ABSTRACT
In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.
Subject(s)
Eye Neoplasms/genetics , Eye Neoplasms/therapy , Genes, Transgenic, Suicide , Genetic Therapy/methods , MicroRNAs/genetics , Retinoblastoma/genetics , Retinoblastoma/therapy , Antigens, Neoplasm/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Cell Adhesion Molecules/genetics , Cell Death/drug effects , Cell Death/genetics , Cell Line , Cell Line, Tumor , Ependymoglial Cells/metabolism , Epithelial Cell Adhesion Molecule , Eye Neoplasms/pathology , Female , Ganciclovir/pharmacology , Gene Expression , Gene Targeting/methods , Humans , MCF-7 Cells , Middle Aged , Promoter Regions, Genetic , RNA, Neoplasm/genetics , Retinoblastoma/pathology , Simplexvirus/enzymology , Simplexvirus/genetics , Thymidine Kinase/genetics , Tissue DistributionABSTRACT
PURPOSE: Quantify cyclin dependent kinase inhibitor 1C (CDKN1C or p57KIP2) mRNA levels in human retinoblastoma tumors (RB) and associate with disease phenotype. METHODS: CDKN1C mRNA expression was quantified in 55 RB, 3 retinoblastoma cell lines, and 12 control retinas by real time PCR. Localization of CDKN1C protein was confirmed by immunohistochemistry. Tumor CDKN1C expression levels were correlated with phenotype. RESULTS: Fifty-three of 55 tumors showed significantly elevated levels of CDKN1C mRNA relative to age-matched or adult retina controls. No significant difference was seen relative to fetal retinal controls or retinoblastoma cell lines. Immunohistochemistry revealed heterogeneous staining of CDKN1C protein in tumor cells, which was mainly nuclear although some protein appeared to be cytoplasmic. No correlation was observed between the CDKN1C mRNA expression levels and tumor phenotype. CONCLUSION: High levels of CDKN1C expression are common in RB. It remains to be determined if elevated expression is a protective response to transformation, provides a selective advantage to tumor cells, or simply reflects the presence of dividing cells.
