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Biomedical subjects

P F Lambert

Publications and source records attributed to P F Lambert.

At least 19 recordsLinked to original sources
PubMed2006

HPV16 E6 confers p53-dependent and p53-independent phenotypes in the epidermis of mice deficient for E6AP.

High-risk human papillomaviruses are the causative agents of cervical and other anogenital cancers. In these cancers, two viral oncogenes, E6 and E7, are expressed. E6 is best known for its ability to inactivate the tumor suppressor p53, which is thought to arise through ubiquitin-mediated degradation of p53 and involve a ternary complex between E6, p53 and the E3 ligase, E6AP. In mice transgenic for wild-type HPV16 E6, its expression leads to epithelial hyperplasia and an abrogation of normal cellular responses to DNA damage. Whereas only the latter phenotype is dependent upon E6's inactivation of p53, both are reduced in transgenic mice expressing an E6 mutant severely reduced in its binding to E6AP and other cellular proteins that bind E6 through a shared alpha-helix motif. Here, we investigated whether E6AP is required for the induction of the above phenotypes through the use of both E6AP-mutant and E6AP-null mice. E6, in the absence of E6AP retains an ability to induce epithelial hyperplasia, abrogate DNA damage responses and inhibit the induction of p53 protein following exposure to ionizing radiation. We conclude that E6 is able to induce both p53-dependent and p53-independent phenotypes through E6AP-independent pathways in the mouse.

Animals↗
PubMed2001

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

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

Cells, Cultured↗
PubMed2001

Cell cycle regulation of human interleukin-8 gene expression by the human immunodeficiency virus type 1 Tat protein.

The human immunodeficiency virus type 1 (HIV-1) Tat protein has been reported to transactivate several cellular genes, including the potent chemotactic factor interleukin-8 (IL-8). Consistent with these in vitro assays, elevated levels of IL-8 protein are found in the serum of HIV-infected individuals. We now extend these observations by demonstrating that Tat induction of IL-8 is linked to the cell cycle. Cells that constitutively express the Tat(1-86) protein (eTat) and control cells (pCEP) were reversibly blocked at the G(1)/S border with hydroxyurea or thymidine. The cells were subsequently released, and IL-8 expression was monitored by RNase protection assays and enzyme-linked immunosorbent assay (ELISA). RNase protection assays demonstrated that IL-8 mRNA expression is transiently induced, approximately fourfold, as the Tat-expressing cells enter S phase. Consistent with the RNase protection assay, an increase in IL-8 protein was observed in the cell supernatant using an IL-8 ELISA. Similar experiments were performed following a reversible block at the G(2)/M border with nocodazole and release into G(1). Using the RNase protection assay and ELISA, little or no increase in IL-8 expression was observed during G(1). Using gel shift as well as an immobilized DNA binding assay, we demonstrate that the increase in IL-8 gene expression correlates with a specific increase in p65 NF-kappa B binding activity only in the nucleus of the Tat-expressing cells. Moreover, the CREB-binding protein coactivator is present in the complex in the Tat cell line. Finally, we demonstrate that the presence of the proteasome inhibitor MG-132 inhibits the induction of NF-kappa B binding, as well as IL-8 expression, supporting the role of NF-kappa B.

Cell Cycle↗
PubMed2000

Interaction of the papillomavirus transcription/replication factor, E2, and the viral capsid protein, L2.

The minor capsid protein L2 of papillomaviruses (PVs) likely plays a role in the selective encapsidation of PV DNA in viral capsids and in the infectivity of PV virions. The L2 protein also can cause the relocalization of the PV early protein, E2TA, to nuclear subdomains known as promyelocytic leukemia oncogenic domains (PODs) in which it is localized. E2TA is a transcriptional transactivator that also plays a critical role in viral DNA replication. In this study, we investigated whether L2, in causing the relocalization of E2TA, alters the activities of E2TA. We provide evidence that L2 inhibits the transcriptional transactivation function of E2, but it does not specifically inhibit the capacity of E2 to support viral DNA replication. We also investigated whether the colocalization of E2 and L2 to PODs and the ability of L2 to inhibit the transcriptional transactivation activity of E2TA might be mediated through a direct interaction between these two proteins. Using an in vitro protein-protein association assay, we found that L2 binds to E2TA. Two regions in E2TA were found to mediate this interaction. One of those domains is present in an alternative E2 gene product, E2TR, which is an antagonist to E2TA. Here we show that the L2 protein also relocalizes the E2 transcriptional repressor, E2TR, to the nuclear subdomains. These data suggest that the ability of L2 to relocalize E2 proteins to PODs is mediated through a direct interaction with L2.

