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Biomedical subjects

A Krishan

Publications and source records attributed to A Krishan.

At least 37 records · Page 2Linked to original sources
PubMed1997

Drug retention, efflux, and resistance in tumor cells.

Multiple drug resistance (MDR) in tumor cells has been related to the expression of transport proteins which alter cellular drug transport and distribution. Three different genes (mdr, MRP, and LRP) and their products have been implicated in MDR. Several fluorescent dyes have been used to monitor the effect of these transport proteins on drug retention, as well as for screening of drugs which block drug efflux and thus enhance cellular drug retention and cytotoxicity. The present review summarizes current knowledge about MDR phenotypic markers and techniques available for study of MDR by flow cytometry.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Flow cytometric analysis of P-glycoprotein expression and drug efflux in human soft tissue and bone sarcomas.

Twenty-two fresh surgical specimens of human sarcomas (soft tissue and bone) from 20 patients were analyzed by flow cytometry for the expression of drug resistance-related P-glycoprotein (P-gp) and cellular daunorubicin (DNR) accumulation with or without the presence of DNR efflux blockers. Single-cell suspensions prepared from the tumor specimens were analyzed by dual-color flow cytometry after reaction with MRK-16 (anti-P-gp) and anti-CD45 (pan-leukocyte) antibodies. MRK-16 reactivity of tumor cells was evaluated after exclusion of CD45-positive cells by electronic gates. Parallel samples were incubated with DNR alone or in combination with DNR efflux blockers, verapamil (VPL), or dipyridamole (DPD) for determination of cellular DNR accumulation and the effect of the efflux blockers. Extensive heterogeneity was observed in both P-gp expression and DNR accumulation of the tumor specimens examined. Eight of the 22 tumor specimens had significant numbers of P-gp-positive cells. In three of the eight P-gp-positive tumors, cellular DNR accumulation was significantly increased by co-incubation with the efflux blockers VPL or DPD. These results indicate that both quantitative and functional analysis of P-gp expression may be essential in determining the cellular drug resistance phenotype of tumor cells and its correlation with therapeutic outcome.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Flow cytometric analysis of the multiple drug resistance phenotype.

Laser flow cytometry is increasingly used for quantitation of cellular fluorescent drug retention, effect of efflux blockers and for expression of drug resistance related cellular surface markers. Several intrinsic and extrinsic factors can affect the results obtained from drug retention functional assays and lead to artifacts. In the present study, we have used a panel of well-characterized parental and drug resistant cell lines, fluorochromes and efflux blockers to identify the possible sources of artefacts in flow cytometric analysis of the multiple drug resistance phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Heterogeneity of anthracycline retention and response to efflux blockers in human tumors.

Rapid cellular efflux of certain natural products used in cancer chemotherapy leads to reduced cytotoxicity and resistance. Multiple Drug Resistance (MDR) gene related glycoproteins are believed to act as the drug efflux pump. Several non-cancer therapy drugs (e.g., verapamil, phenothiazines) compete for the p-glycoprotein pump and thus can block efflux of a chemotherapeutic agent and overcome cellular resistance. Anthracyclines, such as adriamycin, are intrinsically fluorescent and thus their cellular retention can be determined by laser flow cytometry. This method has been used to study heterogeneity in cellular retention of anthracyclines and response to efflux blockers in human tumor cells. These studies have led to generation of clinical protocols where laser flow cytometry is used to monitor and overcome heterogeneity in drug retention of tumor cells from patients treated with a combination of adriamycin and efflux blockers.

Anthracyclines↗
PubMed1995

Expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes during cell cycle traverse.

The expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes was analyzed in cell cycle phase enriched populations of doxorubicin-resistant murine leukemic P388/R-84 cells. Flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation and staining with anti-BrdU antibodies was used to confirm the purity of cell cycle phase enriched populations obtained by centrifugal elutriation. Doxorubicin (DOX) and daunorubicin (DNR) accumulation was significantly lower in S-phase cells, and coincubation with verapamil (VPL) or chlorpromazine (CPZ) enhanced DOX and DNR accumulation more in S-phase than in G1- and G2/M-phase cells. While the cellular content of mdr-1 and topoisomerase II mRNAs changed, GST pi mRNA content remained constant during the cell cycle. S-phase cells had about 3-fold higher mdr-1 mRNA content than G1- and G2/M-phase cells. In G1 cells, P-glycoprotein expression, as determined by C219 monoclonal antibody, was 12% less than that of S and G2/M cells. Topoisomerase II mRNA content increased with the progression of cell cycle and peaked in G2/M cells. These observations suggest that cell cycle stage related changes in expression of drug resistance markers may have a major bearing on chemosensitivity of drug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Prognostic significance of DNA aneuploidy in diffuse malignant mesothelioma.

