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P M Howley

Publications and source records attributed to P M Howley.

At least 73 records · Page 4Linked to original sources
PubMed1992

Conserved cysteine residue in the DNA-binding domain of the bovine papillomavirus type 1 E2 protein confers redox regulation of the DNA-binding activity in vitro.

The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfhydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfhydryl group(s) may be protected by DNA binding. Sensitivity to sulfhydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

Amino Acid Sequence↗
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

Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product.

The adenovirus E1A gene product, the simian virus 40 large tumor antigen, and the human papillomavirus E7 protein share a short amino acid sequence that constitutes a domain required for the transforming activity of these proteins. These sequences are also required for these proteins to bind to the retinoblastoma gene product (pRb). Recent experiments have shown that E1A can dissociate complexes containing the transcription factor E2F bound to pRb, dependent on this conserved sequence element. We now show that the E7 protein and the simian virus 40 large tumor antigen can dissociate the E2F-pRb complex, dependent on this conserved sequence element. We also find that the E2F-pRb complex is absent in various human cervical carcinoma cell lines that either express the E7 protein or harbor an RB1 mutation, suggesting that the loss of the E2F-pRb interaction may be an important aspect in human cervical carcinogenesis. We suggest that the ability of E1A, the simian virus 40 large tumor antigen, and E7 to dissociate the E2F-pRb complex may be a common activity of these viral proteins that has evolved to stimulate quiescent cells into a proliferating state so that viral replication can proceed efficiently. In circumstances in which a lytic infection does not proceed, the consequence of this action may be to initiate the oncogenic process in a manner analogous to the mutation of the RB1 gene.

Adenovirus Early Proteins↗
PubMed1992

Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity.

The "high-risk" human papillomavirus types 16 (HPV-16) and 18 (HPV-18) have been etiologically implicated in the majority of human cervical carcinomas. In these cancers, the viral DNAs are often integrated into the host genome so that expression of the E1 and the E2 genes is lost, suggesting that disruption of these regulatory genes plays an important role in carcinogenic progression. Previous studies defining the viral genes affecting HPV-16 transformation functions have used the "prototype" viral genome, which was cloned from a human cervical carcinoma and later discovered to harbor a mutation in the E1 gene. In this study, we have corrected this mutation and have evaluated the effect of mutations of either the E1 or the E2 gene on the efficiency of HPV-16 immortalization of human keratinocytes. Mutation of either the E1 gene or the E2 gene in the background of a "wild-type" HPV-16 genome markedly increased immortalization capacity. Mutations were also generated in the E2-binding sites located upstream of the P97 promoter, which directs synthesis of the viral E6 and E7 transforming genes. E2 negatively regulates the P97 promoter through binding at adjacent sites. Surprisingly, the mutation of these sites only partially relieved the negative effect of E2 on viral immortalization, implicating additional mechanisms in the E2 repression of viral immortalization functions. Our results provide genetic evidence that the E1 and E2 gene products each can repress HPV-16 immortalization and support the hypothesis that a selective growth advantage is provided by integration of the viral genome in a manner that causes the loss of expression of either E1 or E2.

Base Sequence↗
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

The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein.

The observed interaction between p53 and the oncoproteins encoded by several DNA tumor viruses suggests that these viruses mediate their transforming activities at least in part by altering the normal growth regulatory function of p53. In this study we examined the effect of viral oncoprotein expression on the transcriptional transactivation function of wild-type p53 in human cells. Plasmids expressing human p53 were cotransfected with either SV40 large T-antigen or human papillomavirus (HPV) type 16 E6 expression plasmids and assayed for transactivation function using a reporter gene driven by a p53-responsive promoter containing multiple copies of the consensus p53 DNA binding motif, TGCCT. Both large T-antigen and E6 were able to inhibit transactivation by wild-type p53. Furthermore, SV40 T-antigen mutants that are defective for p53 binding were not able to inhibit transactivation and HPV E6 proteins that were either mutant or derived from non-oncogenic HPV types and unable to bind p53, had no effect on p53 transactivation. These results demonstrate the physiological relevance of the interaction of SV40 T-antigen and HPV E6 oncoproteins with p53 in vivo and suggest that the transforming functions of these viral oncoproteins may be linked to their ability to inhibit p53-mediated transcriptional activation.

Antigens, Polyomavirus Transforming↗
PubMed1992

Transient replication of human papillomavirus DNAs.