Animals↗
PubMed2000

Peripheral tolerance to human papillomavirus E7 oncoprotein occurs by cross-tolerization, is largely Th-2-independent, and is broken by dendritic cell immunization.

The E7 oncoprotein of human papillomavirus 16 functions as a tumor-specific antigen in transformed epithelial cells of the uterine cervix to which immunotherapeutic strategies aimed at CTL induction may be directed. We previously have shown in mice transgenic for the E7 gene driven off an epithelial specific (keratin-14) promoter, that expression of E7 protein in peripheral epithelium is sufficient to tolerize E7-directed CTL precursors (pCTL; Doan et al, J. Virol., 73: 6166-1670, 1999). Here we show that E7 is presented to T cells for tolerization by cells of bone marrow origin ("cross-tolerization"). We demonstrate that tolerization of E7-directed pCTLs occurs within 2 weeks of exposure to E7 in epithelium. It is maintained in the near absence of CD4+ cells and in the absence of the thymus, and is independent of a coexisting E7-directed Th2-type antibody response. Tolerance was broken by immunization with E7 CTL epitope-pulsed dendritic cells. These findings have implications for immunotherapy of patients with human papillomavirus 16-associated cervical carcinoma, whose immune systems may have experienced long-term exposure to E7-expressing epithelial cells.

Adoptive Transfer↗
PubMed2000

Human papillomavirus types 16 E6 and E7 contribute differently to carcinogenesis.

High-risk human papillomaviruses (HPVs) are etiologically implicated in human cervical cancer. Two viral genes, E6 and E7, are commonly found expressed in these cancer cells. We have previously shown that mice transgenic for the HPV-16 E6 gene or E7 gene, in which the E6 or E7 was expressed in the basal layer of epithelia, developed skin tumors. The spectrum of tumors derived from E6 and E7 mice differed, however; although most tumors derived from the E7-transgenic mice were benign, the majority of the tumors from the E6-transgenic mice were malignant. These findings led us to hypothesize that E6 and E7 play different roles in carcinogenesis. To assess at what stages in carcinogenesis E6 and E7 act, we treated the skin of K14E6- and K14E7-transgenic mice with chemical carcinogens known to contribute to distinct stages in carcinogenesis. Both E6 and E7 were found to synergize with chemical carcinogens in causing tumor formation. E6 was found to act weakly at the promotion stage of carcinogenesis in the formation of benign tumors but strongly at the progression stage which involves the malignant conversion of benign tumors. In contrast, E7 primarily affected the promotion stage of carcinogenesis. These results provide direct evidence that E6 and E7 contribute differently to carcinogenesis; E7 promotes the formation of benign tumors, and E6 acts primarily to accelerate progression of these benign tumors to the malignant stage. Consistent with this model, we found E6 and E7 to cooperate in inducing tumor formation in mice expressing both oncogenes.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2000

A novel and rapid PCR-based method for genotyping human papillomaviruses in clinical samples.

Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5(+)-GP6(+) consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil N-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a "T-sequencing ladder" was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.

Base Sequence↗
PubMed2000

The human papillomavirus type 16 E7 oncogene is required for the productive stage of the viral life cycle.

