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K Münger

Publications and source records attributed to K Münger.

At least 37 records · Page 2Linked to original sources
PubMed1997

The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2.

The high risk human papillomaviruses (HPVs) are associated etiologically with the majority of human cervical carcinomas. These HPVs encode two viral oncoproteins, E6 and E7, which are expressed consistently in cervical cancers. The function of these viral oncoproteins during a productive infection is to ensure viral replication in cells that have normally withdrawn from the cell division cycle and are committed to terminal differentiation. Expression of the E7 oncoprotein has been shown to lead to the abrogation of various negative growth regulatory signals, including a p53-mediated G1 growth arrest, TGFbeta-mediated growth inhibition, and quiescence of suprabasal keratinocytes. Here we describe a novel mechanism by which E7 can uncouple cellular proliferation and differentiation. In contrast to normal, differentiating keratinocytes, HPV-16 E7-expressing keratinocytes show delayed cellular differentiation and elevated cdk2 kinase activity despite high levels of p21(Cip1) and association of p21(Cip1) with cdk2. We show that the HPV E7 protein can interact with p21(Cip1) and abrogate p21(Cip1)-mediated inhibition of cyclin A and E-associated kinase activities. Based on these findings, we propose that this capacity of the HPV E7 oncoprotein to overcome p21(Cip1)-mediated inhibition of cdk2 activity during keratinocyte differentiation contributes to the ability of E7 to allow for cellular DNA synthesis in differentiated keratinocytes.

Animals↗
PubMed1997

The carboxyl-terminal zinc-binding domain of the human papillomavirus E7 protein can be functionally replaced by the homologous sequences of the E6 protein.

The carboxyl-terminus is necessary for the functional and structural integrity of the human papillomavirus (HPV) E7 oncoprotein. Since many mutations in this domain of E7 result in the formation of unstable proteins, we have evaluated the importance of this region by replacing it with structurally related domains derived from HPV E6 proteins. Biological analysis of these mutant chimeric E7/E6 proteins showed that they retained E7-specific biological activities including cooperation with the ras oncogene to transform primary baby rat kidney cells and transcriptional activation of an E2F responsive reporter plasmid. One of the chimeric proteins was impaired in its ability to physically disrupt pRB/E2F complexes in vitro suggesting that there are defined molecular determinants in the carboxyl-terminus of E7 for this activity. In contrast, none of these proteins exhibited E6-like properties including binding to p53 and/or degradation of associated proteins.

Amino Acid Sequence↗
PubMed1997

Analysis of the p53-mediated G1 growth arrest pathway in cells expressing the human papillomavirus type 16 E7 oncoprotein.

Cells expressing human papillomavirus type 16 (HPV-16) E7, similar to those which express HPV-16 E6, are resistant to a p53-mediated G1 growth arrest. We examined the p53-mediated DNA damage response pathway in E7-expressing cells to determine the mechanism by which E7-containing cells continue to cycle. In response to DNA damage, no dramatic difference was detected in G1- or S-phase cyclin or cyclin-dependent kinase (Cdk) levels when E7-expressing cells were compared to the parental cell line, RKO. Furthermore, Cdk2 kinase activity was inhibited in both RKO cells and E7-expressing cells, while Cdk2 remained active in E6-expressing cells. However, the steady-state levels of pRB and p107 protein were substantially lower in E7-expressing cells than in the parental RKO cells or E6-expressing cells. There was no reduction in pRB mRNA levels, but the half-life of pRB in E7-expressing cells was markedly shorter. Infection of primary human foreskin keratinocytes with recombinant retroviruses expressing HPV-16 E7 resulted in a decrease in pRB protein levels, indicating this phenomenon is a consequence of E7 expression, not of immortalization or transformation. These data strongly suggest E7 interferes with the stability of pRB and p107 protein. We propose that the removal of these components of the p53-mediated G1 growth arrest pathway in E7-expressing cells contributes to the ability of E7 to overcome a p53-mediated G1 growth arrest.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Elevated activity of cyclin-dependent kinase 6 in human squamous cell carcinoma lines.

We have investigated the functional integrity of the retinoblastoma tumor suppressor pathway in five human squamous cell carcinoma lines. Elevated activity of cyclin-dependent kinase 6 (cdk6), a pRB kinase, was detected in all five squamous cell carcinoma lines. Overexpression of the cdk6 protein was detected in one of the five cell lines. The cdk6-specific inhibitor p18ink4C is expressed and associated with cdk6 in all five squamous cell carcinoma lines. In contrast, only very low levels of p16ink4A were detected in these cell lines. This may contribute to the elevated activity of cdk6 in these lines. Elevated activity of cdk6 may result in hyperphosphorylation of the retinoblastoma protein and, therefore, compromise its negative growth-regulatory activity.

