2％,而对照组为0％,其诱导凋亡作用呈现剂量依赖性;细胞积聚在G0／G1期,同时S期细胞比例减少;Akt、PARP、CDK6表达下调。结论：片仔癀可以抑制OVCAR-3细胞增殖及诱导细胞凋亡作用,并能阻滞细胞于G0／G1期,有望成为卵巢癌治疗药。     

A conserved Asn in TM7 of the thyrotropin receptor is a common requirement for activation by both mutations and its natural agonist

Arsenic trioxide (As2O3) inhibits cell growth and induces apoptosis in certain types of cancer cells including acute promyelocytic leukemia, prostate and ovarian carcinomas, but its effect on response of tumor cells to ionizing radiation has never been explored before. Here we demonstrate that As2O3 can sensitize human cervical cancer cells to ionizing radiation both in vitro and in vivo. As2O3 in combination with ionizing radiation have a synergistic effect in decreasing clonogenic survival and in the regression of established human cervical tumor xenografts. Pretreatment of the cells with As2O3 also synergistically enhanced radiation-induced apoptosis. Apoptosis of the cells by combined treatment of As2O3 and radiation was associated with reactive oxygen species generation and loss of mitochondrial membrane potential, resulting in the activation of caspase-9 and caspase-3. The combined treatment also resulted in an increased G2/M cell cycle distribution at the concentration of As2O3 which did not alter cell cycle when applied alone. These results indicate that As2O3 can synergistically enhance radiosensitivity of human cervix carcinoma cells in vitro and in vivo, suggesting a potential clinical applicability of combination treatment of As2O3 and ionizing radiation in cancer therapies.     

The use of dimethylsulfoxide as a vehicle in cell culture experiments using ovarian carcinoma cell lines.

Dimethylsulfoxide (DMSO) is a well-known solvent that is commonly used in the laboratory. We selected DMSO as the vehicle for an experiment designed to determine if several nonsteroidal anti-inflammatory agents inhibit the growth of Caov-3, OVCAR-3, and SK-OV-3 ovarian carcinoma cell lines. Using the tetrazolium conversion assay, however, we observed some variability in the number of cells present in each ovarian carcinoma cell line with varying concentrations of DMSO (10(-6)-10(-2) M) compared to medium alone. Similarly, when Caov-3, OVCAR-3, and SK-OV-3 cells were treated with 10(-4) M DMSO plus medium (Dulbecco's Modified Eagle Medium with 10% fetal bovine serum) and plated on coverslips, the total number of cells present in 60 random fields increased significantly (P < 0.0001) for each ovarian carcinoma cell line treated with DMSO compared to medium alone. Ethanol did not demonstrate such prominent effects on cellular growth. Our observations are important to consider when selecting an appropriate solvent, especially for growth inhibition studies using Caov-3, OVCAR-3, and SK-OV-3 cell lines.     

Interleukin-10 in serous ovarian carcinoma cell lines

 Interleukin-10, one of the most potent anti-inflammatory cytokines, is expressed in ovarian carcinomas in vivo. In contrast to the high levels of IL-10 in ascites and tumour tissue, the expression of this cytokine appears to be a rare event in ovarian carcinoma cell lines in vitro. Virtually nothing is known about the regulation of IL-10 expression in ovarian carcinoma cell lines. We investigated the expression of IL-10 in four cell lines originally derived from ovarian serous adenocarcinoma: OVCAR-3, SKOV-3, CAOV-3 and OAW-42. IL-10-specific mRNA was detected in OVCAR-3 and only this cell line produced IL-10 constitutively under serum-free conditions as well as in serum-containing medium. Our studies on the regulation of IL-10 secretion in OVCAR-3 revealed that (1) proinflammatory stimuli IL-1β and TNF-α, but not LPS, enhance IL-10 secretion, (2) IL-6 has no influence on the release of IL-10, (3) prostaglandin E2 influences neither the spontaneous nor the TNF-α- or IL-1β-stimulated IL-10 production and (4) interferon-γ inhibits IL-10 secretion. We conclude that only a minority of serous ovarian carcinoma cells maintain the ability to produce IL-10 in vitro. Our data on the regulation of IL-10 production in OVCAR-3 indicate that ovarian carcinoma cells share some, but not all, of the regulatory features typical for the monocytic IL-10 secretion. Received: 1 February 2001 / Accepted: 29 March 2001     

Action of tiazofurin and 8-Cl-cAMP in human colon and pancreatic cancer cells.

