Frank Rösl

The role of human papillomavirus oncoproteins E6 and E7 in apoptosis

The oncogenic potential of high risk human papillomaviruses can be mainly attributed to two small proteins called E6 and E7. Even these oncoproteins have a low molecular size, they are highly promiscuous and are capable to interact with a whole variety of host cellular regulator proteins to elicit cellular immortalization and ultimately complete malignant transformation. To avoid reiterations in summarizing the biochemical and molecular biological properties of E6/E7 in terms of their influence on cell cycle control, the present review is mainly an attempt to describe some regulatory principles by which human papillomavirus (HPV) oncoproteins can interfere with apoptosis in order to escape immunological surveillance during progression to cervical cancer. The models derived from these basic cellular and molecular studies are relevant to our understanding of HPV-induced carcinogenesis. Conversely, experimental procedures aimed at relieving apoptosis resistance, can facilitate the eradication of immunologically suspicious cells and may prevent the accumulation of cervical intraepithelial cell abnormalities in future prophylactic or therapeutic approaches.

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.

Conversion of HPV 18 positive non-tumorigenic HeLa-fibroblast hybrids to invasive growth involves loss of TNF-α mediated repression of viral transcription and modification of the AP-1 transcription complex

AP-1 represents a transcription factor, which plays a pivotal role in initiating and maintaining the expression of human papillomavirus (HPV) oncoproteins E6 and E7 during HPV-linked carcinogenesis of the uterine cervix. AP-1 stands as a synonym for different proteins such as c-Jun, JunB, JunD, c-Fos, FosB as well as the Fos-related antigens Fra-1 and Fra-2, which can either homo- or heterodimerize to build up a functional transcription complex. AP-1 is mainly considered as a positive regulator, which binds to cognate DNA sequences within the viral upstream regulatory region. By using non-tumorigenic HeLa-fibroblast hybrids (`444), their tumorigenic segregants (`CGL3) as well as HPV 18 positive HeLa cells as a experimental model system, evidence is provided that AP-1 composition differs considerably between these cell lines. In nuclear extracts obtained from non-tumorigenic cells, Jun-family members (in the order c-Jun>JunD>JunB) were mainly heterodimerized with Fra-1, a protein, known to be involved in the abrogation of AP-1 activity under certain experimental conditions. In contrast, Fra-1 concentration is low in extracts from tumorigenic cells. Conversely, c-Fos, the canonical dimerization partner of Jun proteins is expressed in substantial quantity in HeLa- and `CGL3 cells, but it is completely absent in AP-1 complexes from non-tumorigenic `444 cells. Ectopical expression of c-fos under a heterologous promoter in `444-cells induces tumorigenicity and a change of the Jun/Fra-1 ratio towards a constellation initially detected in `CGL3-and HeLa cells. Furthermore, conversion to tumorigenicity is accompanied with a resistance against TNF-α, a cytokine, capable to selectively suppress HPV 18 transcription in formerly non-malignant cells. These data propose a novel role for AP-1 as an essential component of an inter- and intracellular surveillance mechanism negatively controlling HPV transcription in non-tumorigenic cells.

The effect of DNA methylation on gene regulation of human papillomaviruses

Integration of human papillomaviruses (HPVs) into the host genome is considered to be an early and important event in HPV-linked cervical carcinogenesis. Consequently, the viral DNA potentially becomes a target for cellular control mechanisms normally acting on the corresponding integration site. Besides resulting position effects, host-specific DNA methylation may play a functional role in HPV gene regulation. To elucidate the influence of such a kind of epigenetic modification on viral transcription, in vitro methylation studies on HPV-18 upstream regulatory region (URR)-controlled reporter plasmids were carried out. Selective methylation of the viral URR results in a down-regulation of the transcriptional activity, which can be attributed to nonrandom distribution of methyl-acceptor sites clustered within the constitutive enhancer region. In vivo competition experiments show that suppression is not directly mediated by steric hindrance of methyl residues with transcription factors, but rather is due to the association with methyl-CpG DNA-binding proteins. Using a restriction enzyme accessibility assay on both the DNA and chromatin levels, it could be demonstrated that, in vivo, extensively methylated viral DNA is nucleosomally organized, characteristic of transcriptionally inactive chromatin. These data suggest that DNA methylation is an important regulatory pathway in the modulation of HPV expression and as a consequence the proliferation rate of virus-infected cells.

