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Biomedical subjects

A Krishan

Publications and source records attributed to A Krishan.

At least 55 records · Page 3Linked to original sources
PubMed1990

Differential expression of glutathione S-transferase, glutathione peroxidase and glutathione reductase in normal and malignant human breast tissues.

In the present study we have compared the levels of glutathione (GSH) S-transferase, GSH peroxidase and GSH reductase in human breast tumors and adjacent normal tissues obtained from the same individuals. We have also quantitated GST pi type antigen in these samples by western blotting. GST pi activity towards 1-chloro-2,4-dinitrobenzene was found to be elevated in tumors from three out of six patients (patient nos. 2, 4 and 5), whereas this activity was suppressed in tumor from patient no. 1. Results of Western blotting using antibodies raised against GST pi of human placenta were in agreement with the GST activity data. GSH peroxidase activity with cumene hydroperoxide as substrate was found to be elevated in four tumor samples (patient nos. 2, 4, 5, and 6) but suppressed in tumor from patient no. 1. On the other hand, GSH reductase activity was elevated in three samples (patients nos. 2, 4 and 5) and downregulated in the remaining three samples (patients nos. 1, 3 and 6). These results indicate that GSH-related enzymes are differentially altered in human breast tumors and GST pi type isoenzyme(s), unlike certain other human carcinomas such as colonic, are not uniformly elevated in human breast tumors.

Adult↗
PubMed1990

Anthracycline resistance in murine leukemic P388 cells. Role of drug efflux and glutathione related enzymes.

Energy-dependent drug efflux is a major factor in cellular resistance of P388/R84 mouse leukemic cells to anthracyclines such as doxorubicin (DOX), and blocking of efflux increases sensitivity. However, efflux does not play a significant role in resistance to N-trifluoroacetyladriamycin-14-valerate (AD 32), a DOX analog. Since drug efflux alone cannot account for resistance to anthracyclines, we have, in the present study, measured cellular glutathione (GSH) content and activity of GSH cycle related enzymes to determine their role in resistance. Cellular GSH content was similar in DOX-sensitive and -resistant mouse leukemic cells (P388 and P388/R84). GSH peroxidase, glucose-6-phosphate dehydrogenase and glutathione reductase activities were 1.36-, 1.58- and 1.14-fold higher in P388/R84 cells. Incubation of P388/R84 cells with 100 microM buthionine-S,R-sulfoximine (BSO) for 24 hr reduced cellular GSH content to 6% of control and reduced their resistance to DOX [dose modification factor (DMF) 3.9]. GSH depletion had no significant effect on the cytotoxicity of AD 32 (DMF 1.5). Exposure of P388/R84 cells to BSO (for GSH depletion) and trifluoperazine (for efflux blocking) further reduced their resistance to DOX (DMF 14). These results indicate that DOX resistance in P388/R84 cells is multifactorial and that changes in GSH cycle related enzymes such as GSH peroxidase may also contribute to their resistance.

Animals↗
PubMed1989

Glutathione S-transferases and glutathione peroxidases in doxorubicin-resistant murine leukemic P388 cells.

Energy-dependent rapid drug efflux is believed to be a major factor in cellular resistance to doxorubicin (DOX). However, several recent studies have demonstrated that cellular DOX retention alone does not always correlate with its cytotoxicity and suggest that mechanisms other than rapid drug efflux may also be important. In the present study, we have compared glutathione (GSH) S-transferase (GST), selenium-dependent GSH peroxidase and selenium-independent GSH peroxidase II activities in DOX-sensitive (P388/S) and resistant (P388/R) mouse leukemic cells. The GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) was markedly higher in P388/R cells compared to P388/S cells. Purification of GST by GSH-affinity chromatography from an equal number of P388/S and P388/R cells revealed an increased amount of GST protein in P388/R cells. Immunological studies indicated that alpha and pi type GST isoenzymes were 1.27- and 2.2-fold higher, respectively, in P388/R cells compared to P388/S cells. Selenium-dependent GSH peroxidase activity was similar in both the cell lines, whereas selenium-independent GSH peroxidase II activity was approximately 1.36-fold higher in P388/R cells compared to P388/S cells. These results suggest that increased GSH peroxidase II activity in P388/R cells may contribute to cellular DOX resistance by enhancing free radical detoxification in this cell line.

