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Biomedical subjects

P F Lambert

Publications and source records attributed to P F Lambert.

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

Presentation of the HPV16E7 protein by skin grafts is insufficient to allow graft rejection in an E7-primed animal.

The E7 transforming protein of Human Papillomavirus type 16 (HPV16) is expressed in the skin of a line of FVB mice transgenic for the E6 and E7 open reading frames of HPV16 driven from the alpha A crystallin promoter (FVB alpha AcryHPV16E6E7). We have transferred skin from FVB alpha AcryHPV16E6E7 mice to naive or E7-primed syngeneic FVB recipients to assess whether the E7 protein of HPV16 can function as a minor transplantation antigen (MTA) and promote skin graft rejection. FVB mice did not reject E7 expressing tail or flank skin grafts. E7 immunized FVB x C57BL/6J mice recipients of FVB alpha-AcryHPV16E6E7 x C57BL/6J skin generated humoral and DTH responses to E7 in vivo and E7-specific CTL precursors in the spleen, but failed to reject E7 expressing tail skin grafts by 100 days posttransfer. Thus although HPV16 E7 + ve mesenchymal and endodermal tumors can be eliminated by an E7-specific immune response, the same protein is unable to act as a MTA and promote graft rejection when expressed in skin cells. Lack of rejection of grafts expressing MTAs such as E7 may be relevant to the immunology of epithelial tumors expressing tumor-specific antigens and to our understanding of the immunology of diseases of the skin.

Animals↗
PubMed1997

The nfkb1 promoter is controlled by proteins of the Ets family.

The gene encoding NFKB1 is autoregulated, responding to NF-kappa B/Rel activation through NF-kappa B binding sites in its promoter, which also contains putative sites for Ets proteins. One of the Ets sites, which we refer to as EBS4, is located next to an NF-kappa B/Rel binding site, kB3, which is absolutely required for activity of the promoter in Jurkat T cells in response to activation by phorbol 12-myristate 13-acetate (PMA), PMA/ionomycin, or the Tax protein from human T cell leukemia virus type I. We show that EBS4 is, required for the full response of the nfkb1 promoter to PMA or PMA/ionomycin in Jurkat cells. EBS4 is bound by Ets-1, Elf-1, and other species. Overexpression of Ets-1 augments the response to PMA/ionomycin and this is reduced by mutation of EBS4. Elf-1 has less effect in conjunction with PMA/ionomycin, but by itself activates the promoter 12-fold. This activation is only partly affected by mutation of EBS4, and a mutant promoter that binds Ets-1, but not Elf-1, at the EBS4 site responds to PMA/ionomycin as efficiently as the wild-type. Ets proteins may be responsible for fine-tuning the activity of the nfkb1 gene in a cell-type-specific manner.

Animals↗
PubMed1997

Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle.

The study of human papillomavirus type 16 (HPV-16) replication has been impaired because of the lack of a cell culture system that stably maintains viral replication. Recently, cervical epithelial cell populations that stably maintain HPV-16 replicons at a copy number of approximately 1,000 per cell were derived from an HPV-16-infected patient (W12 cell clone 20863 [W12-E cells]). We used neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques to characterize HPV-16 DNA replication in these cells. When W12-E cells were maintained in an undifferentiated state mimicking the nonproductive stage of the life cycle, HPV-16 DNA was found to replicate primarily by theta structures in a bidirectional manner. The initiation site of HPV-16 DNA replication was mapped to approximately nucleotide 100, and the termination site was mapped to between nucleotides 3398 and 5990. To study the productive stage of HPV-16 DNA replication, W12-E cells were grown under culture conditions that promote differentiation of epithelial cell types. Under these conditions, where virus-like particles were detected, the mode of viral DNA replication changed from theta structure to what is apparently a rolling circle mode. Additionally, CIN 612-9E cells, which were derived from an HPV-31-infected patient and harbor HPV-31 extrachromosomally, exhibited the same switch in the mode of DNA replication upon induction of differentiation. These data argue that a fundamental switch in the mechanism of viral DNA replication occurs during the life cycle of the papillomavirus.

Cell Differentiation↗
PubMed1997

Bovine papillomavirus type 1 E1 and simian virus 40 large T antigen share regions of sequence similarity required for multiple functions.

