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Biomedical subjects

U Bhatia

Publications and source records attributed to U Bhatia.

7 recordsLinked to original sources
PubMed1995

Induction of apoptosis by 5-azacytidine: drug concentration-dependent differences in cell cycle specificity.

There are conflicting data in the literature as to whether cytotoxicity of the cytidine antimetabolite 5'-azacytidine (AZC) is a consequence of its incorporation into RNA, DNA, or both. Because apoptosis appears to be the predominant mode of tumor cell death after treatment with most antitumor drugs, and in the case of some drugs, the proclivity of the cell to undergo apoptosis varies depending on the cell cycle position, this study was aimed toward elucidating whether induction of apoptosis by AZC is cell cycle phase specific. Human promyelocytic leukemic HL-60 cells were treated with varying concentrations of AZC, and flow cytometric methods that identify apoptotic cels and provide information about the cell cycle distribution of the apoptotic and nonapoptotic cell populations were used. At 2-6 microM concentrations of AZC, the cells in the G1 phase preferentially underwent apoptosis, whereas the cells in G2-M were particularly resistant. Although incorporation of bromouridine into RNA was suppressed at that low AZC concentration, the rate of 5'-bromo-2-deoxyuridine incorporation into DNA was not significantly affected. At an AZC concentration of 8-40 microM, no cell cycle phase specificity in induction of apoptosis was apparent, but both the rate of 5'-bromo-2-deoxyuridine incorporation into DNA and bromouridine into RNA were reduced in proportion to drug concentration. The data suggest that the mechanism of cell killing by AZC may be different, depending on its concentration. Namely, whereas incorporation of AZC into RNA may play a predominant role in the induction of cytotoxicity of G1 cells at low drug concentrations, the perturbation of both RNA and DNA metabolism may be responsible for triggering cell death in the G1 and S phases, as is seen at higher concentrations of this antimetabolite.

Apoptosis↗
PubMed1995

Expression of cyclins A, D2 and D3 in individual normal mitogen stimulated lymphocytes and in MOLT-4 leukemic cells analyzed by multiparameter flow cytometry.

Cyclins are regulatory subunits of the cyclin dependent kinases (CDKs), the enzymes that drive the cell through the respective phases and check-points of the cell cycle. The expression of cyclins in non-tumor cells, regulated by timely induction of their synthesis and proteolysis, is scheduled, occurring at discrete periods of the cell cycle. Using multiparameter flow cytometry we have recently observed that expression of cyclins B1 and E in individual normal lymphocytes mitogenically stimulated by phytohemagglutinin (PHA) and lymphocytic leukemic MOLT-4 cells was similar, restricted to particular phases of the cycle: cyclin B1 was detected only in G2+M- and cyclin E in late G1 and early S-phase cells. In the present study we have measured the expression of cyclins A, D2 and D3 in these cells. The presence of cyclin A was restricted to late S and G2 phases, both in the case of lymphocytes and of MOLT-4 cells. Over 95% of the non-stimulated lymphocytes were both cyclin D2 and D3 negative. Mitogenic stimulation with PHA-induced expression of cyclins D2 and D3 in over 50% cells, which corresponds to the percentage of cells that respond to this mitogen in cultures. Expression of these proteins peaked between 8 and 24 h after addition of PHA, and then decreased at the time of cell entrance to S. During exponential growth (48-72 h after stimulation with PHA) expression of the D-type cyclins was diminished: only between 5-10% of the lymphocytes had levels of cyclin D3 as high as G1 cells between 8-24 h after PHA stimulation. Populations of proliferating lymphocytes and MOLT-4 cells were very heterogeneous in terms of expression of D-type cyclins by individual cells. While expression of cyclin D2 in exponentially growing MOLT-4 cells was similar to that of proliferating lymphocytes, the percent of cells expressing cyclin D3 as well as the degree of expression, was higher in MOLT-4 cells, regardless of the phase of the cycle. These results, with our earlier observations of the untimely expression of cyclins B1 and E in several other tumor lines, suggest that altered expression of cyclins may be a frequent feature of malignancy.

Analysis of Variance↗
PubMed1995

Induction of cell differentiation potentiates apoptosis triggered by prior exposure to DNA-damaging drugs.

At the end of their life span, differentiated cells die by apoptosis. Subsets of cells also die, in some cell systems, shortly after exposure to differentiating agents. This suggests that early during differentiation the cells may undergo "priming," during which synthesis and/or activation and accumulation of effectors of apoptosis occurs. The objective of the present study was to test the hypothesis that the signal for apoptosis provided by DNA-damaging drugs given prior to induction of differentiation will be more effective in triggering apoptosis than when given following induction of differentiation. Human promyelocytic HL-60 cells were treated with the topoisomerase I inhibitor camptothecin, the alkylating agent nitrogen mustard, or 5'-azacytidine, an antimetabolite affecting predominantly RNA metabolism. Following drug removal, the cells were postincubated with n-butyrate, which induces differentiation of HL-60 cells along the monocytic pathway, or with all-trans-retinoic acid, which triggers myelocytic differentiation. Multiparameter flow cytometry using two different methods of analysis of apoptosis-associated DNA breakage in situ, as well as evaluation of cell morphology and DNA gel electrophoresis, were used to ascertain the mode of cell death. Increases of 100-200% in the percentage of apoptotic cells were seen when cells were first treated with camptothecin or nitrogen mustard, followed by n-butyrate or retinoic acid, compared to the combined percentage of apoptotic cells when these agents were used individually.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimetabolites, Antineoplastic↗
PubMed1995

Induction of apoptosis and cell cycle-specific change in expression of p53 in normal lymphocytes and MOLT-4 leukemic cells by nitrogen mustard.

DNA damage in the cell activates expression of the p53 tumor suppressor gene, whose role is associated with cell arrest in G1 or apoptosis. The aim of this study was to examine the cell cycle position-related changes in expression of p53, as well as induction of apoptosis, in mitogen-stimulated normal human lymphocytes and in human leukemic MOLT-4 cells (which express mutated p53), following DNA damage by the alkylating agent nitrogen mustard. Measurement of p53 expression and DNA content by flow cytometry followed by bivariate analysis of the data made it possible to correlate the drug-induced changes in p53 expression in individual cells with their cell cycle position without the need for cell synchronization. Expression of p53 was detected immunocytochemically using the AB-6 mAb, which reacts with the product of the wild-type p53 tumor suppressor gene and with most of its mutated forms. Exposure of normal lymphocytes to 5 microM nitrogen mustard caused their arrest in G1, an increase in p53 expression which was maximal in such cells, and significant apoptosis in cells located beyond the arrest point (S and G2 + M cells). In contrast, neither arrest in G1 nor significant apoptosis of MOLT-4 cells was seen after administration of either 0.5 or 5 microM nitrogen mustard for up to 24 h, although the drug reduced the rate of cell progression in the S-phase at both concentrations. Expression of p53 was highest for S and G2 + M MOLT-4 cells in response to the nitrogen mustard. Although a severalfold lower level of p53 was detected in lymphocytes compared to MOLT-4 cells prior to drug treatment, the relative increase in p53 expression in response to the drug was 2-fold higher in lymphocytes. These data suggest that DNA damage caused by nitrogen mustard provides a signal that results in stabilization of wild-type p53, preferentially in G1 cells, causes cell arrest in G1, and induces apoptosis of the cells that either were in the S-phase at the time of drug administration and/or escaped G1 arrest. The increase in expression of mutated p53, in response to DNA damage, is unrelated to the cell cycle position, and neither provides a signal for cell arrest in G1 nor a trigger for immediate apoptosis.

Apoptosis↗