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Biomedical subjects

K Münger

Publications and source records attributed to K Münger.

At least 19 recordsLinked to original sources
PubMed2001

Biological activities and molecular targets of the human papillomavirus E7 oncoprotein.

The human papillomavirus (HPV) E7 protein is one of only two viral proteins that remain expressed in HPV-associated human cancers. HPV E7 proteins share structural and functional similarities with oncoproteins encoded by other small DNA tumor viruses such as adenovirus E1A and SV40 large tumor antigen. The HPV E7 protein plays an important role in the viral life cycle by subverting the tight link between cellular differentiation and proliferation in normal epithelium, thus allowing the virus to replicate in differentiating epithelial cells that would have normally withdrawn from the cell division cycle. The transforming activities of E7 largely reflect this important function.

Cell Transformation, Neoplastic↗
PubMed2001

The HPV E7 oncoprotein inhibits tumor necrosis factor alpha-mediated apoptosis in normal human fibroblasts.

Tumor necrosis factor-alpha (TNF) is a cytokine that induces programmed cell death, apoptosis, in a number of cell types and is employed by cytotoxic T cells to eliminate virus infected cells. Consequently, many viruses have acquired mechanisms to undermine these host cell defense mechanisms and cause resistance to TNF-mediated apoptosis. Here we show that normal human diploid fibroblasts that express the human papillomavirus type 16 E7 oncoprotein have a decreased propensity to undergo apoptosis in response to TNF treatment. The ability of E7 to undermine TNF-mediated apoptosis correlates with cellular transformation. While E7 does not generally subvert signaling by tumor necrosis factor receptor 1, pro-caspase 8 activation is decreased in E7-expressing cells. E7 also provides some protection from apoptosis caused by stimulation of the TNF receptor 1-related cytokine receptor Fas, where induction of apoptosis occurs much slower in this cell type. Hence, E7-expressing normal human fibroblasts exhibit a specific defect that obstructs cytokine-mediated activation of pro-caspase 8 and apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The cytokines tumor necrosis factor-alpha (TNF-alpha ) and TNF-related apoptosis-inducing ligand differentially modulate proliferation and apoptotic pathways in human keratinocytes expressing the human papillomavirus-16 E7 oncoprotein.

Keratinocytes are the natural target cells for infection by human papillomaviruses (HPVs), most of which cause benign epithelial hyperplasias (warts). However, a subset of papillomaviruses, the "high risk" HPVs, cause lesions that can progress to carcinomas. Inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) and TNF-related apoptosis-inducing ligand (TRAIL) are produced by cells in response to a viral infection. To determine the effects of TNF-alpha and TRAIL on keratinocytes expressing the high risk HPV-16 oncoprotein E7, human foreskin keratinocytes stably expressing E7 were treated with TNF-alpha and TRAIL. Treatment with TNF-alpha alone, but not TRAIL, induced growth arrest and differentiation in keratinocytes that was almost completely overcome by expression of HPV-16 E7. Both cytokines induced apoptosis when administered in combination with the protein synthesis inhibitor cycloheximide, but the apoptotic response to TRAIL was significantly more rapid and efficient compared with the response seen after TNF-alpha treatment. HPV-16 E7-expressing keratinocytes were more prone to both TNF-alpha- and TRAIL-mediated apoptosis compared with vector-infected controls.

Apoptosis Regulatory Proteins↗
PubMed2001

Human papillomavirus type 16 E7 oncoprotein-induced abnormal centrosome synthesis is an early event in the evolving malignant phenotype.

Genomic instability is a hallmark of malignant growth that frequently involves mitotic defects associated with centrosome abnormalities. However, the question of whether abnormal centrosomes cause genomic instability or develop secondary to other changes has not been conclusively resolved. Here we show that human papillomavirus (HPV)-16 E7 can induce abnormal centrosome synthesis before the development of extensive nuclear abnormalities. In contrast, expression of HPV-16 E6 is associated with marked nuclear atypia and concomitant accumulation of centrosomes. Our results demonstrate that HPV-16 E7-induced centrosome abnormalities represent an early event during neoplastic progression potentially driving genomic destabilization.

Cell Nucleus↗
PubMed2001

Centrosome abnormalities, genomic instability and carcinogenic progression.

