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Biomedical subjects

P M Howley

Publications and source records attributed to P M Howley.

At least 55 records · Page 3Linked to original sources
PubMed1996

In vivo ubiquitination and proteasome-mediated degradation of p53(1).

The levels of the tumor suppressor protein p53 are generally quite low in normal cells, due in part to its rapid turnover. Previous studies have implicated ubiquitin-dependent proteolysis in the turnover of wild-type p53 but have not established whether or not p53 is itself a substrate of the ubiquitin system. In this study, inhibitors of the 26S proteasome have been used to further explore the role of ubiquitin proteolysis in regulating p53 turnover. Increased levels of the tumor suppressor protein p53 were observed in normal cells, as well as in cells expressing the human papillomavirus 16 E6 oncoprotein, on exposure of the cells to proteasome inhibitors. Pulse-chase experiments indicated that the increased p53 levels resulted from stabilization of the protein. Furthermore, ubiquitin-p53 conjugates were detected in untreated as well as gamma-irradiated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of p53. Increased levels of the cyclin:cyclin-dependent kinase inhibitor p21, a downstream effector of p53 function, were also observed in proteasome inhibitor-treated cells, and this increase was due in part to an increase in p2l mRNA.

Acetylcysteine↗
PubMed1996

Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity.

Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity.

Animals↗
PubMed1996

Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus type 16 transgenic mice.

High-risk human papillomaviruses (HPVs), including type 16, have been identified as factors in cervical carcinogenesis. However, the presence and expression of the virus per se appear to be insufficient for carcinogenesis. Rather, cofactors most likely are necessary in addition to viral gene expression to initiate neoplasia. One candidate cofactor is prolonged exposure to sex hormones. To examine the possible effects of estrogen on HPV-associated neoplasia, we treated transgenic mice expressing the oncogenes of HPV16 under control of the human keratin-14 promoter (K14-HPV16 transgenic mice) and nontransgenic control mice with slow release pellets of 17beta-estradiol. Squamous carcinomas developed in a multistage pathway exclusively in the vagina and cervix of K14-HPV16 transgenic mice. Estrogen-induced carcinogenesis was accompanied by an incremental increase in the incidence and distribution of proliferating cells solely within the cervical and vaginal squamous epithelium of K14-HPV16 mice. Expression of the HPV transgenes in untreated transgenic mice was detectable only during estrus; estrogen treatment resulted in transgene expression that was persistent but not further upregulated, remaining at low levels at all stages of carcinogenesis. The data demonstrate a novel mechanism of synergistic cooperation between chronic estrogen exposure and the oncogenes of HPV16 that coordinates squamous carcinogenesis in the female reproductive tract of K14-HPV16 transgenic mice.

Animals↗
PubMed1996

Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions.

The E2 gene products of papillomavirus play key roles in viral replication, both as regulators of viral transcription and as auxiliary factors that act with E1 in viral DNA replication. We have carried out a detailed structure-function analysis of conserved amino acids within the N-terminal domain of the human papillomavirus type 16 (HPV16) E2 protein. These mutants were tested for their transcriptional activation activities as well as transient DNA replication and E1 binding activities. Analysis of the stably expressed mutants revealed that the transcriptional activation and replication activities of HPV16 E2 could be dissociated. The 173A mutant was defective for the transcriptional activation function but retained wild-type DNA replication activity, whereas the E39A mutant wild-type transcriptional activation function but was defective in transient DNA replication assays. The E39A mutant was also defective for HPV16 E1 binding in vitro, suggesting that the ability of E2 protein to form a complex with E1 appears to be essential for its function as an auxiliary replication factor.

Amino Acid Sequence↗
PubMed1995

A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase.

E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitin-mediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an approximately 350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).

Amino Acid Sequence↗
PubMed1995

Negative regulation of the bovine papillomavirus E5, E6, and E7 oncogenes by the viral E1 and E2 genes.

Papillomaviruses induce benign squamous epithelial lesions that infrequently are associated with uncontrolled growth or malignant conversion. The virus-encoded oncogenes are clearly under negative regulation since papillomaviruses can latently infect cells and since different levels of viral oncogene expression are seen within the layers of differentiating infected epitheliomas. We used bovine papillomavirus type 1 (BPV-1) to investigate the mechanisms involved in the negative regulation of transformation. We found that the following two distinct and interacting mechanisms negatively regulate BPV-1 transformation effected by virally encoded trans-acting factors: (i) E2 repressors suppress transformation by the E6 and E7 oncogenes, and (ii) E1 and the E2 transactivator suppress transformation by the E6, E7, and E5 oncogenes. These systems interact in that the E2 repressors function to relieve the transformation suppression effected by the E1 and E2 transactivator genes. A BPV-1 mutant that lacked E2 repressors and E1 had greatly augmented transformation capacity. Analysis of this mutant revealed that the enhanced transformation was due to expression of the E6 and E7 genes in the absence of E5, revealing a previously unappreciated potency and synergy for the BPV-1 E6 and E7 oncogenes.

Animals↗
PubMed1995

Suppression of cellular proliferation by the papillomavirus E2 protein.

