Biomedical subjects

Michael M Gottesman

Publications and source records attributed to Michael M Gottesman.

45 records · Page 3Linked to original sources

PubMed2002

Drug selection with paclitaxel restores expression of linked IL-2 receptor gamma -chain and multidrug resistance (MDR1) transgenes in canine bone marrow.

Unstable expression of transferred genes is a major obstacle to successful gene therapy of hematopoietic diseases. We have investigated in a canine large-animal model whether expression of transduced genes can be recovered in vivo. Mixed-breed dogs had undergone autologous bone marrow transplantation (BMT) with stem cell factor and granulocyte-colony-stimulating factor-mobilized retrovirally marked hematopoietic cells. The bicistronic retroviral vector construct allowed for coexpression of MDR1 and human IL-2 receptor common gamma-chain cDNAs. The latter gene is deficient in X-linked severe combined immunodeficiency. After initial high-level expression, P-glycoprotein and the gamma-chain were undetectable in blood and bone marrow 17 months post-BMT. Six months later, one dog was treated i.v. with 125 mg/m2 paclitaxel. Three administrations restored expression of the two linked genes to high levels in blood and bone marrow. Two dogs treated with higher paclitaxel doses died from myelosuppression after the first administration. As determined by flow cytometry, both genes were expressed in granulocytes, monocytes, and lymphocytes of the surviving animal. PCR analysis of DNA from peripheral blood confirmed that the retroviral cDNA was increased after paclitaxel treatment, suggesting enrichment of transduced cells. P-glycoprotein was detectable for more than 1 year after cessation of paclitaxel. Repeated analyses of blood and bone marrow aspirates gave no indication of hematopoietic disturbance after BMT with transduced cells and paclitaxel treatment. In summary, we have shown that with the use of a drug-selectable marker gene, chemotherapy can select for cells that express an otherwise nonselected therapeutic gene in blood and bone marrow.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

In vitro-packaged SV40 pseudovirions as highly efficient vectors for gene transfer.

A procedure for in vitro packaging of plasmid DNA in recombinant SV40 capsid proteins was developed by Sandalon et al. (1997). Here, we report the highly efficient transduction into different human, murine and monkey cell lines using a scaled-up protocol for producing SV40 pseudovirions, packaged in vitro, carrying the human multidrug-resistance gene MDR1 encoding P-glycoprotein (P-gp) or the green fluorescent protein reporter gene (GFP) under control of SV40 and cytomegalovirus (CMV) promoters. The percentage of expressing cells was proportional to the number of transducing particles, with close to 100% of cells transduced at optimal ratios of transducing particles to cells. The ability to confer multidrug resistance was evaluated by measuring dye efflux and cell-surface expression in infected cells. The relative level of expression of P-gp driven by the different promoters varied among different cell lines. In human lymphoblastoid cells, which express high levels of major histocompatibility complex (MHC) class I (a surface receptor for SV40), constructs that carry an intron yield the highest expression. Our experiments further demonstrate that MDR1 and GFP expression driven by these promoters is transient; however, transduced cells remain MDR1-positive if selected in colchicine. Thus, the SV40 vectors are well suited to situations in which only short-term expression is required or expression is selected, such as for bone marrow protection during chemotherapy.

Animals↗

PubMed2002

Multidrug resistance in cancer: role of ATP-dependent transporters.

Chemotherapeutics are the most effective treatment for metastatic tumours. However, the ability of cancer cells to become simultaneously resistant to different drugs--a trait known as multidrug resistance--remains a significant impediment to successful chemotherapy. Three decades of multidrug-resistance research have identified a myriad of ways in which cancer cells can elude chemotherapy, and it has become apparent that resistance exists against every effective drug, even our newest agents. Therefore, the ability to predict and circumvent drug resistance is likely to improve chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Functional characterization of coding polymorphisms in the human MDR1 gene using a vaccinia virus expression system.

