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Modulating drug resistance by targeting BCRP/ABCG2 using retrovirus-mediated RNA interference.

Xie N, Mou L, Yuan J, Liu W, Deng T, Li Z, Jing Y, Jin Y, Hu Z - PLoS ONE (2014)

Bottom Line: To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged.The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry.The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma.

View Article: PubMed Central - PubMed

Affiliation: Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, China.

ABSTRACT

Background: The BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications.

Methodology/principal findings: To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor.

Conclusions/significance: The goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi) to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors.

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Knockdown of BCRP/ABCG2 expression by V-BCRPi in JAR cells using immunofluorescence analysis (×100).The results for experimental groups 1 to 8 are shown: 1: mock cells with MoAb or 2: PBS or cells infected with 3: V-BCRP1i, 4: V-BCRP1i-c, 5: V-BCRP2i, 6: V-BCRP2i-c, 7: V-BCRP3i, or 8: V-BCRP3i-c. The fluorescence intensity of cells subjected to V-BCRP3i treatment was the lowest.
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pone-0103463-g003: Knockdown of BCRP/ABCG2 expression by V-BCRPi in JAR cells using immunofluorescence analysis (×100).The results for experimental groups 1 to 8 are shown: 1: mock cells with MoAb or 2: PBS or cells infected with 3: V-BCRP1i, 4: V-BCRP1i-c, 5: V-BCRP2i, 6: V-BCRP2i-c, 7: V-BCRP3i, or 8: V-BCRP3i-c. The fluorescence intensity of cells subjected to V-BCRP3i treatment was the lowest.

Mentions: After 48 hours of infection by V-BCRPi, immunofluorescence was used to analyze the cell morphology. Using DAPI staining for comparison, the fluorescence intensity of BCRP/ABCG2 in each V-BCRPi treatment group was reduced; the fluorescence intensity of uninfected cells incubated with the primary antibody was the strongest, and the fluorescence intensity of uninfected cells with PBS instead of the primary antibody was weakest. The fluorescence intensity of the V-BCRPc treatment group was higher than those of all the other V-BCRPi groups, and the fluorescence intensity of the V-BCRP3i treatment group was the lowest (Fig. 3).


Modulating drug resistance by targeting BCRP/ABCG2 using retrovirus-mediated RNA interference.

Xie N, Mou L, Yuan J, Liu W, Deng T, Li Z, Jing Y, Jin Y, Hu Z - PLoS ONE (2014)

Knockdown of BCRP/ABCG2 expression by V-BCRPi in JAR cells using immunofluorescence analysis (×100).The results for experimental groups 1 to 8 are shown: 1: mock cells with MoAb or 2: PBS or cells infected with 3: V-BCRP1i, 4: V-BCRP1i-c, 5: V-BCRP2i, 6: V-BCRP2i-c, 7: V-BCRP3i, or 8: V-BCRP3i-c. The fluorescence intensity of cells subjected to V-BCRP3i treatment was the lowest.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116202&req=5

pone-0103463-g003: Knockdown of BCRP/ABCG2 expression by V-BCRPi in JAR cells using immunofluorescence analysis (×100).The results for experimental groups 1 to 8 are shown: 1: mock cells with MoAb or 2: PBS or cells infected with 3: V-BCRP1i, 4: V-BCRP1i-c, 5: V-BCRP2i, 6: V-BCRP2i-c, 7: V-BCRP3i, or 8: V-BCRP3i-c. The fluorescence intensity of cells subjected to V-BCRP3i treatment was the lowest.
Mentions: After 48 hours of infection by V-BCRPi, immunofluorescence was used to analyze the cell morphology. Using DAPI staining for comparison, the fluorescence intensity of BCRP/ABCG2 in each V-BCRPi treatment group was reduced; the fluorescence intensity of uninfected cells incubated with the primary antibody was the strongest, and the fluorescence intensity of uninfected cells with PBS instead of the primary antibody was weakest. The fluorescence intensity of the V-BCRPc treatment group was higher than those of all the other V-BCRPi groups, and the fluorescence intensity of the V-BCRP3i treatment group was the lowest (Fig. 3).

Bottom Line: To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged.The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry.The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma.

View Article: PubMed Central - PubMed

Affiliation: Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, China.

ABSTRACT

Background: The BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications.

Methodology/principal findings: To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor.

Conclusions/significance: The goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi) to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors.

Show MeSH
Related in: MedlinePlus