E2F1-Induced expression of transactivating and dominant-negative forms of p73 transcripts

Abstract

Cell cycle, one of the most important life processes, is controlled by a regulated balance between proliferative and anti-proliferative signals. Dysregulation of these signals leads to tumor development. Retinoblastoma (Rb) gene is the principle regulator of the cell cycle. Rb was identified initially as a gene deleted in a rare form of early child eye tumor, called retinoblastoma, and it was later shown to be a tumor-suppressor. Cellular functions of Rb are inactivated in many cancer types, either directly by Rb gene mutation, or indirectly by inactivation of the pRb protein that is mediated by different viral oncogenes. pRB exists in nonproliferating (quiescent) cells as a complex with E2F transcription factors. Upon phosphorylation of pRb by cyclin-dependent kinases, E2Fs are released and can transactivate their target genes. E2F1, the first E2F to be identified, activates mostly proliferative genes, but also anti-proliferative genes such as p14ARF that acts as an inducer of p53 stabilization. In turn, p53 induces either cell cycle arrest or programmed cell death (apoptosis). Recently, it was reported that E2F1 also induces p53-independent apoptosis by transactivating the expression of the p53 homologue p73 gene. However, p73 encodes not only apoptosis-inducing transciptionally active(TA)-p73, but also dominant negative (DN)-p73 transcript forms which antagonize TA-p73. Our aim was to investigate whether the E2F1 activates the expression of TA-p73, DN-p73 or both. We over-expressed E2F1 and E2F4 in different human cell lines, by transient transfection using appropriate expression vectors, and analyzed p73 transcript levels by semi-quantitative RT-PCR. We demonstrate that, in different cell lines, E2F1 induced the expression of not only TA-p73, but also its two dominant-negative forms, namely p73Deltaexon2 and DN-v p73. Time course studies indicated that TA-p73 and p73DeltaExon2 forms are induced initially, and DN-p73 induction is delayed about 4 hours. Induced expression of dominant-negative forms, in addition to transcriptionally active p73 transcripts by E2F1 may explain how some cancer cells are able to tolerate p73 activation in response to oncogenes such as E2F1.