The human MDR1-encoded transporter is a 170-kDa plasma membrane glycoprotein [P-glycoprotein (P-gp)] capable of binding and energy-dependent extrusion of structurally diverse organic compounds and drugs. P-gp seems to play a significant role in uptake, distribution, and excretion of many different drugs. To determine whether common polymorphic forms of P-gp are likely to alter function of P-gp, we characterized five known MDR1 coding polymorphisms (N21D, F103L, S400N, A893S, and A998T) using a vaccinia virus-based transient expression system. Cell surface expression of wild-type P-gp was time-dependent over a time course of 5.5 to 34.5 h; highest expression was obtained by 22 to 26.5 h after infection/transfection, indicating that a semiquantitative assay for P-gp expression levels was possible. HeLa cells stained with the P-gp specific monoclonal antibodies MRK-16 and Western blots probed with C219 revealed similar cell surface expression for the polymorphisms and for wild-type protein. Time-dependent P-gp pump function maximal at 22 h after infection/transfection was demonstrated for the following MDR1 fluorescence substrates: 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, succinimidyl ester (bodipy-FL)-verapamil, bodipy-FL-vinblastine, calcein-AM, bodipy-FL-prazosin, bisantrene, and bodipy-FL-forskolin, but not for daunorubicin. Transport studies of all tested substrates indicated that the substrate specificity of the pump was not substantially affected by any of the tested polymorphisms. Cell surface expression and function of double mutants including the more common polymorphisms (N21D-S400N, N21D-A893S, and S400N-A893S) showed no differences from wild-type. These results demonstrate that the common MDR1 coding polymorphisms result in P-gps with a cell surface distribution and function similar to wild-type P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Mechanisms of cancer drug resistance.

The design of cancer chemotherapy has become increasingly sophisticated, yet there is no cancer treatment that is 100% effective against disseminated cancer. Resistance to treatment with anticancer drugs results from a variety of factors including individual variations in patients and somatic cell genetic differences in tumors, even those from the same tissue of origin. Frequently resistance is intrinsic to the cancer, but as therapy becomes more and more effective, acquired resistance has also become common. The most common reason for acquisition of resistance to a broad range of anticancer drugs is expression of one or more energy-dependent transporters that detect and eject anticancer drugs from cells, but other mechanisms of resistance including insensitivity to drug-induced apoptosis and induction of drug-detoxifying mechanisms probably play an important role in acquired anticancer drug resistance. Studies on mechanisms of cancer drug resistance have yielded important information about how to circumvent this resistance to improve cancer chemotherapy and have implications for pharmacokinetics of many commonly used drugs.

Animals↗

PubMedDate not supplied

Comparison of drug transporter levels in normal colon, colon cancer, and Caco-2 cells: impact on drug disposition and discovery.

A critical step in early phase drug development is the determination of oral bioavailability. In part, the ability to predict whether a drug will be effectively transported across the gastrointestinal mucosa can be estimated from the physicochemical properties of the compound. Although advancements through rational drug design have more correctly predicted bioavailability, considerable variability remains to be explained. Transporter expression throughout the gastrointestinal tract may explain much of this variation. ATP-binding cassette (ABC) transporters were the first family of transporters identified to modify bioavailability. More recently, the solute carrier family has also been shown to alter the pharmacokinetic profile of drugs. Currently, the Caco-2 human colon carcinoma cell line is often used by the pharmaceutical industry to evaluate intestinal absorption of drugs; however, in vivo/in vitro permeabilities with carrier mediated drugs do not correlate well, suggesting that Caco-2 transporter expression varies from that of the small intestine. With this is mind, we integrated U133A GeneChip expression data from the NCBIs Gene Expression Omnibus (GEO) collection and then compared the expression pattern of Caco-2 cells to normal colon to determine if the Caco-2 cell line is a reliable model for colonic delivery. Furthermore, transporter expression of Caco-2 cells was compared to that of human colon tumors to assess whether this cell line could be useful to predict drug absorption for colon cancer. Our analysis shows that the expression pattern for Caco-2 cells closely resembles the gene expression profile of transporters within the normal colon, suggesting that this cell line may serve as an in vitro model of colonic drug adsorption. However, the molecular "fingerprint" of Caco-2 was distinctly different from tumor samples, indicating that the Caco-2 model would unlikely predict accurate drug absorption for colon cancer sites.

ATP-Binding Cassette Transporters↗

PubMedDate not supplied

Low and high concentrations of the topo II inhibitor daunorubicin in NIH3T3 cells: reversible G2/M versus irreversible G1 and S arrest.

