PubMed Health⌕ Search

Biomedical subjects

K Münger

Publications and source records attributed to K Münger.

At least 55 records · Page 3Linked to original sources
PubMed1992

Transforming growth factor beta 1 regulation of c-myc expression, pRB phosphorylation, and cell cycle progression in keratinocytes.

Transforming growth factor beta 1 (TGF-beta 1) is a potent inhibitor of cellular proliferation in a variety of cell types, including skin keratinocytes. TGF-beta 1 suppression of c-myc transcription has been implicated in the mechanism of TGF-beta 1 inhibition of keratinocytes, and evidence suggests that the protein product of the retinoblastoma gene (pRB) is a necessary component in this pathway. Following growth factor stimulation of quiescent keratinocytes, TGF-beta 1 can inhibit cell cycle progression into S phase at any point prior to the G1-S transition but does not inhibit progression through the S phase of the cell cycle. Since pRB is hypophosphorylated during G1 and hyperphosphorylated during S and G2, the G1-S-specific phosphorylation of pRB becomes an attractive target for the growth-inhibitory activities of TGF-beta 1. However, in TGF-beta 1-treated primary human keratinocytes and in a series of human papilloma virus and SV40 immortalized human keratinocyte cell lines, the phosphorylation status of pRB strictly correlated with cell growth. No evidence was found for a direct effect of TGF-beta 1 on the phosphorylation state of pRB in these cells. It was further demonstrated that synthesis of c-myc protein can be rapidly inhibited by TGF-beta 1 addition throughout G1 and S phases, indicating that the phosphorylation state of pRB, at least as it varies during the cell cycle, does not alter the ability of TGF-beta 1 to suppress c-myc expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor beta 1 and by the retinoblastoma gene product.

Previous studies have shown that transforming growth factor beta 1 (TGF-beta 1) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter as effectively as TGF-beta 1. The same c-myc promoter region was required for regulation by both TGF-beta 1 and pRB. These sequences, termed the TGF-beta control element (TCE), lie between positions -86 and -63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-beta 1 treatment of TGF-beta-sensitive but not TGF-beta-insensitive cells. These data indicate that pRB can suppress c-myc transcription and suggest the involvement of cellular factors in addition to pRB in the TGF-beta 1 pathway for the inhibition of c-myc transcription and growth inhibition.

Animals↗
PubMed1991

The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines.

Human cervical carcinoma cell lines that were either positive or negative for human papillomavirus (HPV) DNA sequences were analyzed for evidence of mutation of the p53 and retinoblastoma genes. Each of five HPV-positive cervical cancer cell lines expressed normal pRB and low levels of wild-type p53 proteins, which are presumed to be altered in function as a consequence of association with HPV E7 and E6 oncoproteins, respectively. In contrast, mutations were identified in the p53 and RB genes expressed in the C-33A and HT-3 cervical cancer cell lines, which lack HPV DNA sequences. Mutations in the p53 genes mapped to codon 273 and codon 245 in the C33-A and HT-3 cell lines, respectively, located in the highly conserved regions of p53, where mutations appear in a variety of human cancers. Mutations in RB occurred at splice junctions, resulting in in-frame deletions, affecting exons 13 and 20 in the HT-3 and C-33A cell lines, respectively. These mutations resulted in aberrant proteins that were not phosphorylated and were unable to complex with the adenovirus E1A oncoprotein. These results support the hypothesis that the inactivation of the normal functions of the tumor-suppressor proteins pRB and p53 are important steps in human cervical carcinogenesis, either by mutation or from complex formation with the HPV E6 and E7 oncoproteins.

Base Sequence↗
PubMed1991

c-myc and pRB: role in TGF-beta 1 inhibition of keratinocyte proliferation.