The full-length product of the bovine papillomavirus type 1 (BPV-1) E1 translational open reading frame is required for viral DNA replication in vivo and in vitro. E1 is a multifunctional protein whose properties include ATP binding, acting as an ATPase-dependent DNA helicase, DNA binding to the BPV-1 origin of viral DNA replication, and association with the E2 transcriptional transactivator, E2TA, a second viral protein involved in DNA replication. All of these properties are thought to be important for E1's role in replicating the viral genome. In addition BPV-1 E1 can inhibit activation of the viral P89 promoter by the BPV-1 E2TA. E1 has amino acid homology with eight regions of SV40 large tumor antigen (T-ag), a DNA helicase that is essential for the replication of the SV40 DNA genome. These eight regions of similarity lie within the domain of T-ag that confers DNA helicase activity. We created a series of missense mutations in BPV-1 E1 at codons 295, 344-345, 446, 464, 466, 497-498, 523, and 542, which encode amino acids of identity in seven of the eight regions of similarity between E1 and T-ag, and at codon 370. The activities of these mutant E1 genes were compared to wild-type E1 in multiple assays that measured DNA replication, inhibition of E2TA-dependent transcription, DNA binding, ATP binding, and protein expression. Based upon these analyses, the following conclusions were made: (i) at least five of the eight regions in E1 that are similar to regions in T-ag are functionally important in viral DNA replication; (ii) specific E1 missense mutants, themselves defective for supporting DNA replication, could act in trans to suppress the replication function of wild-type E1; (iii) certain regions of similarity with T-ag that are important for E1's ability to support DNA replication are not necessary for its capacity to inhibit E2TA-dependent transcription; and (iv) efficient DNA binding by E1 is not essential for E1 to inhibit E2TA-dependent transcription.

Amino Acid Sequence↗
PubMed1997

Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice.

High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7, are commonly found to be expressed in these cancers. The presence of HPV-16 E7 is sufficient to induce epidermal hyperplasia and epithelial tumors in transgenic mice. In this study, we have performed experiments in transgenic mice to determine which domains of E7 contribute to these in vivo properties. The human keratin 14 promoter was used to direct expression of mutant E7 genes to stratified squamous epithelia in mice. The E7 mutants chosen had either an in-frame deletion in the conserved region 2 (CR2) domain, which is required for binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain contributes to cellular transformation at a level other than pRb binding. Four lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five lines harboring a CR2 deletion were generated and were observed for overt and histological phenotypes. A detailed time course analysis was performed to monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of high-level expression. In the transgenic mice with the wild-type E7 gene, age-dependent expression of HPV-16 E7 correlated with the severity of epidermal hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes failed to result in any phenotypes. In addition, the transgenic mice with a mutant E7 gene did not develop tumors. These experiments indicate that binding and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are necessary for induction of epidermal hyperplasia and carcinogenesis in mouse skin and also suggest a role for the CR1 domain in the induction of these phenotypes through as-yet-uncharacterized mechanisms.

Animals↗
PubMed1996

A fourth Sp1 site in the human immunodeficiency virus type 1 long terminal repeat is essential for negative-sense transcription.

We report the discovery of a fourth Sp1 binding site at the 5' end of the U3 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (HIV-1 Sp1 IV), localized to HXB2 nucleotides -433 to -441. This site is shown to bind Sp1 protein specifically in electrophoretic mobility shift assays. Sp1 protein appears to bind to HIV-1 Spl IV with 5 to 10 times lower affinity than to a consensus Sp1 site. Mutation of HIV-1 Sp1 IV in an HXB2-derived long terminal repeat-chloramphenicol acetyltransferase reporter construct gave no significant change in positive-sense transcription but abolished both basal and phorbol myristate acetate-activated negative-sense transcription. Taken together, the results further define the HIV-1 negative-sense promoter as an Sp1-dependent, phorbol myristate acetate-responsive, and Tat-inhibited promoter initiating at HXB2 nucleotide -450.

Antisense Elements (Genetics)↗
PubMed1996

Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene.

