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Biomedical subjects

B K Bhuyan

Publications and source records attributed to B K Bhuyan.

At least 19 recordsLinked to original sources
PubMed1997

Assessment of microtubule stabilizers by semiautomated in vitro microtubule protein polymerization and mitotic block assays.

Paclitaxel (Taxol) a clinically active anticancer agent, exerts its cytotoxicity by inducing tubulin polymerization, leading to cellular mitotic block. In contrast, other antimitotic drugs, such as colchicine, podophyllotoxin, and vinblastine, act by depolymerizing microtubules. We report here (a) a semiautomated assay which measures the tubulin-polymerizing activity of paclitaxel analogs and (b) a cellular assay to measure the potential of these compounds to block cells in mitosis. The microtubule-polymerizing assay measured the turbidity of bovine brain microtubule protein (MTP) polymerized by the test compound in a 96-well plate. We maximized the sensitivity of this assay by conducting the polymerization reaction at 20 degrees C, at which temperature the baseline reaction, i.e. the basic ability of the untreated MTP control to polymerize, was minimal. At 20 degrees C, the effect of 0.05 microg/ml of paclitaxel on MTP could be detected, whereas at 37 degrees C, > 1 microg/ml of paclitaxel was required to detect a significant effect relative to untreated MTP. We describe the analysis of the complex curves of MTP polymerization with varying concentrations of test compounds. The polymerization of microtubules leads to cells being blocked in mitosis. This mitotic blocking effect in intact cells was determined using a cell settling chamber which allowed eight samples to be deposited on a slide. This method required a smaller number of cells (10(3) - 10[5]), maintained cell morphology, and allowed for rapid screening of samples. The activity of several new paclitaxel analogs is reported.

Animals↗
PubMed1995

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 high-potency, 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-aza-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.

Animals↗
PubMed1994

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-resistant 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.

Alkylating Agents↗
PubMed1993

Multidrug resistance is a component of V79 cell resistance to the alkylating agent adozelesin.

Adozelesin is a highly potent alkylating agent which has entered clinical trials based on its unique mechanisms of action and broad-spectrum antitumor activity in vivo. V79 cells resistant to adozelesin (V79/AdoR) were not resistant to the alkylating agent cisplatin but showed the phenotypic and genotypic characteristics of multidrug resistance. Thus V79/AdoR was cross-resistant to several structurally and functionally unrelated drugs, resistance was reversed by verapamil, and the resistant cell line expressed mdr mRNA and p170 glycoprotein. Also, adozelesin uptake and the amount of drug alkylated to DNA was much lower in the resistant cell line as compared to the sensitive parent. However, even with the same amount of drug bound to DNA (10 fmol/micrograms DNA) the survival of V79/S approximately 15% survival) was much lower than that of V79/AdoR (approximately 80%). Therefore the resistance of V79/AdoR cannot be explained solely by the multidrug resistance mechanism (i.e., lower drug uptake and less drug alkylation to DNA), which suggests that multiple mechanisms may account for resistance to adozelesin. V79/AdoR showed different levels of cross-resistance to several adozelesin analogues. The analogues could be divided into 2 groups; those with very low partition coefficients (log P < 2 as compared to 2.74 for adozelesin) had low levels of cross-resistance, whereas analogues with higher partition coefficients (log P > 2.4) were cross-resistant to adozelesin.

Alkylating Agents↗
PubMed1993

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 asynchronous 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.

Animals↗
PubMed1992

Lethality, DNA alkylation, and cell cycle effects of adozelesin (U-73975) on rodent and human cells.