The production of the human papillomavirus type 16 (HPV-16) is intimately tied to the differentiation of the host epithelium that it infects. Infection occurs in the basal layer of the epithelium at a site of wounding, where the virus utilizes the host DNA replication machinery to establish itself as a low-copy-number episome. The productive stage of the HPV-16 life cycle occurs in the postmitotic suprabasal layers of the epithelium, where the virus amplifies its DNA to high copy number, synthesizes the capsid proteins (L1 and L2), encapsidates the HPV-16 genome, and releases virion particles as the upper layer of the epithelium is shed. Papillomaviruses are hypothesized to possess a mechanism to overcome the block in DNA synthesis that occurs in the differentiated epithelial cells, and the HPV-16 E7 oncoprotein has been suggested to play a role in this process. To determine whether E7 plays a role in the HPV-16 life cycle, an E7-deficient HPV-16 genome was created by inserting a translational termination linker (TTL) in the E7 gene of the full HPV-16 genome. This DNA was transfected into an immortalized human foreskin keratinocyte cell line shown previously to support the HPV-16 life cycle, and stable cell lines were obtained that harbored the E7-deficient HPV-16 genome episomally, the state of the genome found in normal infections. By culturing these cells under conditions which promote the differentiation of epithelial cells, we found E7 to be necessary for the productive stage of the HPV-16 life cycle. HPV-16 lacking E7 failed to amplify its DNA and expressed reduced amounts of the capsid protein L1, which is required for virus production. E7 appears to create a favorable environment for HPV-16 DNA synthesis by perturbing the keratinocyte differentiation program and inducing the host DNA replication machinery. These data demonstrate that E7 plays an essential role in the papillomavirus life cycle.

Apoptosis↗
PubMed2000

Differences in the ability of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 tax to inhibit p53 function.

We have analyzed the functional activity of the p53 tumor suppressor in human T-cell lymphotropic virus type 2 (HTLV-2)-transformed cells. Abundant levels of the p53 protein were detected in both HTLV-2A and -2B virus-infected cell lines. The p53 was functionally inactive, however, both in transient-transfection assays using a p53 reporter plasmid and in induction of p53-responsive genes in response to gamma irradiation. We further investigated HTLV-2A Tax and HTLV-2B Tax effects on p53 activity. Interestingly, although Tax-2A and -2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B. In transient-cotransfection assays, Tax-1 and Tax-2B inactivated p53 by 80%, while Tax2A reduced p53 activity by 20%. In addition, Tax-2A does not increase the steady-state level of cellular p53 as well as Tax-1 or -2B does in the same assays. Cotransfection assays demonstrated that Tax-2A could efficiently transactivate CREB-responsive promoters to the same level as Tax-1 and Tax-2B, indicating that the protein was functional. This report provides evidence of the first functional difference between the HTLV-2A and -2B subtypes. This comparison of the action of HTLV-1 and HTLV-2 Tax proteins on p53 function will provide important insights into the mechanism of HTLV transformation.

Cell Line, Transformed↗
PubMed1999

Establishment of the human papillomavirus type 16 (HPV-16) life cycle in an immortalized human foreskin keratinocyte cell line.

The study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult. In this study, we have used BC-1-Ep/SL cells, an immortalized human foreskin keratinocyte cell line, to recreate the HPV-16 life cycle. This cell line exhibits many characteristics of the early-passage HFKs including the ability to stratify and terminally differentiate in an organotypic raft culture system. Because of their similarity to early-passage HFKs, these cells were tested for their ability to support the HPV-16 life cycle. The BC-1-Ep/SL cells could stably maintain two HPV genotypes, HPV-16 and HPV-31b, episomally. Additionally, when the BC-1-Ep/SL cell line was stably transfected with HPV-16 and cultured using the organotypic raft culture system (rafts), it sustained the HPV-16 life cycle. Evidence for the productive stage of the HPV-16 life cycle was provided by: DNA in situ hybridization demonstrating HPV-16 DNA amplification in the suprabasal layers of the rafts, immunohistochemical staining for L1 showing the presence of capsid protein in the suprabasal layers of the rafts, and electron microscopy indicating the presence of virus like particles (VLPs) in nuclei from cells in the differentiated layers of the rafts.

Capsid Proteins↗
PubMed1999

Different responses of epidermal and hair follicular cells to radiation correlate with distinct patterns of p53 and p21 induction.

Different parts of the skin respond to ionizing radiation with different sensitivities. To examine the mechanisms underlying these different responses, we investigated various cellular parameters in the skin after exposure of mice to 5 Gy of ionizing radiation. Epidermal cells responded to radiation by undergoing growth arrest, whereas the cells in the matrix of hair follicles underwent apoptosis but not growth arrest. These distinct responses correlated with differential increases in p53 and p21 proteins in these two populations of cells; whereas an increase in p53 protein levels was observed in both epidermis and hair follicular matrix, especially in the latter, the induction of p21 was strong in the epidermis but absent in the follicular matrical cells. Studies using p53-null and p21-null mice demonstrated that the radiation-induced apoptosis in the hair follicles was fully dependent on p53, and growth arrest in the epidermis was only partially dependent on p53 but fully dependent on p21. These results indicate that two epithelial cell types respond to radiation by different pathways that are governed in part by the differential p53- and p21-dependent responses of these cells; high-level induction of p53 in the absence of p21 induction led to apoptosis, whereas intermediate induction of both p53 and p21 led to growth arrest.