Animals↗
PubMed1989

Class I MHC molecules and doxorubicin resistant P388 murine leukemic cells.

The major histocompatibility complex (MHC) encodes cell surface and secreted products involved in immune regulation and function. We have measured Class I MHC expression on doxorubicin (DOX) sensitive (P388/S) and resistant (P388/R84 and R84A) murine tumor cells using a monoclonal antibody to H-2d molecules. The present report shows a correlation between increased Class I MHC (H-2d) expression and drug resistance in P388 cells. Exposure of P388 cells to H-2d antibody diminished H-2d expression, whereas, treatment with murine recombinant gamma-interferon increased H-2d expression. Neither treatment significantly altered cellular DOX resistance or chemosensitivity. Thus, H-2d molecules can be used to identify DOX resistant P388 tumor cells but are probably not involved functionally in drug resistance.

Animals↗
PubMed1988

Anthracycline-induced DNA breaks and resealing in doxorubicin-resistant murine leukemic P388 cells.

Energy-dependent drug efflux is believed to be a major factor in cellular resistance to doxorubicin (DOX). However, recent studies have shown that decreased retention alone cannot account for anthracycline resistance, and possibly other factors, such as drug metabolism, free radical scavengers, and altered DNA damage/repair, may be involved. We have measured DOX-induced DNA damage and its repair in P388 cells sensitive (P388/S) and resistant (P388/R) to DOX. Our studies show 2- to 5-fold less DNA damage, measured as protein-associated single-strand DNA breaks, in P388/R cells when compared to similarly treated P388/S cells. The repair of DNA in whole cells, expressed as percent DNA rejoined, was complete in 4 hr in P388/R, whereas no repair was seen in P388/S cells until 20 hr. No difference in repair of DNA lesions was observed when nuclei were used in repair experiments. The absence of repair in sensitive whole cells may be due to high retention or slow drug efflux. Increase of cellular DOX retention by exposure of cells to trifluoperazine (TFP) or verapamil (VPL) did not result in the increase of DNA damage in P388/R cells. DOX analogs, N-trifluoroacetyladriamycin-14-valerate (AD 32), 4'-O-tetrahydropyranyladriamycin (THP-adriamycin), and N-benzyladriamycin-14-valerate (AD 198), induced 2- to 4-fold more DNA damage than DOX in resistant cells. There was no difference in the poly(ADP-ribose) synthesis of P388/S and P388/R cells exposed to DOX or AD 32. Since ADP-ribose polymer synthesis is associated with free radical-induced DNA damage and is indicative of DNA repair by an excision-repair mechanism, data from these studies suggest that DNA breaks in anthracycline-exposed cells may not be due to free radical production but rather to other mechanisms, such as inhibition of DNA topoisomerase II activity. The present studies, in addition to emphasizing the role of DNA damage in resistance, also underscore the relative importance of DNA topoisomerase II function in anthracycline cytotoxicity.

Animals↗
PubMed1988

Electrophoretic mobility studies on doxorubicin-resistant and -sensitive murine P388 leukemic cells.

The electrokinetic properties of doxorubicin (DOX)-resistant (P388/R) and -sensitive (P388/S) murine leukemic cells were studied in a free-flow electrophoresis (FFE) system. The electrophoretic mobilities (EM) of P388/S and P388/R cells were 1.07 and 1.35 x 10(-4) cm2 V-1 s-1, respectively, suggesting a higher net negative charge on the P388/R cells. Neuraminidase treatment decreased the EM of both the P388/S and P388/R cells by 15-20% but had no effect on cellular doxorubicin retention. Total and cell surface sialic acid contents were similar in both the cell lines. Our studies show that no direct correlations may exist among surface charge, cell surface sialic acid content, and doxorubicin retention in DOX-resistant and -sensitive P388 cells; however, differences in cell surface charge between these cell types were used to separate them by preparative FFE.

