Dataset Information

Retrovirus-mediated multidrug resistance gene (MDR1) overexpression inhibits chemotherapy-induced toxicity of granulosa cells.

ABSTRACT:

Objective

To protect granulosa cells from chemotherapy-induced toxicity by retrovirus-mediated multidrug resistance (MDR1) gene transfection.

Design

Laboratory study.

Setting

Academic research laboratory in a university hospital.

Patient(s)

None.

Intervention(s)

KK15 immortalized murine granulosa cell line transiently transduced with sf91m3 retrovirus vector carrying MDR1 complementary DNA that encodes P-glycoprtoein (P-gp); transduced cells selected with colchicine and treated with doxorubicin or paclitaxel for 24-72 hours; expression and function of MDR1 and the messenger RNA (mRNA) expression of selected steroidogenesis enzymes evaluated by flow cytometry, cell viability assays, Western blot, and reverse-transcriptase polymerase chain reaction (RT-PCR).

Main outcome measure(s)

Viability of sf91m3-transduced KK15 cells after treatment with doxorubicin and paclitaxel.

Result(s)

The sf91m3-transduced KK15 demonstrated high expression of biologically active MDR1, as shown by flow cytometry analysis and immunoblotting using P-gp monoclonal antibody and Rhodamine 123 efflux assays. The sf91m3-transduced KK15 exhibited statistically significant resistance to toxicity of 10 ?M paclitaxel. The MDR1-transduced KK15 cells were also protected from doxorubicin toxicity (10 nM to 2.5 ?M), as shown by cell viability assay. Both flow cytometry and cell viability assays showed that the protection of KK15 from doxorubicin toxicity was lost at 5 ?M of doxorubicin; equivalent to 500 times LD50. The sf91m3-transduced KK15 showed normal mRNA expression of a panel of selected steroidogenesis enzymes.

Conclusion(s)

Retroviral gene delivery of human MDR1 inhibited chemotherapy-induced granulosa cell toxicity and offered chemoprotection in an in vitro model.

SUBMITTER: Salih SM

PROVIDER: S-EPMC3065123 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Publications

Retrovirus-mediated multidrug resistance gene (MDR1) overexpression inhibits chemotherapy-induced toxicity of granulosa cells.

Salih Sana M SM

Fertility and sterility 20110212 4

<h4>Objective</h4>To protect granulosa cells from chemotherapy-induced toxicity by retrovirus-mediated multidrug resistance (MDR1) gene transfection.<h4>Design</h4>Laboratory study.<h4>Setting</h4>Academic research laboratory in a university hospital.<h4>Patient(s)</h4>None.<h4>Intervention(s)</h4>KK15 immortalized murine granulosa cell line transiently transduced with sf91m3 retrovirus vector carrying MDR1 complementary DNA that encodes P-glycoprtoein (P-gp); transduced cells selected with colc ...[more]

PMID: 21316663

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Project description:Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role of CdMDR1 in drug resistance in C. dubliniensis, we constructed an mdr1 null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. Sequential deletion of both CdMDR1 alleles was performed by the MPA(R)-flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the mdr1 mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that CdMDR1 confers resistance to the structurally unrelated drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1 inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression of CdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previously been shown to confer resistance to these compounds. The effect of CdMDR1 inactivation was identical to that seen in two similarly constructed C. albicans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 and CdMDR1 in clinical C. albicans and C. dubliniensis strains seems to confer the same drug resistance profile in both species.

Project description:BackgroundTo investigate the role of P16 (INK4a)-extracellular signal related kinase 1/2 (ERK1/2) signaling pathway in cisplatin (DDP) resistance induced by multidrug resistance protein 1 (MDR1), also known as P-glycoprotein (P-gp), in cervical adenocarcinoma.MethodsA human DDP-resistant HeLa cell line (HeLa/DDP) was constructed using the combination of incremental and intermittent administration of DDP. Cell Counting Kit-8 (CCK-8) assay was used to measure the IC50 and resistance index (RI) of cells. The morphological changes and population doubling time were observed under an inverted microscope. Plate cloning formation assay was performed to evaluate the cell proliferation and tumorigenic ability. Cell invasion and migration were determined by transwell assays. Besides, the expression of P16, phosphorylated extracellular signal related kinase 1 and 2 (pERK1/2), total ERK1/2 and MDR1 were measured using western blot analysis. The ERK-specific inhibitor U0126 and agonist TPA was used to explore the role of ERK.ResultsThe DDP-resistant cervical adenocarcinoma HeLa/DDP cell line was successfully established, which showed stronger cell growth, invasion, and migration. In the HeLa/DDP cells, pERK1/2 was downregulated, P-gp was upregulated and P16 was downregulated. Overexpression of P16 led to a significant decrease in the proliferation rate, migration ability, and invasion ability of the HeLa/DDP cells. Furthermore, overexpression of P16 increased and the decreased expression of pERK1/2 and P-gp in the HeLa/DDP cells, respectively. Treatment of HeLa/DDP cells transfected with P16 plasmid with ERK-specific inhibitor U0126 significantly decreased the expression of pERK1/2 and increased the expression of P-gp from 6 h to 48 h. Moreover, after 72 h, the expression of pERK1/2 was up-regulated and the expression of P-gp was inhibited.ConclusionOverexpression of P16 could partially reverse the MDR1-mediated DDP resistance in the cervical adenocarcinoma by the enhancement of phosphorylation of ERK signaling pathway, which provided a theoretical basis for the treatment of DDP resistance in cervical adenocarcinoma.

Dataset Information

Retrovirus-mediated multidrug resistance gene (MDR1) overexpression inhibits chemotherapy-induced toxicity of granulosa cells.

Objective

Design

Setting

Patient(s)

Intervention(s)

Main outcome measure(s)

Result(s)

Conclusion(s)

Publications

Retrovirus-mediated multidrug resistance gene (MDR1) overexpression inhibits chemotherapy-induced toxicity of granulosa cells.

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