The human papillomavirus type 16 (HPV-16) genome is commonly present in human cervical carcinoma, in which a subset of the viral genes, E6 and E7, are expressed. The HPV-16 E6 and E7 gene products can associated with and inactivate the tumor suppressor proteins p53 and Rb (the retinoblastoma susceptibility gene product), and in tissue culture cells, these viral genes display oncogenic properties. These findings have led to the hypothesis that E6 and E7 contribute to cervical carcinogenesis. This hypothesis has recently been tested by using transgenic mice as an animal model. HPV-16 E6 and E7 together were found to induce cancers in multiple tissues in which they were expressed, including squamous cell carcinoma, the cancer type most commonly associated with HPV-16 in the human cervix. We have extended these studies to investigate the in vivo activities of HPV-16 E7 when expressed in squamous epithelia of transgenic mice. Grossly, E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In lines of mice expressing higher levels of E7, we observed stunted growth and mortality at an early age, potentially caused by an incapacity to feed. Histological analysis demonstrated that E7 causes epidermal hyperplasia in multiple transgenic lineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratin 10-positive layer of cells and was associated with hyperkeratosis. Hyperplasia was found at multiple sites in the animals in addition to the skin, including the mouth palate, esophagus, forestomach, and exocervix. In multiple transgenic lineages, adult animals developed skin tumors late in life with low penetrance. These tumors arose from the squamous epithelia and from sebaceous glands and were characterized histologically to be highly differentiated, locally invasive, and aggressive in their growth properties. On the basis of these phenotypes, we conclude that HPV-16 E7 can alter epithelial cell growth parameters sufficiently to potentiate tumorigenesis in mice.

Animals↗
PubMed1995

Immunological responses in human papillomavirus 16 E6/E7-transgenic mice to E7 protein correlate with the presence of skin disease.

The human papillomavirus (HPV) oncogenes, E6 and E7, are believed to contribute to the development of cervical cancers in women infected with certain HPV genotypes, most notably HPV-16 and HPV-18. Given their expression in tumor tissue, E6 and E7 have been implicated as potential tumor-specific antigens. We have examined an HPV-16 E6- and E7-transgenic mouse lineage for immune responses to these viral oncoproteins. Mice in this lineage express the HPV-16 E6 and E7 genes in their skin and eyes, and on aging, these mice frequently develop squamous cell carcinomas and lenticular tumors. Young transgenic mice, which had measurable E7 protein in the eye but not in the skin, were immunologically naive to E7 protein. They mounted an immune response to E7 on immunization comparable to that of nontransgenic controls, suggesting a lack of immune tolerance to this protein. Older line 19 mice, which are susceptible to skin disease associated with transcription of the E6 and E7 open reading frames, had measurable E7 protein in their skin. These older transgenic mice spontaneously developed antibody responses to endogenous E7 protein, particularly in association with skin disease. Also detected in older mice were delayed-type hypersensitivity responses to E7. These finding parallel the humoral immune response to E7 protein in patients with HPV-associated cervical cancer and suggest that line 19 mice may provide a model for studying the immunobiology of HPV-associated cancers.

Amino Acid Sequence↗
PubMed1995

Integration of human papillomavirus type 16 DNA into the human genome leads to increased stability of E6 and E7 mRNAs: implications for cervical carcinogenesis.

In many cervical cancers, human papillomavirus type 16 (HPV-16) DNA genomes are found to be integrated into the host chromosome. In this study, we demonstrate that integration of HPV-16 DNA leads to increased steady-state levels of mRNAs encoding the viral oncogenes E6 and E7. This increase is shown to result, at least in part, from an increased stability of E6 and E7 mRNAs that arise specifically from those integrated viral genomes disrupted in the 3' untranslated region of the viral early region. Further, we demonstrate that the A+U-rich element within this viral early 3' untranslated region confers instability on a heterologous mRNA. We conclude that integration of HPV-16 DNA, as occurs in cervical cancers, can result in the increased expression of the viral E6 and E7 oncogenes through altered mRNA stability.

Extrachromosomal Inheritance↗
PubMed1995

Bovine papillomavirus type 1 E2 transcriptional regulators directly bind two cellular transcription factors, TFIID and TFIIB.