Animals↗
PubMed1987

Patterns of anthracycline retention modulation in human tumor cells.

Laser excitation of cellular doxorubicin and daunomycin content (with or without incubation in the presence of efflux blockers such as phenothiazines or verapamil) was studied in cells from leukemic peripheral blood, bone marrow aspirates, and ascites and pleural fluid of solid tumor patients. Selected examples are presented to show that heterogeneity in cellular anthracycline retention as well as sensitivity to efflux modulators is seen in human tumor cells. Several tumor subpopulations differing in their cellular retention of anthracyclines or sensitivity to modulators were seen. In serial tumor samples from patients (pre- and post-treatment with anthracycline-containing protocols), initial drug retention and sensitivity to efflux modulators was followed by lack of drug retention and insensitivity to modulators. The present study shows that in view of the variables encountered in drug-retention characteristics and sensitivity to efflux modulators, one needs to screen tumor samples before recommending use of any particular transport modulator to enhance drug retention and sensitivity of drug-resistant cells.

Bone Marrow↗
PubMed1987

Effect of drug efflux blockers on vital staining of cellular DNA with Hoechst 33342.

The present study shows that staining of certain live cells, e.g., adriamycin-resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one-third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug-sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells. These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.

Animals↗
PubMed1986

Reversal of intrinsic resistance to adriamycin in normal cells by verapamil.

Intracellular accumulation of adriamycin (ADM) was found to be increased in human breast carcinoma cell line (MCF-7) and cardiac-muscle cells as compared to an epithelial cell line derived from normal monkey kidney (CV-1) and non-muscle cells (fibroblasts) derived from the heart. This increase correlates with greater sensitivity of the carcinoma cell line to ADM. In CV-1, efflux of ADM contributes to low drug accumulation which correlates with the intrinsic drug resistance of the cells. Blockage of ADM efflux in this resistant cell line and subsequent increases in intracellular accumulation and sensitivity can be achieved by co-treatment with verapamil. ADM accumulation and sensitivity in MCF-7 however cannot be significantly increased by verapamil. These data demonstrate selectively for ADM in human breast carcinoma cell line, MCF-7 and cardiac-muscle cells in vitro. The reversal by verapamil of the normal cells' natural resistance, may have importance in the clinical use of verapamil as a resistance modulating agent.

Cell Line↗
PubMed1986

Membrane-associated proteins of adriamycin sensitive and resistant murine leukemic P388 cells.

We have isolated an 84-fold adriamycin resistant subline, P388/R84, from mouse leukemia P388 cells by serial cultivation in methylcellulose in the presence of increasing drug concentrations. Electrophoresis of detergent soluble fractions of radiolabeled sensitive and resistant cells suggested marked alterations in the protein fractions of 160, 100, 60, 45, and 30 kd. In resistant clones labeled with 125I an increase in 160 and 100 kd proteins was accompanied by concomitant reduction in the 60, 45, and 30 kd proteins. In 35S methionine-labeled resistant cells, similar increases in the 160 and 100 kd components were observed but in contrast to 125I-labeled cells the 30 kd component was also higher. Alterations in surface proteins were confirmed in experiments where the cell extracts were adsorbed to concanavalin A polymers and extracted with 0.26 M methyl-alpha-D-mannopyranoside. Our data confirm earlier reported observations on cell-surface protein changes in cells resistant to anthracyclines and alkaloids.

Animals↗
PubMed1986

Auromomycin-induced DNA damage and repair in human leukemic lymphoblasts (CCRF-CEM cells).

An alkaline elution procedure was used to study the nature of DNA damage induced by auromomycin, an antitumor protein, in human leukemic lymphoblasts (CCRF-CEM cells). The filter elution of drug-treated cells at pH 12.2 and 9.6 showed induction of both single and double strand DNA breaks. The DNA strand scission activities were linear in relation to drug concentration. The frequency of single strand breaks was higher than that of the double strand breaks. Protein-associated DNA single strand breaks were also detected in alkaline elution of drug-treated cells when a proteinase K digestion step was included in the assay protocol. The auromomycin-induced single strand breaks were repaired to almost completion within 8 h of postincubation of DNA-damaged cells whereas the repair of double strand breaks was not detected.