Adozelesin (U-73975) is an extremely potent cytotoxic agent which causes 90% lethality, after 2 h exposure in vitro, of Chinese hamster ovary and lung (CHO and V79), mouse melanoma (B16), and human ovarian carcinoma (A2780) cells at 0.33, 0.19, 0.2, and 0.025 ng/ml, respectively. Under similar conditions, Adriamycin and cisplatin had 90% lethality values in CHO cells of 150 ng/ml (= 249 nM) and 6800 ng/ml (= 2266 nM), respectively. The relative drug sensitivity of the cell lines (A2780 > V79, B16, CHO) was correlated to the relative amounts of [3H]adozelesin alkylated to DNA. The greater sensitivity of A2780 was due to (a) greater DNA alkylation at different drug doses and (b) greater intrinsic sensitivity of A2780 which resulted in greater cell kill at comparable DNA alkylation. Phase specific toxicity studies show that adozelesin was least lethal to CHO cells in mitosis and very early G1. Lethality increased as cells progressed through G1 and was maximal in late G1 and early S. Mitotic cells had lower drug uptake and correspondingly less drug binding to DNA than G1 or S-phase cells. However, based on the amount of drug alkylated per micrograms of DNA, cells in M, G1, and S were equally sensitive. Therefore, the lower sensitivity of M-phase cells was due to lower drug uptake. Adozelesin had three different effects on progression of CHO, V79, B16, and A2780 through the cell cycle: (a) slowed progression through S which resulted in significantly increasing the percentage of S-phase cells. This effect was transient; (b) cell progression was blocked in G2 for a long time period; (c) the response of the cell lines to the G2 block differed. CHO and V79 cells escaped G2 block by dividing and entered the diploid DNA cycle or did not undergo cytokinesis and became tetraploid. On the contrary, B16 and A2780 cells remained blocked in G2 and did not become tetraploid. Cell progression was inhibited in a similar manner when a synchronized population of M, G1, or S-phase cells were exposed to adozelesin.

Alkylation↗
PubMed1992

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

Adozelesin (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.

Alkylating Agents↗
PubMed1991

Cytotoxicity of tetraplatin and cisplatin for human and rodent cell lines cultured as monolayers and multicellular spheroids.

The cytotoxicity of tetraplatin (dl-trans), its d- and l-isomers, and cisplatin for four human tumor cell lines (myeloma 8226, ovarian 2008, A2780, and OVCAR-3), their cisplatin-resistant variants, and three rodent cell lines (V79, EMT6/Ro, and L1210) were compared. Tetraplatin was more, or equally as, potent as cisplatin for the human cell lines and for L1210 but was clearly less potent for V79 and EMT6/Ro. The d-trans tetraplatin was more potent than the l-trans. Cisplatin resistant human tumor cells were less resistant to tetraplatin. On comparing sensitivity of V79 and EMT6/Ro cells in two growth models, we observed that all of the platinum compounds were more cytotoxic to cells in multicellular spheroids than in exponentially growing monolayers. Uptake studies, however, showed that tetraplatin was more cytotoxic to spheroids because spheroids accumulated more drug than monolayers.

Animals↗
PubMed1990

Characterization of B16 melanoma cells resistant to the CC-1065 analogue U-71,184.