DNA ploidy of pepsin digested preparations of 48 paraffin-embedded specimens from 19 patients with histologically confirmed malignant mesothelioma was determined by laser flow cytometry. Eight of the 19 tumors (42%) were diploid and 11 (58%) were aneuploid. Of the aneuploid tumors, only one showed multiploidy. The median survival time of the patients with diploid tumors was 19, 16, and 14 months from the onset of symptoms, diagnosis, and treatment, respectively. The median survival in patients with aneuploid tumors was 8, 7, and 7 months from the onset of first symptoms, diagnosis, and treatment. Thus, patients with diploid tumors lived longer than patients with aneuploid tumors. These results suggest that DNA ploidy analysis may be of prognostic value in malignant mesothelioma.

Adult↗
PubMed1994

Doxorubicin retention and chemoresistance in human mesothelioma cell lines.

Eight cell lines were established from the pleural effusion of 4 patients with malignant mesothelioma. The most sensitive (FCCMES-4) and the most resistant (FCCMES-2) mesothelioma cell lines had IC50 of 0.66 and 1.85 microM for doxorubicin in clonogenic assays, respectively. In comparison with murine leukemic P388 cells, mesothelioma cell lines were 7.5- to 21-fold more resistant to doxorubicin. Co-incubation with verapamil significantly increased doxorubicin retention in one of the cell lines (FCCMES-2) expressing P-glycoprotein in 16.8% of the cells. These results indicate that doxorubicin resistance may be intrinsic in refractory mesothelioma patients and P-glycoprotein-mediated drug efflux may be involved in resistance of some of the mesotheliomas.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Phase I and pharmacokinetics studies of prochlorperazine 2-h i.v. infusion as a doxorubicin-efflux blocker.

In an earlier phase I study, we reported that the maximal tolerated dose (MTD) of prochlorperazine (PCZ) given as a 15-min i.v. infusion was 75 mg/m2. The highest peak plasma PCZ concentration achieved was 1100 ng/ml. The present study was conducted to determine if PCZ levels high enough to block doxorubicin (DOX) efflux in vitro could be achieved and sustained in vivo by increasing the duration of i.v. infusion from 15 min to 2 h. The treatment schedule consisted of i.v. prehydration with at least 500 ml normal saline (NS) and administration of a fixed standard dose of 60 mg/m2 DOX as an i.v. bolus over 15 min followed by i.v. doses of 75, 105, 135, or 180 mg/m2 PCZ in 250 ml NS over 2 h. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose. Toxicities attributable to PCZ were sedation, dryness of mouth, anxiety, akathisia, hypotension, cramps, and confusion. The MTD of PCZ was 180 mg/m2. Large interpatient variation in peak PCZ plasma levels (91-3215 ng/ml) was seen, with the plasma half-life (t1/2 alpha) being approximately 57 min in patients given 135-180 mg/m2 PCZ. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) were 350.1 +/- 183.8 1/m2, 260.7 +/- 142.7 l m2 h-1 and 1539 +/- 922 ng ml h-1, respectively, in patients given 180 mg/m2 PCZ and the respective values for patients receiving 135 mg/m2 were 48.9 +/- 23.76 l/m2, 33.2 +/- 2.62 l m2 h-1, and 4117 +/- 302 ng ml h-1. High PCZ plasma levels (> 600 ng/ml) were sustained in all patients treated with 135 mg/m2 PCZ for up to 24 h. DOX plasma elimination was biphasic at 135 and 180 mg/m2 PCZ, and a > 10-ng/ml DOX plasma level was maintained for 24 h. Partial responses were seen in three of six patients with malignant mesothelioma, in two of ten patients with non-small-cell lung carcinoma, and in the single patient with hepatoma. Our data show that PCZ can be safely given as a 2-h infusion at 135 mg/m2 with clinically manageable toxicities. The antitumor activity of the combination of DOX and PCZ needs to be confirmed in phase II trials.

Adult↗
PubMed1993

Thaliblastine, a plant alkaloid, circumvents multidrug resistance by direct binding to P-glycoprotein.

