Analysis of enhanced retinoblastoma protein (pRb) degradation in HPV-positive cells after histone deacetylase inhibition

ABSTRACT Human papillomaviruses have been identified as the major aetiological factor in cervical carcinogenesis. Constitutive expression of the high-risk HPV E6 and E7 oncoproteins are important for the malignant transformation during the course of infection. In particular, E6 binds to p53 and E7 binds to pRb to mediate their proteasomal degradation which is a crucial step for the HPV procured cell cycle progression. In order to analyse the biological outcomes in context of the HPV transformed cells, anti-tumour agents known as HDAC inhibitors (HDACi) were used. In the work presented here, the interest was precisely focused to analyse the role of E7 in tumour suppressor protein pRb degradation following HDAC inhibition. Initial observation in HPV18 positive cervical carcinoma cells HeLa and SW756, demonstrated an E7 dependent degradation of pRb upon SB and TSA. This observation was confirmed further by using the HPV negative cervical cancer cells C33-A and HT-3. Furthermore, to prove whether tumorigenic phenotype was a critical determinant, the effect of HDACi on the Stanbridge cell system (non-tumorigenic 444 and the tumorigenic CGL-3 cells) was analysed. No discernible difference in pRb degradation was observed between the different cell segregates. Moreover the use of the individual E6 and E7 immortalised keratinocytes significantly demonstrate the functional link between E7 and pRb degradation. The pRb protein is able to repress the transcription of the cell cycle regulatory proteins (cyclin E, DNA polymerase-α, dihydrofolate reductase (DHFR) etc.), involved in the G1 to S phase by binding the transcription factor E2F and recruiting the HDAC1 molecules to their promoters. To investigate whether pRb degradation was accompanied with the upregulation of the key molecules involved in G1 to S transition the protein level of cyclin E was examined. Western Blot analysis was performed in HeLa, 444, CGL3 cells and in E6- and E7- immortalised keratinocytes. Essentially, cyclin E upregulation was observed while the E2F protein level was unchanged in HPV 18 containing cells and in E7 immortalised keratinocytes. These results demonstrate that the presence of E7 oncoprotein is a prerequisite for pRb degradation upon HDAC inhibition. In order to establish more functional link between E7 and pRb degradation upon HDAC inhibition, two experimental approaches were used. At first, the E7 oncoprotein was knocked down by using siRNA in the HeLa cells. Secondly, E7 oncoprotein was introduced in TE-671 which are HPV negative, but pRb positive cells. In both systems, we observed that E7 has an indispensable role on pRb degradation upon HDACi treatment. In order to dissect the role of the individual oncoprotein in HDACi induced pRb degradation, E6 and E7 immortalised keratinocytes were used. Treatment of the cells with proteasome-inhibitor MG132 (2 µM, 16 h) shows that the degradation of pRb occurs via 26S proteasome pathway in E7 immortalised keratinocytes. In order to accentuate the importance of the direct interaction between E7 and pRb, a serine protease inhibitor TLCK was used. In particular, modification of the E7 protein by using the TLCK, rescued pRb from HDACi induced degradation. Furthermore, cyclin E which is negatively regulated by pRb was downregulated again. These results emphasized the importance of the physical association between E7 and pRb and the crucial role of the LXCXE motif of E7 protein. In addition, co-treatment of SB with TLCK interferes with the SB mediated G1 arrest, which was shown by the cell cycle distribution analysis and downregulation of the cyclin-dependent kinase inibitor p21. Furthermore, p21 is activated in response of HDACi which is responsible for the inactivation of the cyclin/CDK complexes in G1 phase. In order to analyse the HDACi induced apoptosis in the context of HPV, Hela cells were treated with SB and TSA and apoptotic cells were analysed by flow cytometry. Consistently, HeLa cells expressed apoptotic markers within at 24 hours while the HPV negative cervical cancer cells C33-A were not sensitive for apoptosis in response to HDACi treatment. Furthermore this confirmed the crucial role of the E7-pRb interaction for the HDACi mediated apoptosis. HDACi induced cell death mainly by activating the intrinsic apoptotic pathway. In order to address the target apoptotic gene expression in the HPV context and individual oncogenes, Hela cells and the E6- and E7-immortalised keratinocytes were used. After treatment with SB, RT-PCR (real-time) analyses were performed for p73 expression. The E2F mediated apoptotic protein p73 was upregulated in response to HDACi treatment. This effect was reversed by TLCK treatment in HeLa as well as in E7 immortalised keratinocytes. Thus p73 transcription was reduced in HeLa cells as well as in E7 immortalised keratinocytes. These data confirm the presence of HPV E7, which sensitizes the cells for SB mediated apoptosis, and which can be reversible by posttranslational modification of the HPV E7. Finally the HDAC inhibitor mediated apoptosis can be inhibited by TLCK. Subsequently this study bears new future aspects in understanding the role of serine proteases during HDAC inhibition.