Information on papillomavirus DNA replication has primarily derived from studies with bovine papillomavirus type 1 (BPV-1). Our knowledge of DNA replication of the human papillomaviruses (HPVs) is quite limited, in part because of the lack of a cell culture system capable of supporting the stable replication of HPV DNA. This study demonstrates that the full-length genomic DNAs of HPV types 11 and 18 (HPV-11 and HPV-18), but not HPV-16, are able to replicate transiently after transfection into several different human squamous cell carcinoma cell lines. This system was used to identify the viral cis and trans elements required for DNA replication. The viral origins of replication were localized to a region of the viral long control region. Like BPV-1, E1 and E2 were the only viral factors required in trans for the replication of plasmids containing the origin. Cotransfection of a plasmid expressing the E1 open reading frame (ORF) from HPV-11 with a plasmid that expresses the E2 ORF from HPV-6, HPV-11, HPV-16, or HPV-18 supported the replication of plasmid DNAs containing the origin regions of HPV-11, HPV-16, or HPV-18, indicating that there are functions shared among the corresponding E1 and E2 proteins and origins of these viruses. Although HPV-16 genomic DNA did not replicate by itself under experimental conditions that supported the replication of HPV-11 and HPV-18 genomic DNAs, expression of the HPV-16 early region functions from a strong heterologous promoter supported the replication of a cotransfected plasmid containing the HPV-16 origin of replication. This finding suggests that the inability of the HPV-16 genomic DNA to replicate transiently in the cell lines tested was most likely due to insufficient expression of the viral E1 and/or E2 genes required for DNA replication.

Base Sequence↗
PubMed1992

The bovine papillomavirus constitutive enhancer is essential for viral transformation, DNA replication, and the maintenance of latency.

Bovine papillomavirus type 1 (BPV-1) has served as the prototype papillomavirus for the study of viral transcription, DNA replication, and latency. However, no cis essential transcription control regions which are necessary for both transformation and replication of BPV-1 or any other papillomavirus have yet been defined. We have found that BPV-1 mutants with deletions in the long control region were defective for transformation and replication, with the essential region in the 5' long control region corresponding to the previously defined BPV-1 constitutive enhancer (S. B. Vande Pol and P. M. Howley, J. Virol. 64:5420-5429, 1990). BPV-1 mutants deleted of the constitutive enhancer could be complemented in trans by the full-length virally encoded E2 transactivator and replication factor (E2TA) and in cis by the simian virus 40 enhancer. The constitutive enhancer induced the production of E2TA by activating all the major viral early promoters upstream of the E2 open reading frame. Complementation experiments using a temperature-sensitive E2TA mutant indicated that the constitutive enhancer was necessary for the maintenance of viral DNA replication within latently infected cells and implied that viral transcription under the regulation of the constitutive enhancer may be controlled during the cell cycle. The constitutive enhancer is a master regulatory control region for establishing and maintaining BPV-1 latency, and its characteristics reveal some analogies with cell type-specific enhancer elements recognized in the human papillomaviruses.

Animals↗
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

Interaction of the human papillomavirus type 16 E6 oncoprotein with wild-type and mutant human p53 proteins.

The E6 oncoproteins encoded by the cancer-associated human papillomaviruses (HPVs) can associate with and promote the degradation of wild-type p53 in vitro. To gain further insight into this process, the ability of HPV-16 E6 to complex with and promote the degradation of mutant forms of p53 was studied. A correlation between binding and the targeted degradation of p53 was established. Mutant p53 proteins that bound HPV-16 E6 were targeted for degradation, whereas those that did not complex HPV-16 E6 were not degraded. Since the HPV-16 E6-promoted degradation involves the ubiquitin-dependent proteolysis pathway, specific mutations were made in the amino terminus of p53 to examine whether the E6 targeted degradation involved the N-end rule pathway. No requirement for destabilizing amino acids at the N terminus of p53 was found, nor was evidence found that HPV-16 E6 could provide this determinant in trans, indicating that the N-terminal rule pathway is not involved in the E6-promoted degradation of p53.

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↗
PubMed1992

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.

Amino Acid Sequence↗
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

Role of the human papillomaviruses in human cancer.

The papillomaviruses associated with human anogenital carcinomas encode two transforming genes, E6 and E7. The oncoprotein products of these two genes complex with the tumor suppressor gene products p53 and pRB, respectively. The loss of the normal function of these tumor suppressor gene products, either as a consequence of their association with E6 and E7 or by mutation, appears to be a common event in human cervical carcinogenesis.

Female↗
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

A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18.

The E6 protein of human papillomavirus types 16 and 18 (HPV-16 and HPV-18) can stably associate with the p53 protein in vitro. In the presence of rabbit reticulocyte lysate, this association leads to the specific degradation of p53 through the ubiquitin-dependent proteolysis system. We have examined the E6-p53 complex in more detail and have found that association of E6 with p53 is mediated by an additional cellular factor. This factor is present in rabbit reticulocyte lysate, primary human keratinocytes and in each of five human cell lines examined. The factor is designated E6-AP, for E6-associated protein, based on the observation that the E6 proteins of HPV-16 and 18 can form a stable complex with the factor in the absence of p53, whereas p53 association with the factor can be detected only in the presence of E6. Gel filtration and coprecipitation experiments indicate that E6-AP is a monomeric protein of approximately 100 kDa.

Animals↗