Centrosome abnormalities are a frequent finding in various malignant tumors. Since centrosomes form the poles of the mitotic spindle, these abnormalities have been implicated in chromosome missegregation and the generation of aneuploid cells which is commonly found in many human neoplasms. It is a matter of debate, however, whether centrosome alterations can drive cells into aneuploidy or simply reflect loss of genomic integrity by other mechanisms. Since these two models have fundamentally different implications for the diagnostic and prognostic value of centrosome abnormalities, we will discuss the relevance of abnormal centrosomes in the context of different oncogenic events as exemplified by high-risk human papillomavirus-associated carcinogenesis.

Aneuploidy↗
PubMed2001

Degradation of the retinoblastoma tumor suppressor by the human papillomavirus type 16 E7 oncoprotein is important for functional inactivation and is separable from proteasomal degradation of E7.

The steady-state level and metabolic half-life of retinoblastoma tumor suppressor protein pRB are decreased in cells that express high-risk human papillomavirus (HPV) E7 proteins. Here we show that pRB degradation is a direct activity of E7 and does not reflect a property of cell lines acquired during the selection process for E7 expression. An amino-terminal domain of E7 that does not directly contribute to pRB binding but is required for transformation is also necessary for E7-mediated pRB degradation. Treatment with inhibitors of the 26S proteasome not only blocks E7-mediated pRB degradation but also causes the stabilization of E7. Mutagenic analyses, however, reveal that the processes of proteasomal degradation of E7 and pRB are not linked processes. HPV type 16 E7 also targets the pRB-related proteins p107 and p130 for destabilization by a proteasome-dependent mechanism. Using the SAOS2 flat-cell assay as a biological indicator for pRB function, we demonstrate that pRB degradation, not solely binding, is important for the E7-induced inactivation of pRB.

Cysteine Endopeptidases↗
PubMed2001

Centrosome abnormalities and genomic instability by episomal expression of human papillomavirus type 16 in raft cultures of human keratinocytes.

Primary human keratinocytes with ectopic expression of high-risk human papillomavirus (HPV) E6 and E7 oncoproteins display abnormal centrosome numbers, multipolar mitoses, and aneusomy. However, it has not been explored whether these abnormalities can occur in cells containing HPV episomes where E6 and E7 expression is under viral transcriptional control. Here, we demonstrate that centrosome abnormalities and genomic instability occur in organotypic raft cultures of human keratinocytes with episomal HPV-16 even at low copy numbers. We conclude that HPV-16 DNA, when maintained as an episome, can disturb centrosome homeostasis and subvert genomic integrity of the host cell during early stages of the viral infection.

Cells, Cultured↗
PubMed1999

Immortalization of primary human keratinocytes by the helix-loop-helix protein, Id-1.

Basic helix-loop-helix (bHLH) DNA-binding proteins have been demonstrated to regulate tissue-specific transcription within multiple cell lineages. The Id family of helix-loop-helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins. Overexpression of Id proteins within a variety of cell types has been shown to inhibit their ability to differentiate under appropriate conditions. We demonstrate that ectopic expression of Id-1 leads to activation of telomerase activity and immortalization of primary human keratinocytes. These immortalized cells have a decreased capacity to differentiate as well as activate phosphorylation of the retinoblastoma protein. Additionally, these cells acquire an impaired p53-mediated DNA-damage response as a late event in immortalization. We conclude that bHLH proteins play a pivotal role in regulating normal keratinocyte growth and differentiation, which can be disrupted by the immortalizing functions of Id-1 through activation of telomerase activity and inactivation of the retinoblastoma protein.

Catalytic Domain↗
PubMed1999

TID1, a human homolog of the Drosophila tumor suppressor l(2)tid, encodes two mitochondrial modulators of apoptosis with opposing functions.

Mitochondria have emerged as central regulators of apoptosis. Here, we show that TID1, a human homolog of the Drosophila tumor suppressor lethal (2) tumorous imaginal discs, l(2)tid, encodes two mitochondrial matrix proteins, designated hTid-1(L) and hTid-1(S). These splice variants are both highly conserved members of the DnaJ family of proteins, which regulate the activity of and confer substrate specificity to Hsp70 proteins. Both hTid-1(L) and hTid-1(S) coimmunoprecipitate with mitochondrial Hsp70. Expression of hTid-1(L) or hTid-1(S) have no apparent capacity to induce apoptosis but have opposing effects on apoptosis induced by exogenous stimuli. Expression of hTid-1(L) increases apoptosis induced by both the DNA-damaging agent mitomycin c and tumor necrosis factor alpha. This activity is J domain-dependent, because a J domain mutant of hTid-1(L) can dominantly suppress apoptosis. In sharp contrast, expression of hTid-1(S) suppresses apoptosis, whereas expression of a J domain mutant of hTid-1(S) increases apoptosis. Hence, we propose that TID1 gene products act to positively and negatively modulate apoptotic signal transduction or effector structures within the mitochondrial matrix.