Daunorubicin (DNR) blocks the cell cycle by interfering with synthesis and repair of DMA. In both drug-sensitive 3T3 cells and drug-resistant 3T3 cells (NIH-MDR-6185, created by transfection with a human MDR1 cDNA), low concentrations of DNR (up to 80 ng/ml in sensitive cells, 1600 ng/ml in resistant cells) initially slowed S-phase progression for 2 to 3 hours, but the treated cells then continued in progression at a steady rate, close to that of untreated cells, and accumulated in G(2)/M. The 2 to 3 h lag period represents the time taken for fully establishing the G(2)/M block. The time required to bring about cessation of proliferation is the sum of this lag period and the time taken to travel through the cell cycle. This low concentration effect is cytostatic, and fully reversible on washing out the daunorubicin. At higher drug concentrations (above 160 ng/ml in sensitive cells, 3200 ng/ml in resistant cells) the cells became blocked in both G] and S, and did not reach G(2)/M. The high concentration effect was cytotoxic and irreversible, and was followed by cell death. Only cells that were in S phase were subject to this block in S, since cells that had accumulated in G(2)/M by using a low concentration (60 ng/ml DNR for 20 h) were not blocked in S, and did not die, when subsequently treated with high drug concentrations (320 ng/ml, 30 h). The low concentration effect occurred at the same maximal rate (4 %/h) in sensitive or resistant cells, but the external drug concentration required to produce half the maximal rate was, appropriately, twenty-fold higher in the resistant cells (20 ng/ml and 400 ng/ml, respectively).

Antineoplastic Agents↗

PubMedDate not supplied

Modulation by the ATP/GTP ratio of the phosphorylation level of P-glycoprotein and of various plasma membrane proteins of KB-V1 multidrug resistant cells.

The level of protein phosphorylation is known to affect the properties of various membrane proteins. We have previously shown that GTP is capable of greatly enhancing the phosphorylation by [gamma-32P]ATP of P-glycoprotein (Pgp) from KB-V1 cells (3). Investigating the possibility of a general modulation of [gamma-32P]ATP plasma membrane protein phosphorylation, we found that phosphorylation of other membrane proteins are also modulated by various combinations of [ATP + GTP]. The ATP/GTP ratio giving the highest phosphorylation level depended on the protein studied. Modulation of the [gamma-32P]ATP-mediated phosphorylation of numerous membrane proteins requires hydrolysis of both ATP and GTP. ADP and GDP also increased [gamma-32P]ATP-driven phosphorylation but to a lesser extent than GTP. This plasma membrane endogenous phosphorylation activity was neither inhibited by specific inhibitors of protein kinase C, nor by inhibitors of cAMP- or cGMP-dependent protein kinases or of casein kinase II, respectively. Mastoparan, a G-protein regulator, increased the phosphorylation of some proteins that were already enhanced by the presence of [ATP + GTP] mixtures, especially proteins migrating in gels at the same position as P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

Different roles for K+ channels in cisplatin-resistant cell lines argue against a critical role for these channels in cisplatin resistance.

Cisplatin resistance has been associated with altered K+ fluxes. Here, we focused our investigations on the detection of K+ channels in a series of cisplatin-resistant (CP-r) cells with increasing resistance and on the functional relationship of these K+ channels to resistance. Microarray analysis and confocal microscopy indicated that there was overexpression of the ether-a-gogo gene (HERG) and the inwardly rectifying potassium channel gene (TWIK) in a human epidermal KB and human liver BEL-7404 carcinoma cell line series selected for cisplatin resistance. With increased resistance, the plasma membrane potential, but not the mitochondrial membrane potential, also increases in these two series. For these reasons, we conducted cell proliferation studies in the presence of either antibodies directed against the detected K+ channels, omeprazole (a H+ pump inhibitor) or a specific inhibitor of the HERG channel (WAY-123398-A-5). The antibodies and omeprazole influenced cell growth only very slightly. The specific K+ channel blocker did not alter cisplatin resistance. We also observed that manipulation of K+ fluxes with antibodies and the H+ pump with omeprazole resulted in opposite effects on cisplatin resistance in these two cell lines. We conclude that K+ and H+ homeostasis are not critical factors in cisplatin resistance since they affect cisplatin resistance differently in KB and BEL-7404 cells.

Antineoplastic Agents↗