U-71,184 is a CC-1065 analogue which is highly cytotoxic in vitro and has a broad spectrum of antitumor activity in vivo. Against B16 cells, U-71,184 was 8-fold and 253-fold more potent than Actinomycin D and Adriamycin, respectively. U-71,184 killed 90% of B16 cells at 0.01 ng/ml levels of drug in the medium, which was equivalent to an intracellular concentration of about 8 pg/10(6) cell (= 2 x 10(-8) pmol/cell). A B16 cell line resistant to U-71,184 developed after 3 months of in vitro exposure to gradually increasing concentrations of the drug. The sensitive and resistant cell lines were cloned and a B16/R clone was selected which was 60 to 100 times more resistant to U-71,184 than the cloned sensitive parent (B16/S). Cells grown in the absence of U-71,184 for 2 months retained resistance to the drug. B16/R was slightly cross-resistant only to Adriamycin but not to Actinomycin D, vinblastine, or colchicine. Among alkylating agents, it was slightly cross-resistant to Melphalan but not to 1,3-bis(2-chloroethyl)-1-nitrosourea or cisplatin. B16/R did not overexpress mdr mRNA. Therefore, this cell line does not exhibit the multidrug-resistant phenotype. Most karyotypes of B16/R had a marker chromosome which carried an aberrantly staining region apparently containing repetitive replication of the same segment. Resistance can be partly accounted for by the approximately 10-fold lesser uptake of [3H]-U-71,184 in B16/R, as compared to B16/S. B16/R was cross-resistant in varying degrees to several other CC-1065 analogues. The ratio of the 50% lethal dose of U-71,184 for B16/R, as compared to B16/S, was about 60 (i.e., R/S = 60). In comparison, the following compounds had an R/S ratio of less than 20 (i.e., modest level of cross-resistance to U-71,184): U-68,819, U-73,975, U-75,500, U-75,559, and CC-1065. In contrast, the following compounds had an R/S ratio greater than 20 (i.e., highly cross-resistant to U-71,184): U-71,184 analogues U-71,185, U-73,903, and U-75,012; U-73,975 analogues U-75,613, U-75,032, and U-73,896; and CC-1065 enantiomer U-76,915. We cannot yet explain the difference in the level of cross-resistance between these compounds in vitro. B16/S and B16/R cells were tumorigenic in mice and B16/R was resistant to U-71,184 in vivo. There was no clear indication of cross-resistance of B16/R in vivo to Adriamycin, Actinomycin D, cisplatin, or Melphalan. However, U-73,975, a compound with modest cross-resistance in vitro, was significantly cross-resistant in vivo.

Animals↗
PubMed1990

P388 leukaemia cells resistant to the anthracycline menogaril lack multidrug resistant phenotype.

Menogaril is an anthracycline presently in Phase II clinical trials. Menogaril-resistant mouse leukaemia P388 cells were developed in vitro by 4 months of exposure to step-wise increasing concentrations of menogaril after which resistant cells (P388/MEN) were cloned in 320 ng ml-1 menogaril. P388/MEN cells were 40-fold more resistant to menogaril in vitro compared to P388/O and were also resistant in vivo. Resistance to menogaril was stable for at least 2 months in the absence of the drug. The results indicate that P388/MEN, although resistant to an anthracycline, did not display the typical multidrug resistant phenotype. It was not cross-resistant to several structurally unrelated drugs such as actinomycin D, cisplatin, or vinblastine, but it was cross-resistant to the anthracycline, adriamycin. Uptake and efflux of menogaril was similar in sensitive and resistant cell lines. Also, resistance was not reversed by verapamil. No major karyotypic difference was noted between P388/O and P388/MEN. There was no significant amplification or overexpression of the mdr gene in P388/MEN compared to P388/O. In contrast to P388/MEN, P388 cells resistant to adriamycin displayed the typical multidrug resistant phenotype. Glutathione content of P388/MEN cells was similar to that of P388/O and depletion of glutathione did not potentiate menogaril cytotoxicity. Therefore, we conclude that glutathione is not likely to be involved in menogaril resistance to P388/MEN cells.

Animals↗
PubMed1990

Secular trend in birthweight in an industrial hospital in India.

Measurement of birthweight is an indicator of community health. The mean birthweight (MBW) of babies born in 1963, 1983 and 1986 were measured in 2254, 3550 and 3368 babies, respectively. Mean (SD) birthweight was found to be 2652 (553) g in 1963, 2724 (502) g in 1983 and 2726 (478) g in 1986. There was a rise in MBW (72 g) between 1963 and 1983 and a reduction in the percentage incidence of low birthweight from 34.29% to 26.06%. A change in birthweight distribution (was also seen, except in extreme weight groups. The improvement in birthweight is statistically significant (p less than 0.001). A trend for increase has been noticed in birthweights, and the mean birthweight values are the same as the national average.

Age Factors↗
PubMed1990

HPLC and flow cytometric analyses of uptake of adriamycin and menogaril by monolayers and multicell spheroids.