Tiazofurin and 8-Cl-cAMP are novel chemotherapeutic agents shown to be effective against various cancer cells in vitro and in vivo. They act through distinct mechanisms that might modulate the signal transduction pathway, which causes growth inhibition, differentiation and down-regulation of c-ras and c-myc oncogene expression. We examined the effects of tiazofurin and 8-Cl-cAMP on colony formation of HT-29 human colon cancer and BxPC-3 and PANC-1 human pancreatic cancer cell lines. The IC50 of 8-Cl-cAMP was 0.1 and 0.2 microM in the pancreatic and colon cancer cell lines, respectively, and tiazofurin yielded IC50s from 4 (PANC-1) to 18 microM (HT-29). Simultaneous incubation with 8-Cl-cAMP and tiazofurin had additive effects on the inhibition of colony formation in the three examined cell lines. These results indicate possible clinical usefulness of a combination of tiazofurin and 8-Cl-cAMP in the treatment of colon and pancreatic carcinomas.     

IMP dehydrogenase and action of antimetabolites in human cultured blast cells

Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the Km values for IMP and NAD were 29 and 54 microM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5'-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the Ki values of RMP (0.58 microM) and TAD (0.075 microM) were several orders of magnitude lower than those of XMP (85 microM) and NADH (94 microM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells.     

Synergistic action of tiazofurin and difluorodeoxycytidine on differentiation and cytotoxicity.

Tiazofurin (TR), an inhibitor of IMP dehydrogenase, causes remissions and induced differentiation in human leukemia through lowering the concentrations of GTP and dGTP. A deoxycytidine analog, difluorodeoxycytidine (DFDC), is an anti-tumor agent phosphorylated by deoxycytidine kinase, resulting in decreased concentration of dCTP, leading to inhibition of DNA synthesis. In HL-60 cells DFDC induced differentiation and inhibited proliferation in a dose-dependent manner (IC50 = 4 nM); TR provided synergism with DFDC. DFDC inhibited proliferation in OVCAR-5 human ovarian carcinoma cells (IC50 = 25 nM) and colony formation in PANC-1 human pancreatic carcinoma cells (IC50 = 2 nM) and rat hepatoma 3924A cells (IC50 = 22 nM). TR and DFDC are synergistically cytotoxic in hepatoma cells and additive in PANC-1 cells. The two drugs together should be helpful in treating leukemias and solid tumors in humans.     

Radiotherapy combined with gemcitabine and oxaliplatin in pancreatic cancer cells

Clinical evidence suggests that gemcitabine (Gem) plus oxaliplatin (Ox) is superior to gemcitabine alone in advanced pancreatic carcinoma. The addition of radiation to gemcitabine improves response and is a standard treatment for locally advanced disease. We investigated the effect of oxaliplatin on gemcitabine-based chemoradiation by determining whether gemcitabine and oxaliplatin produced synergistic cytotoxicity using median effect analysis and radiosensitization using clonogenic survival assays. We analyzed the effects of gemcitabine and oxaliplatin on cell cycle distribution by DNA content and on radiation-induced DNA damage repair by phosphorylated H2AX (gamma-H2AX). Gemcitabine and oxaliplatin produced schedule-dependent synergistic cytotoxicity in BxPC-3 and Panc-1 cells (combination indices: 0.76 +/- 0.05, 0.61 +/- 0.11). In BxPC-3 cells, oxaliplatin did not affect gemcitabine-mediated radiosensitization (Gem 1.99 +/- 0.27; Gem + Ox 2.38 +/- 0.30). In Panc-1 cells, oxaliplatin significantly enhanced gemcitabine-mediated radiosensitization (Gem 1.31 +/- 0.05; Gem + Ox 2.90 +/- 0.31). Radiosensitization by gemcitabine was accompanied by early S-phase arrest and induction/persistence of gamma-H2AX protein, which were unaltered by oxaliplatin. Addition of oxaliplatin to gemcitabine produces radiosensitization equal to or greater than gemcitabine alone, supporting our clinical investigation of oxaliplatin with gemcitabine-radiation in pancreatic cancer aimed at improving systemic disease control while maintaining local tumor radiosensitization.     