Biomarkers, Tumor↗
PubMed1996

Interactions of the human papillomavirus E7 protein with cell cycle regulators.

Human papillomavirus (HPVs) critically depend on the cellular machinery for the replication of their genome. Viral replication is restricted to the differentiated strata of the skin that are normally growth arrested. Hence, the HPVs have developed strategies to subvert cellular growth regulatory pathways and are able to uncouple cellular proliferation and differentiation. The HPV E7 protein can overcome the activity of some cyclin-dependent kinase inhibitors, associate with cyclin/cyclin dependent kinase complexes and bind to and destabilize the retinoblastoma tumor suppressor protein. These biological activities contribute to the carcinogenic potential of the high risk HPV E7 proteins which are consistently expressed in HPV-positive cervical carcinomas.

Amino Acid Sequence↗
PubMed1995

Dimerization of the human papillomavirus E7 oncoprotein in vivo.

We have used a yeast two-hybrid system to show that human papillomavirus E7 proteins can form oligomeric complexes in vivo. The carboxyl-terminal cysteine-rich metal-binding domain is critical for this activity although amino-terminal sequences also contribute to oligomerization. Our experiments also reveal that E7 possesses an intrinsic transcription activation activity in yeast, which resides in the amino terminus of the protein.

Amino Acid Sequence↗
PubMed1995

The molecular biology of cervical cancer.

Infections with specific high-risk of human papillomavirus constitute a major risk factor in the development of precancerous and cancerous lesions of the uterine cervix. Laboratory studies suggest that the human papillomavirus has a mechanistic role in development of these lesions. The two viral proteins consistently expressed in cervical carcinomas functionally abrogate critical cell cycle regulatory pathways, including those governed by the p53 tumor suppressor protein and the product of the retinoblastoma susceptibility gene, pRB. Subversion of these pathways by viral proteins causes genomic instability, resulting in the accumulation of chromosomal abnormalities followed by clonal expansion of malignant cells. Since continued expression of the papillomavirus proteins is critical for maintenance of the transformed state, they are attractive targets for prevention and therapy of precursor as well as cancerous lesions of the cervix.

Disease Progression↗
PubMed1994

A mutational analysis of the amino terminal domain of the human papillomavirus type 16 E7 oncoprotein.

The human papillomavirus type 16 (HPV-16) E7 oncoprotein shares structural and functional similarity with the adenovirus (Ad) E1A protein and the SV40 large tumor antigen (TAg). Like these other DNA tumor virus oncoproteins, HPV-16 E7 interacts with the "pocket proteins," a family of host cellular proteins that include the retinoblastoma tumor suppressor protein and can cooperate with the ras oncogene to transform primary rodent cells. Mutational analyses have indicated that amino acid sequences outside of the pRB binding region are also important for the cellular transformation property of HPV-16 E7. These sequences include an amino terminal domain of the E7 protein that is similar to a portion of conserved region 1 of Ad E1A. In this study it is shown that the homologous amino acid sequences in Ad E1A and SV40 TAg are functionally interchangeable with the amino terminal HPV-16 E7 domain in transformation assays. Deletion analysis across the amino terminus of HPV-16 E7 indicated that the overall integrity of the entire CR1 homology domain is important for the biological activity of the HPV E7 oncoprotein.

Adenovirus E1A Proteins↗
PubMed1994

Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice.

To model human papillomavirus-induced neoplastic progression, expression of the early region of human papillomavirus type 16 (HPV16) was targeted to the basal cells of the squamous epithelium in transgenic mice, using a human keratin 14 (K14) enhancer/promoter. Twenty-one transgenic founder mice were produced, and eight lines carrying either wild-type or mutant HPV16 early regions that did not express the E1 or E2 genes were established. As is characteristic of human cancers, the E6 and E7 genes remained intact in these mutants. The absence of E1 or E2 function did not influence the severity of the phenotype that eventually developed in the transgenic mice. Hyperplasia, papillomatosis, and dysplasia appeared at multiple epidermal and squamous mucosal sites, including ear and truncal skin, face, snout and eyelids, and anus. The ears were the most consistently affected site, with pathology being present in all lines with 100% penetrance. This phenotype also progressed through discernible stages. An initial mild hyperplasia was followed by hyperplasia, which further progressed to dysplasia and papillomatosis. During histopathological progression, there was an incremental increase in cellular DNA synthesis, determined by 5-bromo-2'-deoxyuridine incorporation, and a profound perturbation in keratinocyte terminal differentiation, as revealed by immunohistochemistry to K5, K14, and K10 and filaggrin. These K14-HPV16 transgenic mice present an opportunity to study the role of the HPV16 oncogenes in the neoplastic progression of squamous epithelium and provide a model with which to identify genetic and epigenetic factors necessary for carcinogenesis.