We have used both HPLC and flow cytometry to measure and compare the uptake of two anthracyclines, menogaril (MEN) and Adriamycin (ADR), in V79 Chinese hamster lung fibroblasts grown as monolayers and as 650 microns multicell spheroids. In order to compare intracellular drug accumulation in spheroid cells measured by the two methods, we converted mean channel fluorescence of the flow cytometer to drug uptake expressed as ng/10(6) cells by using a standard curve. The standard curve related the flow cytometric mean channel fluorescence, of monolayer cells exposed to either drug, to the intracellular drug accumulation determined by HPLC. This standard curve was then used to convert the mean channel fluorescence of cells from drug-exposed spheroids to ng/10(6) cells. Our results show that equal intracellular drug accumulation (determined by HPLC) in spheroids and monolayers does not result in equal cellular fluorescence emission (determined by flow cytometry) by these 2 cell populations. For example, monolayer cells with an intracellular MEN accumulation of 650 ng/10(6) cells, emit 40 units of fluorescence as measured by flow cytometry. However, spheroid cells with the same intracellular accumulation emit about 80 units of fluorescence. This results in the intracellular MEN uptake in spheroids measured by flow cytometry being as much as 2- to 3-fold higher than that measured by HPLC. Intracellular ADR accumulation measured by flow cytometry was also higher than that obtained by HPLC. In spite of the quantitative difference between the two methods, qualitatively both methods gave similar results. Thus, both techniques showed that at equal drug concentration in medium drug uptake in monolayers was much greater than in spheroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Interaction of CC-1065 and its analogues with mouse DNA and chromatin.

CC-1065 is a potent antitumor antibiotic which is cytotoxic to P388 and L1210 leukemia cells in vitro and in vivo. CC-1065 covalently binds to calf thymus DNA preferentially to adenine-thymine regions at N3 of adenine. Here, we compare CC-1065 interaction with P388-derived chromatin, DNA, and histones as measured by electronic absorption and circular dichroism. Two CC-1065 analogues (U-71,184 and its enantiomer, U-71,185) which show different biological activities from CC-1065 were also studied. The shape and temporal behavior of the induced circular dichroism curves generated by CC-1065 or its analogues bound to chromatin were similar to CC-1065 plus DNA. This suggested that CC-1065 and its analogues bind to the minor groove of chromatin DNA in a manner similar to calf thymus DNA. However, the binding of CC-1065 and its analogues to DNA induced a more intense circular dichroism band than binding to chromatin. The order of interaction for both chromatin and DNA was CC-1065 greater than U-71,184 greater than U-71,185. In contrast to the essentially irreversible binding to DNA after 24-h incubation, binding to chromatin was primarily a reversible interaction, the degree of reversibility being U-71,185 greater than U-71,184 = CC-1065. CC-1065 binds weakly and nonspecifically to histones.

Animals↗
PubMed1988

Effects of U-71,184 and several other CC-1065 analogues on cell survival and cell cycle of Chinese hamster ovary cells.

CC-1065 is a very potent antitumor antibiotic which selectively binds in the minor groove of DNA with alkylation at N-3 of adenine. Since therapeutic doses of CC-1065 caused delayed deaths in mice, analogues were synthesized, some of which had significant antitumor activity. The effects of several of these analogues on inhibition of CHO cell survival, cell progression, and their phase-specific toxicity are reported. CC-1065, U-66,664, U-66,819, U-66,694, and U-71,184 all have a left hand segment with an intact cyclopropyl group but have different tail segments. Lethality of these compounds after 2 h drug exposure was in the following order (50% lethal dose in nM in parentheses): CC-1065 (0.06) greater than U-71,184 (1.3) greater than U-66,694 (3.2) greater than U-68,819 (171) greater than U-66,664 (greater than 1200). In general, these compounds did not inhibit progression from G1 to S but slowed progression through S and blocked cells in G2-M. The phase-specific toxicity of U-71,184 and U-66,694 was different from that of CC-1065. CC-1065 was most cytotoxic to cells in M and early G1 and toxicity decreased as cells entered late G1 and S. In contrast, U-66,694 and U-71,184 were most toxic to cells in late G1. The biochemical and cellular effects of U-71,184 were then studied in detail since it was the most active among these analogues. After a 2-h exposure to 3 ng/ml U-71,184, 90% cell kill or growth inhibition was observed whereas 100 ng/ml was needed for similar inhibition of DNA and RNA synthesis. This discrepancy between the doses suggested that inhibition of nucleic acid synthesis may not be causally related to lethality. Further studies showed that when drug was removed after 2 h exposure, DNA synthesis continued to be inhibited whereas RNA and protein synthesis reached levels higher than the control. Therefore, it is likely that at cytotoxic doses the low level of inhibition of DNA synthesis combined with the stimulation of RNA and protein synthesis leads to unbalanced growth and cell death.