Selective suppression of monocyte chemoattractant protein-1 expression by human papillomavirus E6 and E7 oncoproteins in human cervical epithelial and epidermal cells.

Infection of cervical keratinocytes by high‐risk HPV is involved in the etiology of cervical carcinoma. Since viral products are immunogenic, development of cancer may require suppression of immune responses directed against infected epithelial cells. Many markers of host immune effector responses decrease as cervical intraepithelial neoplasia progresses. Among these is epithelial cell expression of the chemokine MCP‐1, though the mechanism for its suppression is unclear. Here, we show that the E6 and E7 viral oncogenes from high‐risk HPV, individually and together, suppress MCP‐1 expression in primary epithelial cells derived from the female genital tract. This is not a consequence of global suppression of chemokine expression since other chemokines, including IP‐10, IL‐8 and RANTES, were less affected. Furthermore, 4 of 6 HPV‐positive cervical carcinoma cell lines did not express MCP‐1. Our data indicate that suppression of MCP‐1 expression is part of the program of high‐risk HPV E6/E7‐induced transformation of primary epithelial cells. These observations are consistent with a model in which MCP‐1 expression by infected keratinocytes, which would stimulate an immune attack on HPV‐transformed cells, is suppressed for invasive cervical cancer to appear.

Monocyte-chemo-attractant-protein-1 (MCP-1)-gene expression in cervical intra-epithelial neoplasias and cervical carcinomas

Chemokines play a central role in the chemotactic activation of immunological effector cells. One of the currently best characterized chemokines is the monocyte‐chemo‐attractant protein‐1 (MCP‐1), which is involved in the cross‐talk with cells of the monocyte‐macrophage lineage. Since macrophages and macrophage‐derived cytokines appear to be important in the transcriptional regulation of “high‐risk” types of human papillomaviruses (HPV), we monitored MCP‐1 expression by in situ hybridization (ISH) in histologically distinct stages of cervical intra‐epithelial neoplasms (CIN), cervical cancer and non‐HPV‐associated cases of erosive endocervicitis. Here, we demonstrate that high‐grade dysplasia (CIN III, n = 9) completely lacks both MCP‐1 expression and CD68+‐macrophage infiltration, while MCP‐1‐specific signals were occasionally detectable in one out of 5 CIN‐II and in one out of 3 CIN‐I lesions. Inspection of hyperplastic squamous epithelium adjacent to cervical carcinomas reveals high MCP‐1 expression and accumulation of infiltrating macrophages. In contrast, no macrophages could be detected in corresponding hyperplastic tissue areas surrounding CIN‐II and CIN‐III lesions, although MCP‐1 was found to be highly expressed. Finally, in agreement with our earlier in vitro data, invasive carcinomas of the cervix uteri showed MCP‐1‐specific hybridization signals and macrophage infiltration only in the stroma surrounding the carcinoma cells and in endothelial cells of capillaries, especially at the invasion front of the tumor, while the inner mass of the carcinomas was completely negative. On the other hand, ISH and histochemical evaluation of inflammatory, non‐HPV‐associated cases of erosive endocervicitis indicate strong MCP‐1 expression, which is regularly accompanied by chemotactic appearance of macrophages. These observations indicate that dysregulation of MCP‐1‐gene expression may represent an important step during HPV‐linked carcinogenesis, allowing the escape of virus‐positive cells from local immune response. Int. J. Cancer 82:6–11, 1999.

Restoration of p53 expression sensitizes human papillomavirus type 16 immortalized human keratinocytes to CD95-mediated apoptosis

To understand the function of the individual oncogenes of HPV16 in modulating the cellular response to apoptogenic signals, we used human keratinocytes immortalized with either E6, E7 or E6/E7 oncoproteins as model system. Applying CD95 antibodies or recombinant CD95 ligand, only the E7-immortalized cells underwent extensive apoptosis. In contrast, E6- and E6/E7-expressing keratinocytes were resistant. Dominance of E6 correlated with significant down-regulation of p53, c-Myc, p21 and Bcl-2. CD95 was found to be reduced in resistant HPV-positive cells, while there were no quantitative differences in expression levels of FADD, FLICE/caspase-8 or caspase-3. Notably, in contrast to primary human keratinocytes, all immortalized cells showed a general reduction of c-FLIP, an inhibitory protein which normally prevents unscheduled CD95-induced apoptosis. E6- and E6/E7-positive keratinocytes, however, can be sensitized to CD95 apoptosis by blocking proteasome-mediated proteolysis. CD95-resistant HPV-positive cells underwent apoptosis within 3–5u2009h upon co-incubation with MG132 and agonistic antibodies or CD95 ligand, which was preceded by a strong re-expression of p53 and c-Myc, but not of other half-life controlled proteins such as Bax or IκBα. Blockage of proteasomal activity alone did not result in apoptosis, although the same set of pro-apoptotic proteins was up-regulated. Performing similar experiments with cervical carcinoma cells expressing mutated p53 (C33a) or with p53-‘null’ lung carcinoma cells (H1299), no CD95 cell killing occurred eventhough c-Myc was strong induced. These data indicate that the reduced bioavailability of p53 is a key-regulatory event in perturbation of CD95 signaling in HPV16 immortalized keratinocytes.