Alternative Splicing↗
PubMed1999

Expression of the HPV E7 oncoprotein mimics but does not evoke a p53-dependent cellular DNA damage response pathway.

Acute expression of the human papillomavirus E7 oncoprotein in preimmortal human fibroblasts induces changes in the abundances of multiple cellular regulatory proteins. These alterations include a destabilization of the retinoblastoma tumor suppressor protein pRB, stabilization of the tumor suppressor protein p53, and increases in the level of the cyclin-dependent kinase inhibitor p21(cip1). Since the HPV E7 oncoproteins can interfere with several cell cycle checkpoints and similar alterations in the levels of pRB, p53, and p21(cip1) are also observed in a p53-dependent response to DNA damage, we investigated whether E7 expression triggers this signal transduction pathway. The results demonstrate that E7-mediated destabilization of pRB does not require p53 activity and is independent of the ability of E7 to induce apoptosis. Moreover, E7-mediated increases in p21(cip1) levels are largely p53-independent and involve stabilization of the p21(cip1) protein. In contrast the decreases in pRB expression in response to DNA damage involve transcriptional downregulation of RB gene expression.

Apoptosis↗
PubMed1998

Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2'-deoxycytidine treatment induces a senescence-like state.

We have previously reported that a set of oral squamous cell carcinoma lines express specifically elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 amplification and expresses functional p16ink4a, the other cell lines express undetectable levels of p16ink4a, despite a lack of coding-region mutations. Two of the cell lines, SCC15 and SCC40 have a hypermethylated p16ink4A promoter and a third cell line, SCC9, has a mutation in the p16ink4a promoter. Using the demethylation agent 5-aza-2'-deoxycytidine, we showed that the p16ink4a protein was re-expressed after a 5-day treatment with this chemical. One cell line, SCC15 expressed high levels of p16ink4a. In this line, cdk6 activity was decreased after 5-aza-2'deoxycytidine treatment, and the hypophosphorylated, growth suppressive form of the retinoblastoma tumor suppressor protein pRB was detected. Expression of p16ink4a persisted, even after the drug was removed and the cells expressed senescence-associated beta-galactosidase activity. Ectopic expression of p16ink4a with a recombinant retrovirus in this cell line also induced a similar senescence-like phenotype. Hence, it was possible to restore a functional pRB pathway in an oral squamous cell carcinoma line by inducing re-expression of endogenous p16ink4a in response to treatment with a demethylating agent.

Azacitidine↗
PubMed1998

A novel human DnaJ protein, hTid-1, a homolog of the Drosophila tumor suppressor protein Tid56, can interact with the human papillomavirus type 16 E7 oncoprotein.

We have cloned hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, by virtue of its ability to form complexes with the human papillomavirus E7 oncoprotein. The carboxyl terminal cysteine-rich metal binding domain of E7 is the major determinant for interaction with hTid-1. The carboxyl terminus of E7 is essential for the functional and structural integrity of E7 and has previously been shown to function as a multimerization domain. The hTid-1 protein is a member of the DnaJ-family of chaperones. Its mRNA is widely expressed in human tissues, including the HPV-18-positive cervical carcinoma cell line HeLa and human genital keratinocytes, the normal host cells of the HPVs. The hTid-1 gene has been mapped to the short arm of chromosome 16. The large tumor antigens of polyomaviruses encode functional J-domains that are important for viral replication as well as cellular transformation. The ability of HPV E7 to interact with a cellular DnaJ protein suggests that these two viral oncoproteins may target common regulatory pathways through J-domains.

Amino Acid Sequence↗
PubMed1998

Alterations in cyclin-dependent kinase 2 function during differentiation of primary human keratinocytes.

