engleski

p53 and p21 mediated gene therapy

Introduction: The p53 tumor suppressor gene encodes a nuclear phosphoprotein that function as negative regulator of cell cycle progression. It can also trigger apoptosis in genetically damaged cells. The loss of wild-type p53 function leads to malignant transformation. Reconstitution of normal p53 expression in tumor cells can lead to suppression of cell growth and cell death. This supports a therapeutic strategy based on p53 replacement, since p53 is one of the most commonly mutated genes in human cancer. p53-dependant cell cycle arrest may function through the action of the cyclin dependant kinase (CKI) p21. The p21 gene encodes a CKI that affects the cell cycle progression by the inhibition of PCNA-dependant replication and phosphorilation of the tumor suppressor retinoblastoma gene product. These observations suggest that p21 might function independently as a tumor suppressor protein. In the present study, we evaluated the relative antitumor activities of p53 and p21 using adenovirus-mediated gene transfer. We also analyzed the effect of wild-type p53 overexpression on the cell lines with different intrinsic p53 status (inactivated, mutant or wild type). Materials and Methods: Four human tumor cell lines were used: HeLa (cervical cancer), MCF-7 (breast carcinoma), CaCo-2, and SW 620 (colon carcinoma). The replication-deficient adenoviruses Ad-p53, Ad-p21and Ad-dl312 (control vector) were propagated in 293 cells and purified by two cesium chloride density centrifugation. The cells were infected with different MOI (PFU/cell) and the cell growth inhibition was followed. The expression of the p53 and p21 gene was measured using quantitative RT-PCR, while the expression of p53 and p21 proteins was measured by immunocytochemical method. The induction of apoptosis after the adenoviral infection was measured using the Annexin V staining method, as well as by detection of DNA laddering. Results: Both Ad-p53 and Ad-p21 inhibited the growth of all tumor cell lines tested (Ad-dl 312 did not influence significantly the cell growth). The most pronounced inhibitory effect was shown on the HeLa and SW 620 cell lines which have either inactivated (HeLa) or mutant (SW 620) p53 gene. Somewhat less pronounced inhibitory effect was shown on MCF-7 cell line and the CaCo-2 cell line was only slightly inhibited. Both MCF-7 and CaCo-2 cell lines have wild type p53 gene. Twenty four hours after the infection with Ad-p53 apoptosis was induced in HeLa and SW 620 cell lines only, while the Ad-p21did not induce apoptosis in either of the cell lines tested. However, apoptosis was also induced in HeLa and SW 620 cell lines 48 and 72 hours after the infection with Ad-p21. Conclusion: This data support a role for p53 gene therapy of cancer, including malignances harboring mutations in this tumor suppressor gene. Although the cells that have endogenous wild-type p53 gene are also inhibited by p53 overexpression, the inhibitory effect is much stronger on the cells with mutated or inactivated gene. p21 gene could also be a good candidate for cancer gene therapy, although its role in the induction of apoptosis should be further analyzed. The possibility of p53 gene to induce apoptosis is certainly the major advantage over p21gene in regard the cancer gene therapy.

p53; p21; tumor supressor; gene therapy; adenovirus; vector

nije evidentirano