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. 2008 Aug;7(8):2377-85.
doi: 10.1158/1535-7163.MCT-08-0316. Epub 2008 Aug 7.

Lentiviral Short Hairpin RNA Screen of Genes Associated With Multidrug Resistance Identifies PRP-4 as a New Regulator of Chemoresistance in Human Ovarian Cancer

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Lentiviral Short Hairpin RNA Screen of Genes Associated With Multidrug Resistance Identifies PRP-4 as a New Regulator of Chemoresistance in Human Ovarian Cancer

Zhenfeng Duan et al. Mol Cancer Ther. .
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Abstract

Published reports implicate a variety of mechanisms that may contribute to drug resistance in ovarian cancer. The chief aim of this study is to understand the relationship between overexpression of drug resistance associated genes and multidrug resistance in ovarian cancer. Using lentiviral short hairpin RNA collections targeting 132 genes identified from transcriptional profiling of drug-resistant cancer cell lines, individual knockdown experiments were done in the presence of sublethal doses of paclitaxel. Specific genes whose knockdown was found to be associated with cellular toxicity included MDR1 (ABCB1), survivin, and pre-mRNA processing factor-4 (PRP-4). These genes, when repressed, can reverse paclitaxel resistance in the multidrug-resistant cell line SKOV-3(TR) and OVCAR8(TR). Both MDR1 and survivin have been reported previously to play a role in multidrug resistance and chemotherapy-induced apoptosis; however, the effect of PRP-4 expression on drug sensitivity is currently unrecognized. PRP-4 belongs to the serine/threonine protein kinase family, plays a role in pre-mRNA splicing and cell mitosis, and interacts with CLK1. Northern analysis shows that PRP-4 is overexpressed in several paclitaxel-resistant cell lines and confirms that PRP-4 expression could be significantly repressed by PRP-4 lentiviral short hairpin RNA. Both clonogenic and MTT assays confirm that transcriptional repression of PRP-4 could reverse paclitaxel resistance 5-10-fold in SKOV-3(TR). Finally, overexpression of PRP-4 in drug-sensitive cells could induce a modest level of drug resistance to paclitaxel, doxorubicin, and vincristine.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Flow chart of the functional lentiviral shRNA screen measuring the effect of drug resistant gene knockdown on paclitaxel sensitivity. The drug resistance associated 132-lentiviral shRNA library was established. For the screen, 1.6x104 SKOV-3TR cells per well in 96-well plates were infected with lentiviral particles encoding shRNA against different drug resistance associated genes. The infected cells were subsequently selected with puromycin following a protocol provided by the manufacturer. The number of viable cells was analyzed using the CellTiter 96®AQueous One Solution Cell Cytotoxicity Assay and visualized under the microscope.
Figure 2
Figure 2
Effect of PRP-4 knockdown on paclitaxel sensitivitiy in SKOV-3TR cells. Clonogenic assay transduction of PRP-4 lentiviral shRNA vector confers paclitaxel sensitivity to SKOV-3TR cells. After transfection with the PRP-4 lentiviral shRNA, puromycin selected clones were plated and exposed to varying concentrations of paclitaxel for 6 days. The cells were fixed and stained with crystal violet to visualize the viable cells, with darker staining representing the more viable cells.
Figure 3
Figure 3
Confirmation of PRP-4 depletion in clones and effect on paclitaxel sensitivitiy. (A) MTT assay shows validation of PRP-4 knockdown in the reversal of paclitaxel resistance. Data shows two of three positive target sites of PRP-4 by shRNA. (B). Duplicate sample sets of total RNA isolated from the SKOV-3 TR, SKOV-3TR/TRC719, and SKOV-3TR/TRC722 cell lines analyzed in the MTT assay were subjected to Northern blot analysis using cDNA probe directed against PRP-4 (upper panel) and β-actin (lower panel)
Figure 4
Figure 4
Exogenous expression of PRP-4 confers paclitaxel resistance to the ovarian cancer cell line SKOV-3. (A) Confirmation of PRP-4 gene knockdown by Northern blot. Duplicate sample sets of total RNA isolated from the SKOV-3, SKOV-3pIRES, and SKOV-3pIRESPRP-4 cell lines analyzed in the MTT assay were subjected to Northern blot analysis using cDNA probe directed against PRP-4 (upper panel) and β-actin (lower panel). (B) Confirmation of PRP-4 protein knockdown by Western blot. Total protein was isolated from SKOV-3, SKOV-3pIRES, and SKOV-3pIRESPRP-4 cell lines and analyzed by Western blotting with anti PRP-4, Pgp or anti-actin antibodies. (C) The relative cytotoxicity of paclitaxel, doxorubicin, vincristine and cisplatin in SKOV-3 derived cell lines (SKOV-3pIRESPRP-4) stably transfected with a pIRESPRP-4 expression vector and in the parental (SKOV-3) and empty vector (SKOV-3pIRES) controls were assessed using the MTT assay. All samples were analyzed in triplicate.

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