Terminal differentiation of epithelial cells is intimately linked to cell-cycle withdrawal. The tight coupling of these two processes is critical to maintenance of epidermal tissue homeostasis and is frequently disrupted in squamous cell carcinoma. To identify possible molecular targets of epithelial carcinogenesis, we investigated the regulatory pathways that couple cellular differentiation and proliferation in primary cultures of human keratinocytes and found that the cyclin-dependent kinase inhibitors (CKIs) p21cip1/waf1 and p27kip1 were induced early during differentiation of human keratinocytes, whereas p15ink4B was induced later in differentiation. The induction of p21c1/waf1 was mediated by both transcriptional and non-transcriptional mechanisms, and the activities of cyclin A/cyclin-dependent kinase (cdk) 2 and cyclin E/cdk2 complexes were specifically inhibited during keratinocyte differentiation. In contrast, p21cip1/wafl did not associate with cdk4, and the activities of cdk4 complexes remained unchanged. Hence, our results support the model that multiple CKIs participate in linking cellular proliferation and differentiation in human keratinocytes by specific modulation of cdk2 activity.

Animals↗
PubMed1998

Human papillomaviruses and associated malignancies.

The human papillomaviruses (HPVS) are small DNA tumor viruses that infect epithelial cells and induce proliferative lesions. Substantial epidemiologic data along with in vitro and in vivo studies have led to the implication of particular HPVs with the development of epithelial malignancies. Greater than 90% of all cervical carcinomas are positive for HPV infection. Most of these lesions are caused by infection with mucosal-associated high-risk HPV subtypes. Much work has been undertaken in basic science laboratories to determine the molecular basis for HPV-associated malignancies. Although many significant advances have been made in understanding the biologic properties of these viruses using in vitro analyses, the field has been greatly hindered until recently by the inability to propagate the virus in culture. In this review, we discuss the basic biologic properties of HPVs and the current understanding of the mechanisms of cellular transformation by malignancy-associated viral subtypes. We place particular emphasis on discussion of the HPV oncogenes, E6 and E7. We also discuss premalignant and malignant disorders of squamous and mucosal epithelia, which have been associated with HPV infections, and the current understanding of the mechanism of HPV-associated carcinogenesis in these settings. We focus these discussions on cervical carcinogenesis and briefly review the particulars regarding HPV-associated malignancies in normal and immunocompromised hosts. We end with a discussion of potential targeted molecular therapies for HPV-associated malignancies that may result from the current knowledge of HPV-related cellular growth dysregulation and carcinogenesis.

Cell Transformation, Viral↗
PubMed1997

The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints.

The E6 and E7 proteins of the high risk human papillomaviruses (HPVs) are consistently expressed in HPV-positive cervical carcinomas. We investigated the ability of HPV-16 E6 and E7 to disrupt mitotic checkpoints in normal diploid human cells. Acute expression of HPV-16 E6, but not HPV-16 E7, decreased the fidelity of multiple checkpoints controlling entry into and exit from mitosis. After irradiation, nearly 50% of cells containing HPV-16 E6 readily entered mitosis as opposed to less than 10% of control cells. Consistent with this, asynchronous populations of cells expressing HPV-16 E6 had increased cdc2-associated histone H1 kinase activity relative to control populations. In addition, HPV-16 E6 increased sensitivity to chemically-induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function relative to control populations. HPV-16 E6 mutants with a reduced ability to target p53 for degradation were unable to abrogate mitotic checkpoints, suggesting a possible mechanism by which HPV-16 E6 disrupts mitotic checkpoints. Expression of a mutant p53 gene yielded an intermediate phenotype relative to HPV-16 E6, generating moderate increases in sensitivity to chemically-induced S-phase PCC and mitotic spindle disruption and a heightened propensity to enter mitosis after irradiation.

CDC2 Protein Kinase↗
PubMed1997

Destabilization of the RB tumor suppressor protein and stabilization of p53 contribute to HPV type 16 E7-induced apoptosis.

Cells that express the human papillomavirus (HPV) type 16 E7 oncoprotein are predisposed to undergo apoptosis. Transgenic mice that have E7 expression targeted to either the retinal photoreceptor cells or the lens cells exhibit signs of apoptosis in cells attempting to undergo differentiation. We established a cell culture system to study this process and have determined the domains of E7 that are required for predisposing cells to undergo apoptosis in response to growth arrest signals. Regions within the core pRB binding site of E7 were necessary but not sufficient for inducing apoptosis. Residues within the adenovirus conserved region 1 homology domain and the consensus casein kinase II phosphorylation site are also important for this effect on cell viability. Our data also demonstrate that the ability of E7 to induce destabilization of pRB and stabilization of p53 coincides with E7-mediated transformation and apoptosis.

Animals↗