Patrick Finzer

Inhibitors of histone deacetylase arrest cell cycle and induce apoptosis in cervical carcinoma cells circumventing human papillomavirus oncogene expression

Histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A arrest human papillomavirus (HPV)-positive carcinoma cells in G1 to S transition of the cell cycle, which is paralleled by an up-regulation of the cyclin-dependent kinase inhibitors (CKIs) p21CIP1 and p27KIP1 as well as the complete loss of cdk2 activity. Although HPV expression was hitherto thought to be required to maintain a proliferative phenotype of these cells, cdk2 suppression is achieved even in the presence of ongoing viral transcription. While CKIs normally cannot exert their cdk2-inhibitory function in the presence of the viral oncoprotein E7, co-immunoprecipitation experiments revealed that E7 binding is prevented. Increase of p27KIP1 correlates with down-regulation of p45SKP2, a component of the ubiquitin-protein ligase SCFSKP2 controlling the half-life of regulatory proteins during the cell cycle. HDAC inhibition also triggered an E7-dependent degradation of pRb, while the levels of E2F remained unaffected. The presence of free intracellular E2F and the concomitant up-regulation of CKIs during G1 arrest results in a ‘conflicting growth situation’, which finally renders the cells to undergo apoptosis. These data provide novel molecular insights into how the transforming potential of HPV can be bypassed and open new therapeutical perspectives for the treatment of cervical cancer.

Differential transcriptional regulation of the monocyte-chemoattractant protein-1 (MCP-1) gene in tumorigenic and non-tumorigenic HPV 18 positive cells: the role of the chromatin structure and AP-1 composition.

The expression of the monocyte-chemoattractant-protein-1 (MCP-1) is closely linked with a non-tumorigenic phenotype in somatic cell hybrids made between the human papillomavirus type 18 (HPV 18) positive cervical carcinoma cell line HeLa and normal human fibroblasts. In contrast, MCP-1 transcription is absent in tumorigenic segregants derived from the same hybrids or in parental HeLa cells. Selectivity of MCP-1 transcription, which is regulated at the level of initiation of transcription, is mainly based on differences in the location and extension of DNAse I-hypersensitive regions (DHSR) at both ends of the gene. While TNF-α only moderately increases the sensitivity of pre-existing 5′-DHSRs, a 3′-end DHSR became strongly induced exclusively in non-malignant hybrids. DNA sequencing showed that the 3′-DHSR coincides with an additional AP-1 site located approximately 600 bp downstream of the polyadenylation site. Analyses of AP-1 composition revealed that MCP-1 is only expressed in those cells where jun-family members were mainly heterodimerized with the fos-related protein fra-1. In contrast, in tumorigenic cells the 1 : 1 ratio between jun and fra-1 is disturbed and the MCP-1 gene is no longer expressed. Hence, alterations in the heterodimerization pattern of AP-1 and its selective accessibility to opened chromatin may represent a novel regulatory pathway in the regulation of chemokines in malignant and non-malignant HPV-positive cells.

Genetic complementation to non-tumorigenicity in cervical-carcinoma cells correlates with alterations in AP-1 composition

The transcription factor AP‐1 represents a central key element in the expression of human pathogenic papillomaviruses (HPV). We here propose a novel role for AP‐1 as an essential component of an intracellular surveillance mechanism negatively controlling the proliferation of HPV‐positive cells under in vivo conditions. The dissection of AP‐1 composition in cervical‐carcinoma cells revealed an inverse relationship between the Fos‐related antigen Fra‐1 and the tumorigenic phenotype. Cervical‐carcinoma cell lines were either negative or expressed only low amounts of Fra‐1 (jointly with c‐Fos) within their AP‐1 complexes. Somatic‐cell hybridization technique was used to fuse different HPV‐positive malignant cell lines. This resulted either in tumorigenic hybrids or in cells in which the malignant phenotype of the parental fusion partners was completely suppressed. The monitoring of AP‐1 composition in electrophoretic mobility super‐shift assays showed that the amount of Fra‐1 was substantially increased within the AP‐1 complex of non‐malignant cells. In contrast, Fra‐1 was even diminished in malignant hybrids, while c‐Fos remained expressed. This correlation suggests that the concentration of Fra‐1 within the AP‐1 transcription complex might be an important marker for predicting the in vivo growth properties of HPV‐positive cells. Int. J. Cancer 86:811–817, 2000.

HDAC inhibitors trigger apoptosis in HPV-positive cells by inducing the E2F–p73 pathway

Histone deacetylase (HDAC) inhibitors induce an intrinsic type of apoptosis in human papillomavirus (HPV)-positive cells by disrupting the mitochondrial transmembrane potential (ΔΨm). Loss of ΔΨm was only detected in E7, but not in E6 oncogene-expressing cells. HDAC inhibition led to a time-dependent degradation of the pocket proteins pRb, p107 and p130, releasing ‘free’ E2F-1 following initial G1 arrest. Inhibition of proteasomal proteolysis, but not of caspase activity rescued pRb from degradation and functionally restored its inhibitory effect on the cyclin E gene, known to be suppressed by pRb-E2F-1 in conjunction with HDAC1. Using siRNA targeted against p53, E2F-1 still triggered apoptosis by inducing the E2F-responsive proapoptotic α- and β-isoforms of p73. These data may determine future therapeutic strategies in which HDAC inhibitors can effectively eliminate HPV-positive cells by an apoptotic route that does not rely on the reactivation of the ‘classical’ p53 pathway through a preceding shut-off of viral gene expression.