The bovine papillomavirus type 1 (BPV-1) E2 translational open reading frame encodes three proteins that regulate viral transcription and DNA replication: the E2 transcriptional activator (E2TA), the E2 transcriptional repressor (E2TR) and the E8/E2 transcriptional repressor (E8/E2TR). E2TA is a strong activator of papillomaviral promoters and is required for viral DNA replication. E2TR and E8/E2TR inhibit the activities of E2TA but also possess weak transactivational properties of their own. Two components of the cellular transcription apparatus, TFIID and TFIIB, have previously been shown to associate with other viral and cellular transcriptional activators. We present evidence here that E2TA, the full-length E2 open reading frame gene product, directly binds both of these transcription factors in vitro. Glutathione S-transferase E2TA (GST-E2TA) fusion protein bound in vitro-synthesized TATA-box-binding protein (TBP), a component of TFIID, and in vitro-synthesized TFIIB. Likewise, GST-E2TA bound TFIID and TFIIB present in a nuclear extract from the human cervical cancer-derived cell line, HeLa. The binding of GST-E2TA to TBP and TFIIB required no additional mammalian factors, as shown by direct binding of GST-E2TA to bacterially synthesized recombinant TBP and recombinant TFIIB. The domain of E2TA required for its interaction with both TBP and TFIIB was localized to the C terminus of E2TA, a region also present in E2TR and E8/E2TR. This domain lies within the region of E2TA previously shown to confer cooperative DNA binding by E2TA and TBP and overlaps with the region of E2TA required for DNA binding and dimerization. Our findings, taken in context with previous studies, lead us to conclude that (i) cooperative DNA binding by E2 proteins and TBP is likely mediated by the direct binding of E2 proteins to TBP, (ii) the weak transcriptional transactivation by E2TR and E8/E2TR may result as a consequence of direct TBP and TFIIB binding by these proteins, and (iii) TBP and/or TFIIB binding may be required but is not sufficient for E2TA's strong transactivational activity.

Animals↗
PubMed1995

Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells.

Integration of human papillomavirus type 16 (HPV-16) DNA into a host chromosome has been hypothesized to result in altered expression of two viral transforming genes, E6 and E7, in cervical cancers. In order to investigate the role that changes in viral genomic state and gene expression play in cervical carcinogenesis, we have derived clonal populations of human cervical epithelial cells which harbor multiple copies of either extrachromosomal or integrated viral DNA. The clonal populations harboring extrachromosomal HPV-16 DNA stably maintained approximately 1,000 viral copies for at least 15 passages (approximately 100 cell doublings), which contrasted with the unstable HPV-16 replicons in the parental counterpart. In the clonal populations harboring integrated viral DNA, 3 to 60 copies of HPV-16 DNA were found integrated in either of two forms: type 1, in which all the copies of HPV-16 DNA were disrupted in the E2 open reading frame upon integration, and type 2, in which intact viral copies were flanked by disrupted viral copies and cellular sequences. Despite the lower HPV-16 DNA copy number, the clonal populations with integrated viral DNA had levels of E7 protein that were in most cases higher than those found in the clonal populations harboring extrachromosomal viral DNA. Irrespective of viral genomic state, the clonal populations were capable of undergoing terminal differentiation and unable to form colonies in soft agar, which is indicative of the nontumorigenic nature of these cells. Importantly, a cell population with integrated viral DNA was found to outgrow another with extrachromosomal DNA when these cells were cocultured over a period of time. Thus, integration of human papillomaviral DNA correlates with increased viral gene expression and cellular growth advantage. These observations are consistent with the hypothesis that integration provides a selective advantage to cervical epithelial precursors of cervical carcinoma.

Base Sequence↗
PubMed1994

Primate relaxin: synthesis of gorilla and rhesus monkey relaxins.

The synthesis of the hormone relaxin from the species Gorilla gorilla (gorilla) and Macaca mulatta (rhesus monkey) has been achieved. Each of the two chains which constitute the peptide structures was assembled separately, the A-chains (24 amino acids) by the Boc-polystyrene solid-phase procedure and the B-chains (29 and 28 amino acids) by the Fmoc-polyamide (gorilla) and the Boc-polystyrene (rhesus monkey) solid-phase methods. After cleavage from the solid supports, the separate chains were purified to a high degree of homogeneity. Oxidative combination of the respective A- and B-chains in solution at high pH afforded the synthetic relaxins in low overall yield. Chemical and physiochemical characterization of the products confirmed both their purity and their conformational similarity to the human hormone. The synthetic gorilla and rhesus monkey relaxins were both found to possess potent chronotropic and inotropic activity in the isolated rat cardiac atrium assay.

Amino Acid Sequence↗
PubMed1994

Role of transcriptional repressors in transformation by bovine papillomavirus type 1.

Transformation of rodent cells by bovine papillomavirus type 1 (BPV-1) has been shown to require the direct contribution of the viral oncogenes encoded by the E5, E6, and E7 translational open reading frames (ORFs). It is also known that the viral E1 and E2 ORFs contribute indirectly to cellular transformation through their transcriptional modulation of these viral oncogenes. A mutant BPV-1 disrupted in two of the proteins encoded by the E2 ORF, the E2 transcriptional repressors, has a complex transformation phenotype. In this paper, we provide evidence that (i) this phenotype is not attributable to the absence of other viral genes that overlap the E2 repressor genes, (ii) the acquisition of transformation over time in cells harboring the E2 double-repressor mutant correlates with reversions of the mutations that disrupt one of the E2 repressor genes, E8/E2TR, and (iii) the initial transformation defect in the E2 double-repressor mutant can be rescued by disruption of the full-length protein encoded by the E1 ORF, the E1 transcriptional repressor. We propose a model by which the combination of E1 and E2 transcriptional repressors functions to modulate the transforming capacity of the virus.