Ovarian carcinoma cells synthesize both chondroitin sulfate and heparan sulfate cell surface proteoglycans that mediate cell adhesion to interstitial matrix

Metastatic ovarian carcinoma metastasizes by intra-peritoneal, non-hematogenous dissemination. The adhesion of the ovarian carcinoma cells to extracellular matrix components, such as types I and III collagen and cellular fibronectin, is essential for intra-peritoneal dissemination. The purpose of this study was to determine whether cell surface proteoglycans (a class of matrix receptors) are produced by ovarian carcinoma cells, and whether these proteoglycans have a role in the adhesion of ovarian carcinoma cells to types I and III collagen and fibronectin. Proteoglycans were metabolically labeled for biochemical studies. Both phosphatidylinositol-anchored and integral membrane-type cell surface proteoglycans were found to be present on the SK-OV-3 and NIH:OVCAR-3 cell lines. Three proteoglycan populations of differing hydrodynamic size were detected in both SK-OV-3 and NIH:OVCAR-3 cells. Digestions with heparitinase and chondroitinase ABC showed that cell surface proteoglycans of SK-OV-3 cells had higher proportion of chondroitin sulfate proteoglycans (75:25 of chondroitin sulfate:heparan sulfate ratio), while NIH:OVCAR-3 cells had higher proportion of heparan sulfate proteoglycans (10:90 of chondroitin sulfate:heparan sulfate ratio). RT-PCR indicated the synthesis of a unique assortment of syndecans, glypicans, and CD44 by the two cell lines. In adhesion assays performed on matrix-coated titer plates both cell lines adhered to types I and III collagen and cellular fibronectin, and cell adhesion was inhibited by preincubation of the matrix with heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or chondroitin glycosaminoglycans. Treatment of the cells with heparitinase, chondroitinase ABC, or methylumbelliferyl xyloside also interfered with adhesion confirming the role of both heparan sulfate and chondroitin sulfate cell surface proteoglycans as matrix receptors on ovarian carcinoma cells.     

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP. When guanosine and adenine were added together with tiazofurin, the differentiation of K562 was prevented, the concentration of diacylglycerol was maintained at control values, and the reduction in the concentration of cGMP was partially prevented. Other inducers of differentiation which acted by different mechanisms, caused similar changes in the concentrations of diacylglycerol and cGMP.     

Parthenolide induces apoptosis by activating the mitochondrial and death receptor pathways and inhibits FAK-mediated cell invasion

The natural product parthenolide induces apoptosis in cancer cells. However, the mechanism of apoptosis in ovarian cancer cells exposed to parthenolide is not clear. In addition, it is unclear whether parthenolide-induced apoptosis is mediated by the formation of reactive oxygen species and the depletion of GSH contents, and the effect of parthenolide on the invasion and migration of human epithelial ovarian cancer cells has not been studied. Therefore, we investigated the effects of parthenolide exposure on apoptosis, cell adhesion, and migration using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. The results suggest that parthenolide may induce apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The apoptotic effect of parthenolide appears to be mediated by the formation of reactive oxygen species and the depletion of GSH. Parthenolide inhibited fetal bovine serum-induced cell adhesion and migration of OVCAR-3 cells, possibly through the suppression the focal adhesion kinase-dependent activation of cytoskeletal-associated components. Therefore, parthenolide might be beneficial in the treatment of epithelial ovarian adenocarcinoma and combination therapy.     