The effect of thaliblastine (TBL, NSC-68075), a plant alkaloid, in over-coming multidrug resistance was investigated in doxorubicin (ADR)-resistant murine leukemic P388/R-84 cells. In the soft agar clonogenic assay, a nontoxic concentration of TBL (2 microM) reduced the 50% inhibitory dose of ADR (1-h exposure) from 10.8 to 1.4 microM with a dose modification factor of 7.7. Continuous treatment of P388/R-84 cells with ADR and TBL for 24 h further lowered the 50% inhibitory dose from 3.5 to 0.07 microM, the resistance level being decreased from 233-fold in the absence of TBL to 4.7-fold in the presence of TBL as compared to the parental P388 cells. Although ADR or TBL individually had no detectable effects on cell cycle traverse, the combination of the two drugs caused a significant G2 block. Flow cytometric analysis showed that TBL enhanced ADR retention in P388/R-84 cells in a dose- and time-dependent manner. TBL partially blocked the photolabeling of P-glycoprotein with [3H]azidopine, and this blocking effect was further enhanced in combination with ADR. Our results indicate that TBL can reverse multidrug resistance by direct interaction with P-glycoprotein, thereby increasing cellular ADR retention.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Doxorubicin-induced DNA breaks, topoisomerase II activity and gene expression in human melanoma cells.

We have analyzed five human melanoma cell lines, displaying variable doxorubicin resistance (1- to 6-fold), for drug-induced DNA breaks, topoisomerase II activity and mRNA expression. Enhanced drug efflux was not the reason for doxorubicin resistance of these tumor cells although they overexpressed the transmembrane 170 kDa P-glycoprotein. Doxorubicin-induced DNA lesions (2-fold) and topoisomerase II activity (7-fold) were higher in HM-1 and G361 cells than in the less doxorubicin-sensitive NH and FCCM-9 cells. Topoisomerase II mRNA expression was also 2-fold higher in HM-1 and G361 cells. Doxorubicin-induced DNA breaks and topoisomerase II activity inversely correlated with the degree of doxorubicin sensitivity. Southern blot analysis showed variation in the hybridization pattern of topoisomerase II gene in doxorubicin-resistant cells when compared to sensitive cells. This study portrays the low doxorubicin sensitivity of NH and FCCM-9 cells as "atypical" and emphasizes the importance of DNA damage and topoisomerase II activity in cellular low doxorubicin resistance.

DNA Damage↗
PubMed1993

Doxorubicin resistance in human melanoma cells: MDR-1 and glutathione S-transferase pi gene expression.

Cellular drug resistance is believed to involve P-glycoprotein-related drug efflux as well as xenobiotic detoxification. In the present study, we analyzed five human melanoma cell lines with 1- to 6-fold doxorubicin resistance for doxorubicin retention and MDR-1 and GST pi gene expression. All the cell lines had high doxorubicin retention, and efflux blockers such as trifluoperazine and verapamil did not have a major effect on drug retention or cytotoxicity. Even though all the cell lines carried the MDR-1 and GST pi genes, gene amplification was not associated with drug resistance. Both laser flow cytometry and immunoperoxidase staining showed high expression of C-219 reactive P-glycoprotein in some of the resistant cells which was not accompanied by either high drug efflux or sensitivity to doxorubicin efflux blockers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Prochlorperazine as a doxorubicin-efflux blocker: phase I clinical and pharmacokinetics studies.

Doxorubicin (DOX) efflux in drug-resistant cells is blocked by phenothiazines such as trifluoperazine (TFP) and prochlorperazine (PCZ) in vitro. The present phase I study was conducted in 13 patients with advanced, incurable, nonhematologic tumors to determine whether PCZ plasma levels high enough to block DOX efflux could be achieved in vivo. The treatment schedule consisted of prehydration and i.v. administration of 15, 30, 50, and 75 mg/m2 PCZ followed by a standard dose of 60 mg/m2 DOX. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose used. Toxicities attributable to PCZ were sedation, dryness of the mouth, cramps, chills, and restlessness. The maximal tolerated dose (MTD) of PCZ in this schedule was 75 mg/m2. Pharmacokinetic analysis indicated a large interpatient variation in peak plasma PCZ levels that ranged from 95 to 1100 ng/ml. The three plasma half-lives of PCZ were: t1/2 alpha (+/- SE), 20.9 +/- 5.3 min; t1/2 beta, 1.8 +/- 0.3 h; and t1/2 gamma, 21.9 +/- 5.3 h. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) for PCZ were 2254 +/- 886 l/m2, 60.2 +/- 13.5 l m-2 h-1, and 1624 +/- 686 ng ml-1 h, respectively. DOX retention in tumor cells retrieved from patients during the course of therapy indicated the appearance of cells with enhanced DOX retention. The combination of DOX and high-dose i.v. PCZ appeared to be safe, well tolerated, and active in non-small-cell lung carcinoma.