The TGF-beta s are potent inhibitors of proliferation of most cell types in culture and in vivo. Previous studies have demonstrated that TGF-beta inhibition of skin keratinocyte proliferation involves suppression of c-myc transcription. Evidence derived from use of expression plasmids for certain DNA viral oncoproteins has suggested that the retinoblastoma gene (RB) may be involved in this process. Transient expression of pRB, like TGF-beta 1, in skin keratinocytes represses expression of a human c-myc reporter plasmid, and the same c-myc promoter region (TCE) is required for repression by either TGF-beta 1 or pRB. We showed here that proliferation and c-myc expression in a cell line lacking normal pRB (DU145 human prostate adenocarcinoma cells) are not inhibited by TGF-beta 1. Oligonucleotides containing the TCE were found to bind to a cellular protein of approximately 106 kD (termed p106) in Southwestern assays, utilizing extracts from both the skin keratinocytes and DU145 cells. TCE binding to p106 was diminished by TGF-beta in TGF-beta-sensitive skin keratinocytes but not in TGF-beta-insensitive SV40-transformed keratinocytes. These data support the hypothesis that pRB is required for TGF-beta 1 suppression of c-myc transcription and suggest the involvement of a cellular factor(s) in addition to pRB in the TGF-beta 1 pathway for inhibition of c-myc transcription and growth inhibition.

Animals↗
PubMed1991

Analysis of trans activation by human papillomavirus type 16 E7 and adenovirus 12S E1A suggests a common mechanism.

The human papillomavirus E7 gene product is an oncoprotein with properties similar to those of the adenovirus E1A proteins. The human papillomavirus E7 proteins possess substantial amino acid sequence similarity to portions of conserved regions 1 and 2 of E1A, and the human papillomavirus type 16 E7 protein trans-activates the adenovirus E2 early promoter. Analysis of point mutations in the E2 promoter indicated that the E2F recognition sites were critical to E7 stimulation. In contrast to the activation of the E2 promoter, E7 could not trans-activate various other E1A-inducible promoters. Although the promoter specificity for E7 differs from that of 13S E1A trans activation, it is very similar to activation by the E1A 12S product. Moreover, analysis of the E7 protein has suggested that amino acid sequences critical for trans activation include those shared with E1A within conserved region 2. Biochemical studies demonstrate that the E7 protein, like the 12S E1A product, can alter the interaction of cellular factors with the E2F transcription factor. We therefore conclude that E7 trans activation is functionally related to that mediated by the 12S E1A product.

Adenovirus Early Proteins↗
PubMed1991

Biochemical and biological differences between E7 oncoproteins of the high- and low-risk human papillomavirus types are determined by amino-terminal sequences.

Differences in the biological characteristics of the high-risk human papillomavirus type 16 (HPV-16) and the low-risk HPV-6 E7 proteins were analyzed and shown to correlate with certain biochemical properties. To ascertain which region of E7 conferred these properties, chimeric E7 genes were constructed by the exchange of the amino and carboxyl coding halves of the HPV-6 and HPV-16 E7 genes. The amino-terminal half of E7 determined the affinity for binding to the retinoblastoma protein pRB, the transformation properties, and the ability to abrogate transforming growth factor beta-mediated repression of the c-myc promoter. This region of E7 is therefore responsible for the biological and biochemical differences between the E7 proteins of the low-risk and the high-risk HPVs and consequently is one of the critical determinants distinguishing these two groups of viruses. Transcriptional transactivation of the adenovirus E2 promoter, in contrast, was a property shared by E7 proteins of both low-risk and high-risk HPVs.

Adenovirus Early Proteins↗
PubMed1991

Cellular targets of the oncoproteins encoded by the cancer associated human papillomaviruses.