Animals↗
PubMed1993

The human papillomavirus E7 protein as a transforming and transactivating factor.

The HPV proteins encoded by the early viral genes, including E6 and E7, are thought to subvert the normal regulatory pathways of infected cells to accommodate viral replication. Mechanistically some of this is accomplished by protein-protein interactions between viral proteins and a number of key cellular regulatory proteins that include tumor suppressor gene products. By undermining cellular regulatory pathways the HPV oncogenes cause hyperproliferation and the perturbation of normal cellular differentiation pathways. Although expression of the high-risk HPV-encoded E6 and E7 oncoproteins may be important prerequisites for cellular transformation, it is very likely that additional cellular changes are necessary for carcinogenic progression. The elucidation of the role of the early HPV genes in the initiation and/or maintenance of carcinogenic progression will continue to be a fascinating area of investigation and may reveal new opportunities for antiviral therapy and antitumor intervention.

Amino Acid Sequence↗
PubMed1993

The human papillomavirus E7 oncoprotein and the cellular transcription factor E2F bind to separate sites on the retinoblastoma tumor suppressor protein.

The ability of the high-risk and low-risk human papillomavirus E7 oncoproteins to disrupt complexes of the retinoblastoma tumor suppressor protein pRB and the cellular transcription factor E2F was studied. The ability of E7 to disrupt this transcription factor complex correlated with the different pRB binding efficiencies of the high-risk and low-risk human papillomavirus-encoded E7 proteins. The pRB binding site was the sole determinant for these observed differences. The phosphorylation status of the casein kinase II site that is immediately adjacent to the pRB binding site in E7 had no marked effect on this biochemical property of E7. Peptides consisting of the pRB binding site of E7, however, were not able to disrupt the pRB/E2F complex. These data suggest that additional carboxy-terminal sequences in E7 are also required for the efficient disruption of the pRB/E2F complex and that E7 and E2F may interact with nonidentical sites of pRB.

Base Sequence↗
PubMed1993

Neuroepithelial carcinomas in mice transgenic with human papillomavirus type 16 E6/E7 ORFs.

The effect of the human papillomavirus (HPV) oncogenes E6 and E7 was examined in transgenic mice with a construct containing the human beta-actin promoter regulating HPV16 E6 and E7 open reading frames. In the sole line of mice that transmitted the transgene, neuroepithelial tumors appeared at 2.5 months of life, and by 10 months, 87 of 122 (71%) of the animals were dead from brain tumors. The most frequent type of tumor (74%) was an anaplastic neuroepithelial tumor associated with the ependyma of the third and fourth ventricles, which locally invaded adjacent brain tissue and spread for considerable distances along the ventricular surface. The other two types of tumor were well-differentiated choroid plexus carcinomas (26%) and rare pituitary carcinomas (8.7%). HPV16 E6 RNA and E7 oncoprotein expression were demonstrated in tumor tissue and primary cell lines derived from the tumors. Examination of two tumor suppressor gene products, the retinoblastoma protein and p53, known to bind to HPV16 E7 and E6 oncoproteins, respectively, showed both were expressed in the primary tumor cell lines. These data support a causative role for the HPV oncoproteins in epithelial carcinogenesis.

Animals↗
PubMed1992

Efficiency of binding the retinoblastoma protein correlates with the transforming capacity of the E7 oncoproteins of the human papillomaviruses.

The human papillomaviruses (HPVs) associated with genital tract lesions can be classified as either "high risk" or "low risk" based on their association with human anogenital cancer. The E7 proteins of the high-risk and the low-risk viruses are quite similar in their amino acid composition and structural organization yet differ in their transforming potential and in a number of biochemical properties. A series of chimeric proteins consisting of segments of the high-risk HPV-16 and the low-risk HPV-6 E7 proteins were constructed in order to define which domains within the amino-terminal half of E7 were responsible for the different biological and biochemical properties. The E7 oncogenic capacity, which was determined by assaying transformation of baby rat kidney cells in cooperation with an activated ras oncogene, segregated with the retinoblastoma tumor suppressor protein (pRB) binding domain of the HPV-16 E7 protein. A comparison of the pRB binding sites of the sequenced genital tract HPVs revealed a consistent amino acid difference (aspartic acid/glycine) between the high-risk and low-risk viruses. Single amino acid substitution mutations were generated at this position in the HPV-6 and HPV-16 E7 proteins, and this single amino acid residue was shown to be the principal determinant responsible for the differences in the apparent pRB binding affinity and transformation capacity distinguishing the HPV E7 proteins of the high-risk and low-risk HPVs.