Combined application of camptothecin and the guanylate cyclase activator YC-1: Impact on cell death and apoptosis-related proteins in ovarian carcinoma cell lines

Camptothecin analogs and guanylate cyclase activator YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole] have been shown to induce apoptosis in cancer cells. However, the combined effect of camptothecin analogs and YC-1 on the viability of epithelial ovarian cancer cells remains uncertain. We assessed the combined effect of YC-1 on the camptothecin toxicity in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Camptothecin and YC-1 induced apoptosis in OVCAR-3 and SK-OV-3 cells in a dose- and time-dependent manner. Both compounds induced nuclear damage, decreased Bid and Bcl-2 protein levels, enhanced cytochrome c release, activated caspase-3 and upregulated tumor suppressor p53. Camptothecin decreased Bax protein levels, whereas YC-1 increased Bax levels. YC-1 enhanced the camptothecin-induced changes in the apoptotic protein levels and increased apoptotic effect of camptothecin on ovarian carcinoma cell lines. The results suggested that YC-1 may enhance a camptothecin toxicity against ovarian carcinoma cell lines by increasing activation of the caspase-8 and Bid pathway as well as activation of the mitochondria-mediated apoptotic pathway, leading to cytochrome c release and subsequent caspase-3 activation. Combination of camptothecin analogs and YC-1 may provide a therapeutic benefit against ovarian adenocarcinoma.     

Effects of cadmium on differentiation and cell cycle progression in cultured Xenopus kidney distal epithelial (A6) cells

Cadmium (Cd) is an important industrial and environmental pollutant, and the kidney is the primary organ to be affected. To elucidate the effects of Cd on cell proliferation, an epithelial cell line (A6) originally derived from the distal part of the Xenopus laevis kidney was cultured in media containing 10% fetal bovine serum. The effects of Cd (added as CdCl(2)) on cellular growth and differentiation from single cells to confluent epithelia were investigated by visual inspection and by measurement of the degree to which living cells covered a unit area. Over a concentration range from 5 to 50 microM, Cd did not affect the settling and adherence of single cells to the bottom of the culture well. The addition of 5 microM Cd for 4 days did not affect the ability of the A6 cells to develop confluent epithelia, measured as the area covered by adherent living epithelial cells (99 +/- 4% of the control value). However, 10 microM Cd did effectively inhibit development of confluent epithelia to 13 +/- 5% compared to control. Visual inspection of adherent cells exposed to 50 microM Cd for 7 days revealed no increase in cell number or in cell death, which indicated the induction of cell cycle arrest. Flow cytometric analysis showed that treatment of cells with Cd (0.4mM) for 24 hours induced a significant increase in the proportion of G1 phase cells from 58.6 +/- 3.9 to 80.6 +/- 3.7%, and a corresponding reduction in the proportion of cells in both the S and G2 phases from 24.0 +/- 3.6 to 13.4 +/- 3.3% and 17.2 +/- 1.7 to 5.8 +/- 2.1%, respectively. This study showed that Cd stopped cell proliferation in a very narrow concentration range, between 5 and 10 microM, and cell cycle analysis indicated that Cd arrested the cells in the G1 phase of the cell cycle.     

An Antimitotic and Antivascular Agent BPR0L075 Overcomes Multidrug Resistance and Induces Mitotic Catastrophe in Paclitaxel-Resistant Ovarian Cancer Cells

Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC₅₀ = 2–7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.     