Animals↗
PubMed1988

Mutagenicity of the antitumor antibiotic CC-1065 and its analogues in mammalian (V79) cells and bacteria.

CC-1065 is a very potent antitumor antibiotic which binds in the minor groove of DNA with alkylation at N-3 of adenine. Since CC-1065 caused delayed deaths in mice at therapeutic doses, analogues were prepared whose antitumor and biochemical activities have been reported. In this study, the mutagenicity for V79 cells (6-thioguanine resistance) and Salmonella (histidine auxotrophy or azaguanine resistance) of selected analogues was compared to DNA-binding activity and the structure-activity relationship was determined. CC-1065, U-62,736, U-66,866, U-66,694, U-67,786, and U-68,415 all have an A segment with an intact cyclopropyl group and different B segments. The cyclopropyl group is absent from U-66,226 and U-63,360. Elimination of the cyclopropyl ring diminished the cytotoxic and mutagenic potency of the compounds such that U-63,360 was nearly three orders of magnitude less potent than CC-1065 in V79 cells. For the compounds with an intact cyclopropyl group, the order of cytotoxic and mutagenic potency (molar basis) in V79 cells generally correlated with binding to calf thymus DNA, and increased with the length of the B segment. Thus, the order of cytotoxicity was CC-1065 greater than U-68,415 greater than U-66,694 greater than U-66,866 greater than U-62,736. U-67,786 fell outside this pattern since it was more cytotoxic and mutagenic than U-66,694, although it was of a similar size and had similar DNA-binding activity. These results show that an electrophilic carbon afforded by an intact cyclopropyl group of this type is necessary but not sufficient to account for the high cytotoxic and mutagenic potency of CC-1065 and U-68,415. The size and characteristics of the B segment also affect the potency. At an equitoxic (10 or 50% lethal dose) dose, an inverse relationship exists between cytotoxic and mutagenic potency such that at the 50% lethal dose, the least cytotoxic compound (U-62,736) was more mutagenic than the most cytotoxic compound (CC-1065). We speculate that the more cytotoxic analogues are less mutagenic (at an equitoxic dose) because they may have greater structure-directed binding to less mutable DNA sites in the minor groove.

Animals↗
PubMed1987

Colcemid effects on B16 melanoma cell progression and aberrant mitotic division.

Mitotic cells selectively harvested after several h of colcemid treatment are routinely used to obtain synchronized cell cultures. DNA flow cytometry shows that when colcemid-treated B16 mitotic cells divide, they give rise to daughter cells in G1, some of which contain abnormal amounts of DNA. Two subpopulations appear to exist, one having a DNA content distribution expected of G1 cells, another having a mean DNA content about 0.8 of expected and an SD of DNA content more than 5 times expected. The effect was dependent on dose and duration of exposure to colcemid. Colcemid was more cytotoxic to cells in G2 + M than to G1 + S phase cells, and it slowed the progression of G1 cells to S. These effects of colcemid were much greater in aneuploid B16 melanoma cells than in pseudodiploid Chinese hamster ovary (CHO) cells.

Aneuploidy↗