Anti-Bacterial Agents↗
PubMed1986

Calcium, calmodulin, and protein content of adriamycin-resistant and -sensitive murine leukemic cells.

Anticalmodulin and calcium channel blockers have been shown to reverse Adriamycin resistance by reducing the drug efflux from resistant cells. Since cellular calcium and calmodulin levels are probably related to these effects, we have measured the total, membrane-bound, and intracellular calcium levels in Adriamycin-resistant (P388/R) and -sensitive (P388/S) leukemia cells. In P388/R cells, total calcium was approximately 1.4-fold higher than that of P388/S cells. Membrane-bound and intracellular calcium levels were also higher in P388/R cells. No major difference was observed in the calmodulin content of these cells. The P388/R cells had a higher (approximately 1.4-fold) protein content. When calculated on the basis of per unit protein, P388/S and P388/R cells had similar total calcium but a higher intracellular free calcium and calmodulin content in P388/S cells. Thus our studies suggested that the lower drug efflux and increased drug retention in P388/R cells may not be related to calcium and calmodulin levels but may be due to some other membrane-related factors.

Aminoquinolines↗
PubMed1986

Flow cytometric monitoring of cellular anthracycline accumulation in murine leukemic cells.

Cellular accumulation of daunorubicin (DNR), N-trifluoroacetyl-adriamycin-14-valerate, and THP-Adriamycin (THP-ADR) in doxorubicin sensitive and resistant murine leukemic P388 cells was studied with laser excited flow cytometry. Appearance of DNR fluorescence in P388/S cells was rapid in contrast to that of P388/R cells. A comparison of P388/S and P388/R cells incubated for 20-30 min showed that DNR fluorescence in P388/R cells was one-sixth that of P388/S cells. In contrast, the difference between fluorescence value of P388/S and P388/R cells similarly incubated with N-trifluoroacetyl-adriamycin-14-valerate or THP-ADR was less than 2-fold. Chlorpromazine, verapamil, and trifluoperazine increased the cellular accumulation and cytotoxicity of DNR and THP-ADR but had no major effect on N-trifluoroacetyl-adriamycin-14-valerate fluorescence or cytotoxicity in P388/R cells. Fluorometric and soft agar assays confirmed the data on the effect of these modulators on drug accumulation obtained by the more rapid method of laser flow cytometry.

Animals↗
PubMed1985

Cell surface tubulin in leukemic cells: molecular structure, surface binding, turnover, cell cycle expression, and origin.

We report here new characteristics of cell surface tubulin from a human leukemia cell line. These cells (CEM cells) possess tubulin that is readily iodinated on the surface of living cells, turns over at a rate identical to that of other surface proteins, and is present throughout the cell cycle. When removed with trypsin, it rapidly returns to the surface. Peptide mapping of iodinated surface tubulin indicates that it possesses a similar, but not identical, primary structure to total CEM and rat brain tubulin. Living CEM cells are able to bind specifically a subfraction of CEM tubulin from metabolically labeled high speed supernatants of lysed CEM cells. Surface tubulin is more basic than the total tubulin pool. The binding, which is saturable, is inhibited by unlabeled CEM high speed supernatants but not by excess thrice-cycled rat or bovine brain tubulin. Surface tubulin is also shown to bind to living nontransformed normal rat kidney cells but not to normal, circulating, mononuclear white cells. Activated lymphocytes produce a tubulin that binds to CEM cells. Since CEM tubulin was detected in the media of 6-h cultures of CEM cells, we must conclude that at least some of the surface tubulin comes from the media. We further conclude that these leukemic cells produce an unusual tubulin that may bind specifically to any membrane. The presence of iodinatable surface tubulin, however, appears to require both the production of a unique tubulin and the presence of a "receptor-like" surface binding component.

Animals↗