Adenovirus Early Proteins↗
PubMed1992

Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins.

The E6 and the E7 proteins of the oncogenic human papillomavirus types 16 and 18 can stably associate with p53 and the retinoblastoma protein, respectively. The E6-p53 interaction results in the accelerated degradation of p53 in vitro via the ubiquitin-dependent proteolysis system. In this study we demonstrate that a fusion protein consisting of the N-terminal half of the HPV-16 E7 protein and the full length HPV-16 E6 protein promotes the in vitro degradation of the retinoblastoma protein. This indicates that the property of the HPV-16 E6 protein to stimulate the degradation of p53 can be targeted to other proteins. Unlike the HPV-16 or HPV-18 E6 protein, the E6 proteins of HPV-6 and 11 do not bind to p53 and consequently do not target p53 for degradation. Analogous E7-E6 fusion proteins using the E6 proteins of HPV-6 and HPV-11, however, also have the ability to promote the degradation of the retinoblastoma protein, indicating that the property to target associated proteins for degradation is shared by the anogenital specific HPV E6 proteins.

Base Sequence↗
PubMed1992

Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins.

Studies of adenovirus E1A oncoprotein mutants suggest that the association of E1A with the retinoblastoma protein (pRB) is necessary for E1A-mediated transformation. Mutational analysis of E1A indicates that two regions of pRB are required for E1A to form stable complexes with the retinoblastoma protein. In addition to pRB binding, these regions are necessary for E1A association with several other cellular proteins, including p130, p107, cyclin A, and p33cdk2. Here we show that short synthetic peptides containing the pRB-binding sequences of E1A are sufficient for interaction with p107, cyclin A, and p130. The E7 protein of human papillomavirus type 16 contains an element that binds to pRB and appears to be functionally homologous to the E1A sequences. Peptides containing this region of the E7 protein were able to interact with p107, cyclin A, and p130 in addition to pRB. These findings suggest that the common mechanism of transformation used by these viral oncogenes involves their association with a set of polypeptides.

Adenovirus E1A Proteins↗
PubMed1992

Structure-function analysis of the human papillomavirus type 16 E7 oncoprotein.

The E7 gene of human papillomavirus type 16 encodes a multifunctional nuclear phosphoprotein that is functionally and structurally similar to the adenovirus (Ad) E1A proteins and the T antigens of other papovaviruses. E7 can cooperate with an activated ras oncogene to transform primary rodent cells, trans activate the Ad E2 promoter, and abrogate transforming growth factor beta-mediated repression of c-myc. Recent studies suggest that these functions may in part be a consequence of the ability of E7 to associate with the product of the retinoblastoma tumor suppressor gene (pRB). In this study, a series of site-specific mutations of the human papillomavirus type 16 E7 gene product were constructed and assessed for their effects on intracellular protein stability, ras cooperativity, transcriptional trans activation, pRB association, and phosphorylation. The results of these studies indicate that the transforming and trans-activating domains extensively overlap within a region of the protein analogous to conserved region 2 of Ad E1A, suggesting that pRB binding is necessary for both activities. Deletion of sequences in conserved region 1 abrogates cellular transformation but has only a marginal effect on trans activation. These data suggest that E7 trans activation and cellular transformation are interrelated but separable functions.

Amino Acid Sequence↗
PubMed1992

Interactions of HPV E6 and E7 oncoproteins with tumour suppressor gene products.

The HPVs associated with anogenital cancers encode two oncoproteins, E6 and E7. Both E6 and E7 can form specific complexes with tumour suppressor gene products. The E7 protein binds to the retinoblastoma tumour suppressor gene product pRB, with a preference for the underphosphorylated, "active" form of pRB. The E7 proteins derived from the "high risk" HPVs bind to pRB with a higher affinity than the E7 proteins from the "low risk" HPVs. The "high risk" HPV E6 proteins can associate with the p53 tumour suppressor protein. This interaction promotes the degradation of p53 in vitro, which presumably accounts for the very low levels of p53 in cervical carcinoma cell lines. The functional inactivation of pRB and p53 by the HPV oncoproteins E7 and E6, respectively, are likely to be important steps in cervical carcinogenesis, since mutations in the RB and p53 genes were detected in HPV negative but not HPV positive cervical carcinoma cell lines. Cytogenetic studies strongly suggest, however, that additional chromosomal changes may be necessary for carcinogenic progression of HPV induced anogenital lesions.

DNA, Viral↗