Insight into the mechanisms by which DNA tumor viruses transform cells has come from the recognition that the virus-encoded oncoproteins interact specifically with important cell regulatory proteins. The "high risk" human papillomaviruses such as HPV-16 and HPV-18 which are associated with human anogenital carcinomas encode two transforming genes (E6 and E7) which are expressed in HPV positive cancers and derived cell lines. E7 shares functional and structural features with the adenovirus E1A proteins. Like Ad E1A and the large T proteins of the polyomaviruses, E7 can complex pRB. The E7 proteins of the "high risk" HPVs associate with pRB with approximately a 10-fold higher affinity than do the E7 proteins of the "low risk" HPVs, and important biological differences between the E7 proteins of these two groups of HPVs are determined by amino-terminal sequences which include the pRB binding domain. Like SV40 large T and Ad 5 E1B, the E6 oncoprotein encoded by the "high risk" HPVs can form a complex with p53. In vitro, E6 promotes the degradation of p53 and this degradation involves the ubiquitin-dependent protease system. The selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant acting oncoproteins. The relevance of the inactivation of the normal functions of pRB and p53 in human cervical carcinogenesis has recently been demonstrated by the analysis of these two genes and their products in a series of HPV-positive and HPV-negative cell lines. Each of five HPV-positive cervical cancer cell lines expressed normal pRB and low levels of wild type p53 proteins, which are presumed to be altered in function as a consequence of association with the HPV oncoproteins. In contrast, mutations were identified in the p53 and RB genes expressed in the HPV-negative cervical carcinoma cell lines, C33-A and HT-3. These results support the hypothesis that the inactivation of the normal functions of the tumor suppressor proteins pRB and p53 are important steps in human cervical carcinogenesis, either by mutation or through complex formation with HPV E6 and E7 oncoproteins.

Cell Line↗
PubMed1991

TGF beta regulation of epithelial cell proliferation: role of tumor suppressor genes.

Transforming growth factor beta 1 (TGF beta 1) is the prototype of a large family of polypeptides involved in growth control, extracellular matrix production, and development. The TGF beta s have marked stimulatory effects on connective tissue formation. They are chemotactic for fibroblasts, indirect mitogens for certain mesenchymal cells and stimulators of extracellular matrix deposition. The TGF beta s are also potent inhibitors of proliferation of most cell types in culture, and in vivo studies have indicated that the predominant effect of TGF beta 1 on cell proliferation is inhibition. We have investigated the mechanism of TGF beta 1 inhibition of skin keratinocyte growth. Earlier studies demonstrated that TGF beta 1 inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence suggested that the product of the retinoblastoma tumor susceptibility gene, pRB, may be involved in this process. More recently, we have shown that transient expression of pRB in skin keratinocytes can repress human c-myc promoter/CAT transcription as effectively as TGF beta 1. The same c-myc promoter region, termed the TGF beta control element (TCE), was required for regulation by both TGF beta 1 and pRB. Oligonucleotides containing the TCE bound to several nuclear factors in mobility shift assays and a cellular protein of approximately 106 kD in Southwestern assays. Binding of these factors could be demonstrated in cells with or without normal pRB, and the binding of some factors was rapidly inhibited by TGF beta 1 treatment of TGF beta-sensitive but not TGF beta-insensitive cells. These data indicate that pRB can function to inhibit c-myc transcription and suggest the involvement of cellular factor(s) in addition to pRB in the TGF beta 1 pathway for suppression of c-myc transcription. Studies with other cell types have shown that another tumor suppressor gene, p53, can also regulate transcription of c-myc in transient assays. Whereas wild type p53 markedly suppressed transcription, four different mutant p53 clones caused transactivation. The data support the hypothesis that pRB and p53 can both cause growth inhibition by blocking the expression of the protooncogene, c-myc, and indicate that tumor suppressor genes may function in the response pathway for diffusible negative growth regulators such as TGF beta.

Animals↗
PubMed1990

TGF-beta 1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains.

TGF-beta 1 is demonstrated to inhibit skin keratinocyte proliferation when added during the G1 phase of the cell cycle. Human foreskin keratinocytes transformed with either HPV-16 or -18 or SV40, however, were resistant to the growth inhibitory effects of TGF-beta 1. Since TGF-beta 1 appears to inhibit keratinocyte growth through down-regulation of c-myc, it was hypothesized that these DNA tumor viruses might be modulating the response to TGF-beta 1 via this pathway. Transient expression of proteins HPV-16 E7, adenovirus type 5 E1A, and SV40 large T antigen is demonstrated to block TGF-beta 1 suppression of c-myc transcription. This effect was not observed with DNA tumor virus transforming proteins mutated in their pRB binding domain. These observations indicate that pRB or another protein that interacts with this binding domain mediates TGF-beta 1 regulation of c-myc gene expression and growth inhibition.