Carcinogenic progression of a human papillomavirus (HPV)-infected cell is often associated with integration of the viral genome in a manner which results in the loss of expression of the viral regulatory protein E2. One function of E2 is the regulation of expression of the viral oncogenes, E6 and E7. Introduction of the bovine papillomavirus type 1 (BPV-1) E2 transactivator (E2-TA) in HeLa cells, an HPV type 18 (HPV-18)-positive cervical carcinoma cell line results in growth arrest. In this study, we have found that the HPV-16 and HPV-18 E2 proteins share with BPV-1 E2-TA the ability to suppress HeLa cell growth. This property was not observed for the BPV-1 E2 transcriptional repressor (E2-TR). Analysis of various mutant E2 proteins for growth suppression revealed a requirement for the intact transactivation and DNA binding domains. A HeLa cell line (HeLa-tsE2) which expressed a conditional mutant E2 protein that was functional only at the permissive temperature (32 degrees C) was established, permitting an analysis of the molecular and cellular consequences of E2 expression. Our data indicate that one mechanism by which E2 suppresses cellular growth is through repression of E6 and E7 expression, thereby enabling the cellular targets of E6 and E7 to resume regulation of the cell cycle.

Animals↗
PubMed1995

Amino-terminal domains of the bovine papillomavirus type 1 E1 and E2 proteins participate in complex formation.

Interaction between the E1 and E2 papillomavirus proteins appear to play an important role in viral DNA replication, although the exact domains of each protein involved in this interaction have not been identified. Using bovine papillomavirus type 1 (BPV-1) as a model for examining interactions between E1 and E2, we have used the two-hybrid and glutathione S-transferase (GST) fusion systems to map domains of BPV-1 E1 and E2 that interact in vivo and in vitro. In the two-hybrid system experiments, portions of BPV-1 E2 were expressed in Saccharomyces cerevisiae as LexA fusion proteins, which were tested for interaction with various domains of BPV-1 E1. These assays indicated that domains sufficient for E1-E2 interaction are present within the amino-terminal 250 amino acids of E1 and within the first 91 amino acids of E2. Interestingly, a LexA fusion protein that included amino acid residues 53 to 161 of BPV E2 demonstrated transcriptional activation in this system. In vitro binding assays using combinations of BPV-1 E1-GST fusion proteins and BPV-1 E2 expressed by in vitro translation confirmed the observations from the yeast system; a GST fusion protein containing the first 222 amino acids of BPV-1 E1 bound specifically to full-length BPV-1 E2 in vitro. Furthermore, E1(1-222)-GST bound to forms of E2 deleted of the carboxy-terminal DNA binding-dimerization domain, suggesting that E2 dimerization is not required for this interaction. Finally, in vitro interaction between E1-GST and E2 was observed at 22 degrees C but not at 4 degrees C.

Bacterial Proteins↗
PubMed1994

Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53.

The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway. The E6 protein binds to a cellular protein of 100 kDa termed E6-AP. The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2). This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level. In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily. We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53.

Amino Acid Sequence↗
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 HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53.

The ubiquitin-dependent proteolytic pathway plays a major role in selective protein degradation. Ubiquitination of proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor protein p53. The HPV E6 oncoprotein binds to a cellular protein of 100 kd, termed E6-associated protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-protein ligase activity in the absence of E6.

Cell Line↗
PubMed1993

Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53.

The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.

Amino Acid Sequence↗
PubMed1993

Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins.

E6-AP is a 100-kDa cellular protein that mediates the interaction of the human papillomavirus type 16 and 18 E6 proteins with p53. The association of p53 with E6 and E6-AP promotes the specific ubiquitination and subsequent proteolytic degradation of p53 in vitro. We recently isolated a cDNA encoding E6-AP and have now mapped functional domains of E6-AP involved in binding E6, association with p53, and ubiquitination of p53. The E6 binding domain consists of an 18-amino-acid region within the central portion of the molecule. Deletion of these 18 amino acids from E6-AP results in loss of both E6 and p53 binding activities. The region that directs p53 binding spans the E6 binding domain and consists of approximately 500 amino acids. E6-AP sequences in addition to those required for formation of a stable ternary complex with E6 and p53 are necessary to stimulate the ubiquitination of p53. These sequences lie within the C-terminal 84 amino acids of E6-AP. The entire region required for E6-dependent ubiquitination of p53 is also required for the ubiquitination of an artificial E6 fusion protein.

Binding Sites↗
PubMed1993

Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression.

The wild-type (wt) p53 protein has transcriptional activation functions which may be linked to its tumor suppressor activity. Many mutant p53 proteins expressed in cancers have lost the ability to function as transcriptional activators and furthermore may inhibit wt p53 function. To study the mechanisms by which mutant forms of p53 have lost their transactivation function and can act in a dominant negative manner, a structure-function analysis of both mutant and engineered truncated forms of p53 was carried out. We show that different mutant p53 proteins found in cancers vary in the ability to inhibit the transcriptional transactivation and specific DNA binding activities of wt human p53. This transdominant effect was mediated through the carboxy-terminal oligomerization region. The role of the transactivation activity in transformation suppression by wt p53 was also examined by constructing an N-terminal deletion mutant lacking the transactivation domain. This mutant was unable to transactivate but could bind specifically to DNA. Although it was impaired in its ability to suppress transformation of primary rat embryo fibroblasts by adenovirus E1A plus activated ras, the N-terminal deletion mutant still had some suppression activity, suggesting that additional functions of p53 may contribute to transformation suppression.

Animals↗
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↗