Aged↗
PubMed1993

MDR-1 gene expression, anthracycline retention and cytotoxicity in human lung-tumor cells from refractory patients.

Lung-tumor cells from pleural effusion of four refractory patients and in cell lines established from them were analyzed for anthracycline retention, cytotoxicity, and MDR-1 gene and P-glycoprotein expression. Murine leukemic P388 and doxorubicin-resistant P388/R84 lines were used as controls. The 50% growth-inhibitory concentration (IC50) for doxorubicin among lung-tumor lines varied from 0.16 to 0.31 microM in soft agar. Heterogeneity in doxorubicin or daunorubicin retention and response to the efflux-blocking action of 25 microM prochlorperazine was noted in pleural effusion of FCCL-1, -4, and -8. Among the cell lines established, an efflux-blocking effect in a subpopulation was noticed only in FCCL-1 and -4. Although the MDR-1 gene was present in all cell lines, including P388, its expression was pronounced only in P388/R84 and FCCL-1. In situ hybridization of antisense RNA probe to tumor cells showed high heterogeneity for MDR-1 message in the human lung-tumor cells as compared with the murine cells. Northern and slot blot hybridization confirmed in situ hybridization in lines with high levels of MDR-1 expression. The synthesis of MDR-1 mRNA and P-glycoprotein in tumor lines was correlated. The results suggest that because of extensive tumor-cell heterogeneity in human tumors, monitoring of MDR expression by in situ hybridization, quantitation of P-glycoprotein content by laser flow cytometry (and/or immunohistochemical methods), and drug efflux (by laser flow cytometry) may be the best ways to monitor multidrug resistance in human tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Flow cytometric monitoring of glutathione content and anthracycline retention in tumor cells.

We have used an enzymatic (spectro-photometric) and a flow cytometric (GSH-MBCL) method to compare the glutathione (GSH) content of doxorubicin sensitive (P388) and resistant (P388/R-84) murine leukemic and human lung cancer cells. The flow cytometric analysis revealed that GSH-MBCL conjugate formation was dependent on glutathione-S-transferase (GST) activity. The human solid tumor cell lines exhibited extensive heterogeneity, high GSH content, and GST activity. In contrast to the enzymatic method, the flow cytometric method did not accurately reflect the 95% reduction in GSH content of cells treated for 24 h with 100 microM BSO. Possible reaction of MBCL with other sulfhydryl groups (other than GSH) in BSO-treated cells may be responsible for this discordance. We have also shown the feasibility of using dual parameter flow cytometry to monitor cellular anthracycline (daunorubicin) retention and GSH-MBCL conjugate fluorescence in human tumor cells. These two parameters, which measure drug retention and cellular detoxification, are believed to be the important determinants of chemoresistance in tumor cells.

Adenocarcinoma↗
PubMed1991

Comparison of three commercially available antibodies for flow cytometric monitoring of P-glycoprotein expression in tumor cells.

Cellular drug resistance to natural products is often due to the presence of an efflux pump which reduces intracellular drug content and chemosensitivity. A 170 kD cell surface resident P-glycoprotein is believed to act as the efflux pump. In the present report, we have compared three commercially available antibodies C-219, JSB-1, and mdr(Ab-1) for use in flow cytometric detection of P-glycoprotein positive cells. Our data show that C219 gives uniformly good results in a variety of murine and human tumor cell lines for detection of P-glycoprotein positive cells. We have also compared data of C219 stained cells analyzed in parallel on a flow cytometer equipped with a small laser (15 mW) and a large laser (5 watt) cell sorter. Data obtained on these two instruments are comparable. A staining protocol and data on dual staining of cells for DNA content by propidium iodide and P-glycoprotein expression after FITC labeling are also presented.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Cytochrome P450 reductase, antioxidant enzymes and cellular resistance to doxorubicin.

Our data suggest that DOX resistance in P388/R-84 cells may result, at least in part, from reduced free radical formation by both suppression of flavin reductase(s) and overexpression of certain antioxidant enzymes such as GSH peroxidase and catalase. In addition, our results, in conjunction with other studies, indicate that flavin reductase(s) and antioxidant enzymes are differentially altered in cancer cells with acquired or de novo resistance to DOX. Further studies are needed, however, to elucidate the mechanism(s) by which the gene expression of these enzymes is regulated in drug-sensitive and -resistant cells.

Animals↗