Antitumor activity of a thioether-linked immunotoxin: OVB3-PE

A thioether-linked immunotoxin was made between Pseudomonas exotoxin and the monoclonal antibody OVB3. This conjugate, OVB3-PE, was cytotoxic for the human ovarium cancer cell line OVCAR-3 (ID of 2.5 x 10(-12) M) and it was therefore tested for antitumor activity in a nude mouse model of ovarian cancer. This model employs the injection of a lethal number of OVCAR-3 cells into the peritoneal cavity of nude mice. When 0.2-1 micrograms of OVB3-PE was injected intraperitoneally on three successive days beginning 3-5 days after OVCAR-3 cell implantation, the survival of the tumor-bearing mice was increased 2-4-fold compared to that of untreated control mice. Median survival times for control mice ranged from 44 to 50 days while survival times of 150 days or greater were seen in mice treated with OVB3-PE. When OVB3-PE administration was delayed until 2-4 weeks after tumor cell implantation, OVB3-PE treatment also showed antitumor activity, but the duration of survival was less than with the early treatments. OVB3-PE was also cytotoxic for MCF-7 breast carcinoma cells, HT-29 colon carcinoma cells, and A431 epidermoid carcinoma cells.     

Centrosome and spindle pole microtubules are main targets of a fluorescent taxoid inducing cell death

Microtubules offer a very large local concentration of binding sites for cytotoxic taxoids or for hypothetical endogenous regulators. Several compounds from diverse sources stabilize microtubules and arrest cell division similarly to the antitumour drug Taxol. We have investigated the subcellular location of the Taxol binding sites, employing a fluorescent taxoid (FLUTAX) that reversibly interacts with the Taxol binding sites of microtubules and induces cellular effects similar to Taxol. The specific binding of FLUTAX to a fraction of the available cellular binding sites effectively inhibits division of cultured human tumour cells at G(2)/M, and triggers apoptotic death. The loci of reversible binding, directly imaged in intact U937 cells treated with cytotoxic doses of fluorescent taxoid are the centrosomes, with a few associated microtubules in interphase cells, and the spindle pole microtubules in mitotic cells, instead of uniformly labelling the microtubule cytoskeleton. Cytoskeletal lesions induced and visualized with FLUTAX consist of microtubule bundles and abnormal mitoses, including monopolar spindles and highly fluorescent multipolar spindles. The multiple asters and monopolar spindles mark arrested U937 leukaemia and OVCAR-3 ovarian carcinoma cells on their path to apoptosis (as well as K562, HeLa, and MCF-7 cells). Depending on the FLUTAX treatment, OVCAR-3 cells died from abnormal mitosis or from a subsequent interphase with double chromatin content and lobulated nuclei (micronuclei), indicating impairment of centrosome separation. Fragmented centrosomes could be observed in FLUTAX-treated non-transformed 3T3.A31 cells, which developed micronuclei but were resistant to apoptosis. These results strongly suggest that centrosomal impairment by taxoid binding during interphase, in addition to the suppression of the kinetochore microtubule dynamics in the mitotic spindle, is a primary cause of cell cycle de-regulation and cell death.     

The use of dimethylsulfoxide as a vehicle in cell culture experiments using ovarian carcinoma cell lines

Dimethylsulfoxide (DMSO) is a well-known solvent that is commonly used in the laboratory. We selected DMSO as the vehicle for an experiment designed to determine if several nonsteroidal antiin?ammatory agents inhibit the growth of Caov-3, OVCAR-3, and SK-OV-3 ovarian carcinoma cell lines. Using the tetrazolium conversion assay, however, we observed some variability in the number of cells present in each ovarian carcinoma cell line with varying concentrations of DMSO (10^-6-10^-2 M) compared to medium alone. Similarly, when Caov-3, OVCAR-3, and SK-OV-3 cells were treated with 10^-4 M DMSO plus medium (Dulbecco's Modified Eagle Medium with 10% fetal bovine serum) and plated on coverslips, the total number of cells present in 60 random fields increased significantly (P 50.0001) for each ovarian carcinoma cell line treated with DMSO compared to medium alone. Ethanol did not demonstrate such prominent effects on cellular growth. Our observations are important to consider when selecting an appropriate solvent, especially for growth inhibition studies using Caov-3, OVCAR-3, and SK-OV-3 cell lines.