The chemopreventive compound curcumin is an efficient inhibitor of Epstein‐Barr virus BZLF1 transcription in Raji DR‐LUC cells*

To characterize the effects of inhibitors of Epstein‐Barr virus (EBV) reactivation, we established Raji DR‐LUC cells as a new test system. These cells contain the firefly luciferase (LUC) gene under the control of an immediate‐early gene promoter (duplicated right region [DR]) of EBV on a self‐replicating episome. Luciferase induction thus serves as an intrinsic marker indicative for EBV reactivation from latency. The tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) induced the viral key activator BamH fragment Z left frame 1 (BZLF1) protein (“ZEBRA”) in this system, as demonstrated by induction of the BZLF1 protein‐responsive DR promoter upstream of the luciferase gene. Conversely, both BZLF1 protein and luciferase induction were inhibited effectively by the chemopreventive agent curcumin. Semiquantitative reverse transcriptase (RT)‐polymerase chain reaction (PCR) further demonstrated that the EBV inducers TPA, sodium butyrate, and transforming growth factor‐β (TGF‐β) increased levels of the mRNA of BZLF1 mRNA at 12, 24, and 48 h after treatment in these cells. TPA treatment also induced luciferase mRNA with similar kinetics. Curcumin was found to be highly effective in decreasing TPA‐, butyrate‐, and TGF‐β‐induced levels of BZLF1 mRNA, and of TPA‐induced luciferase mRNA, indicating that three major pathways of EBV are inhibited by curcumin. Electrophoretic mobility shift assays (EMSA) showed that activator protein 1 (AP‐1) binding to a cognate AP‐1 sequence was detected at 6 h and could be blocked by curcumin. Protein binding to the complete BZLF1 promoter ZIII site (ZIIIA+ZIIIB) demonstrated several specific complexes that gave weak signals at 6 h and 12 h but strong signals at 24 h, all of which were reduced after application of curcumin. Autostimulation of BZLF1 mRNA induction through binding to the ZIII site at 24 h was confirmed by antibody‐induced supershift analysis. The present results confirm our previous finding that curcumin is an effective agent for inhibition of EBV reactivation in Raji DR‐CAT cells (carrying DR‐dependent chloramphenicol acetyltransferase), and they show for the first time that curcumin inhibits EBV reactivation mainly through inhibition of BZLF1 gene transcription.

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 600u2009bp 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 1u2009:u20091 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.

Selective suppression of human papillomavirus transcription in non-tumorigenic cells by 5-azacytidine.

The transcription of human papillomavirus type 18 (HPV 18) is selectively suppressed in non‐tumorigenic HeLa x fibroblast or HeLa x keratinocyte cell hybrids by 5‐azacytidine. In contrast, viral gene expression is not influenced by 5‐azacytidine in both tumorigenic hybrid segregants and in the parental HeLa cells. The suppression mechanism seems to operate at the level of initiation of transcription since nuclear run‐on experiments show the absence of elongated nascent viral RNA, whereas the transcription of cellular reference genes remains unaffected. Down‐regulation of HPV 18 mRNA correlates directly with cessation of cellular growth and can be abolished using the protein synthesis inhibitor cycloheximide. Furthermore human keratinocytes immortalized by HPV 16 but still retaining the non‐tumorigenic phenotype reveal the same inhibitory effect on viral transcription after treatment with 5‐azacytidine. These results support a model of a postulated intracellular control mechanism, directed against papillomavirus transcription, which can be induced by 5‐azacytidine and appears to correlate with the presence of specific chromosomes in non‐tumorigenic cells.