Adenovirus Early Proteins↗
PubMed1989

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line.

Amino Acid Sequence↗
PubMed1989

Luminescence emission from Neurospora copper metallothionein. Time-resolved studies.

The luminescence lifetime of Cu-metallothionein from the fungus Neurospora crassa has been studied by the frequency-domain emission technique. Lifetimes of 10.3 and 3.4 microseconds have been found for the protein in the absence and in the presence of oxygen respectively. Binding of Hg(II) results in a quenching of the luminescence correlated to the shortening of lifetime to 0.3-0.4 microsecond. No quenching by oxygen is found for the Hg(II)-Cu-metallothionein adduct. By analogy to model compounds, luminescence emission is attributed to a triplet excited state of a Cu(I)-to-sulphur charge-transfer complex. The comparison of bimolecular quenching constants for O2 and acrylamide indicates a highly compact structure of the protein.

Luminescent Measurements↗
PubMed1989

The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product.

Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.

Adenovirus Early Proteins↗
PubMed1989

The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes.

The early human papillomavirus type 16 genes that directly participate in the in vitro transformation of primary human keratinocytes have been defined. In the context of the full viral genome, mutations in either the E6 or E7 open reading frame completely abrogated transformation of these cells. Mutations in the E1, E2, and E2-E4 open reading frames, on the other hand, had no effect. Thus, both the full-length E6 and E7 genes were required for the induction of keratinocyte immortalization and resistance to terminal differentiation. The E6 and E7 genes expressed together from the human beta-actin promoter were sufficient for this transformation; mutation of either gene in the context of this recombinant plasmid eliminated the ability to induce stable differentiation-resistant transformants.

Cell Transformation, Viral↗
PubMed1989

Molecular mechanisms of transformation by the human papillomaviruses.

A variety of studies have now indicated that the papillomaviruses associated with cervical carcinoma, HPV-16 and HPV-18, contain two genes (E6 and E7) which have transforming properties. These genes are generally expressed in cervical carcinoma cells. The HPV-16 E7 protein has recently been shown to be a multi-functional protein possessing both transcriptional modulatory and cellular transforming properties similar to those described for adenovirus E1A proteins (1). E7 is able to transactivate the adenovirus E2 promoter and can cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The N-terminal 37 amino acids of all of the E7 proteins of the genital associated HPVs contain regions which are highly conserved and which are quite similar to portions of conserved domains 1 and 2 of adenovirus E1A. These domains in E1A are critical for cellular transformation properties and contain the amino acid sequences involved in binding the product of the retinoblastoma tumor suppressor gene (pRB). Results from a collaborative study with Ed Harlow and Nick Dyson (Cold Spring Harbor Laboratory) have shown that the E7 oncoprotein of HPV-16 can associate with the retinoblastoma gene product in vitro (2). The ability of the E7 proteins encoded by various HPVs to bind pRB has been examined using an in vitro complexing assay. E7 is not sufficient for transformation of human keratinocytes. The co-operation of the HPV-16 E6 and E7 genes has been shown to be important for transformation of these cells (3). Potential intracellular protein targets for E6 are being assessed.

Animals↗
PubMed1988

The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A.

Clinical and epidemiological data have implicated the human papillomaviruses (HPVs) as having an etiologic role in some anogenital malignancies, with HPV-16 being most frequently (greater than 60%) detected in cervical carcinoma. HPV-16 is actively transcribed in the cancers; the most abundant transcripts map to the E6 and E7 early open reading frames. Evidence is presented that the HPV-16 E7 open reading frame encodes transcriptional transactivation and cellular transformation functions analogous to those of adenovirus E1A proteins. Specifically, the HPV-16 E7 gene product could transactivate the adenovirus E2 promoter and cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The E7 transforming function differed somewhat from that of adenovirus E1A in that E7 was also able to transform established mouse cells. Examination of the amino acid sequence of HPV-16 E7 revealed striking similarities with conserved domains 1 and 2 of adenovirus E1A proteins.

Adenoviruses, Human↗