Animals↗
PubMed1994

Role of papillomavirus oncogenes in human cervical cancer: transgenic animal studies.

Human papillomaviruses are believed to be etiologic agents for the majority of human cervical carcinoma, a common cancer that is a leading cause of death by cancer among women worldwide. In cervical carcinoma, a subset of papillomaviral genes, namely E6 and E7, are expressed. In vitro tissue culture studies indicate that HPV E6 and E7 are oncogenes, and that their oncogenicity is due in part to their capacity to inactivate cellular tumor suppressor genes. The behavior of E6 and E7 in vitro and the genetic evidence from analysis of human cancers suggest that the E6 and E7 genes play a significant role in the development of cervical cancer. This hypothesis is now being tested using animal models. In this review, we summarize our current knowledge of the oncogenicity of papillomavirus genes that has been generated through their study in transgenic mice.

Animals↗
PubMed1993

Epidermal cancer associated with expression of human papillomavirus type 16 E6 and E7 oncogenes in the skin of transgenic mice.

Certain "high-risk" anogenital human papillomaviruses (HPVs) have been associated with the majority of human cervical carcinomas. In these cancers, two papillomaviral genes, E6 and E7, are commonly expressed. In this study we provide evidence that expression of the E6 and E7 genes from the high-risk HPV-16 in the skin of transgenic mice potentiated the development of preneoplastic lesions, and a high percentage of these epidermal lesions subsequently developed into locally invasive cancers. High levels of E6/E7 expression were found in these tumors relative to the preneoplastic lesions, and expression was localized to the proliferating, poorly differentiated epidermal cells. Also, the p53 and Rb genes were found to be intact, not mutationally inactivated, in representative skin tumors. These findings demonstrate that the E6 and E7 genes from a papillomavirus etiologically associated with human cervical cancer can contribute to the development of epidermal cancers in an animal model.

Animals↗
PubMed1993

Tumorigenicity by human papillomavirus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation.

The human papillomavirus type 16 (HPV-16) E6 and E7 oncogenes are thought to play a role in the development of most human cervical cancers. These E6 and E7 oncoproteins affect cell growth control at least in part through their association with and inactivation of the cellular tumor suppressor gene products, p53 and Rb. To study the biological activities of the HPV-16 E6 and E7 genes in epithelial cells in vivo, transgenic mice were generated in which expression of E6 and E7 was targeted to the ocular lens. Expression of the transgenes correlated with bilateral microphthalmia and cataracts (100% penetrance) resulting from an efficient impairment of lens fiber cell differentiation and coincident induction of cell proliferation. Lens tumors formed in 40% of adult mice from the mouse lineage with the highest level of E6 and E7 expression. Additionally, when lens cells from neonatal transgenic animals were placed in tissue culture, immortalized cell populations grew out and acquired a tumorigenic phenotype with continuous passage. These observations indicate that genetic changes in addition to the transgenes are likely necessary for tumor formation. These transgenic mice and cell lines provide the basis for further studies into the mechanism of action of E6 and E7 in eliciting the observed pathology and into the genetic alterations required for HPV-16-associated tumor progression.

Animals↗
PubMed1993

The 23-kilodalton E1 phosphoprotein of bovine papillomavirus type 1 is nonessential for stable plasmid replication in murine C127 cells.

The 23-kDa protein encoded by the 5' segment of the E1 open reading frame of bovine papillomavirus type 1 (BPV1) was previously ascribed a negative regulatory function for the replication of viral plasmid DNA. However, results from recent functional and biochemical studies do not readily support this genetic assignment. Therefore, we have reassessed the role of this protein in papillomavirus DNA replication by using a mutant of BPV1 which is unable to express this E1 protein. This mutant viral DNA was found to replicate extrachromosomally with stability and copy number per cell similar to those of wild-type plasmid DNA. Thus, the absence of expression of the 23-kDa E1 protein did not lead to deregulated viral plasmid replication. We conclude that the 23-kDa E1 protein is nonessential for stable plasmid replication.

Amino Acid Sequence↗