Animals↗
PubMed1999

The human papillomavirus type 16 E6 gene alone is sufficient to induce carcinomas in transgenic animals.

High-risk human papillomaviruses (HPVs) are the causative agents of certain human cancers. HPV type 16 (HPV16) is the papillomavirus most frequently associated with cervical cancer in women. The E6 and E7 genes of HPV are expressed in cells derived from these cancers and can transform cells in tissue culture. Animal experiments have demonstrated that E6 and E7 together cause tumors. We showed previously that E6 and E7 together or E7 alone could induce skin tumors in mice when these genes were expressed in the basal epithelia of the skin. In this study, we investigated the role that the E6 gene plays in carcinogenesis. We generated K14E6 transgenic mice, in which the HPV16 E6 gene was directed in its expression by the human keratin 14 promoter (hK14) to the basal layer of the epidermis. We found that E6 induced cellular hyperproliferation and epidermal hyperplasia and caused skin tumors in adult mice. Interestingly, the tumors derived from E6 were mostly malignant, as opposed to the tumors from E7 mice, which were mostly benign. This result leads us to hypothesize that E6 may contribute differently than E7 to HPV-associated carcinogenesis; whereas E7 primarily contributes to the early stages of carcinogenesis that lead to the formation of benign tumors, E6 primarily contributes to the late stages of carcinogenesis that lead to malignancy.

Animals↗
PubMed1999

T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice.

One of three lines of mice transgenic for the E6 and E7 genes of human papillomavirus type 16 (HPV16) expressed from an alphaA-crystallin promoter also expresses the transgene ectopically in the skin. This line, designated alphaACE6E7#19, develops skin disease from 3 months of age, characterised by epidermal hyperplasia and eventual skin loss. Administration of complete Freund's adjuvant (CFA) to alphaACE6E7#19 mice, but not to non-transgenic littermate controls, induced local epidermal hyperplasia which was histologically similar to the spontaneously arising skin pathology. Local application of 2,4-dinitrochlorobenzene (DNCB) to DNCB-sensitised alphaACE6E7#19 mice, but not DNCB-sensitised controls, also induced hyperplasia. Treatment with cyclosporin A (CsA) or systemic depletion of CD4+ cells significantly reduced the incidence of skin disease. These data suggest that local inflammation, and cytokines produced by T helper cells, contribute to the induction of hyperplastic skin disease in alphaACE6E7#19 mice. Spontaneous skin disease with similar histological appearance, frequency, age of onset and severity in alphaACE6E7#19 mice was observed in scid-/- alphaACE6E7#19 mice, despite immune paresis. Antigen-specific immune responses and T-cell cytokines are therefore not necessary for the induction of skin disease. We propose that epidermal hyperplasia associated with HPV16 E6 and E7 expression in skin is accelerated by local secretion of pro-inflammatory cytokines, whose production can be enhanced by activated CD4+ T cells.

Age Factors↗
PubMed1998

Phosphorylation of p53 serine 15 increases interaction with CBP.

p53 exerts its cell cycle regulatory effects through its ability to function as a sequence-specific DNA binding transcription factor. CREB-binding protein (CBP)/p300, through its interaction with the N terminus of p53, acts as a coactivator for p53 and increases the sequence-specific DNA-binding activity of p53 by acetylating its C terminus. The same N-terminal domain of p53 has recently been shown to be phosphorylated at Ser15 in response to gamma-irradiation. Remarkably, we now demonstrate that phosphorylation of p53 at Ser15 increases its ability to recruit CBP/p300. The increase in CBP/p300 binding was followed by an increase in the overall level of acetylation of the C terminus of p53. These results provide a mechanism for the activation of p53-regulated genes following DNA damage, through a signaling pathway linking p53 N-terminal kinase and C-terminal acetyltransferase activities.

Acetylation↗
PubMed1998

Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways.

E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors in vivo. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis to determine how these two proteins modulate DNA damage responses in vivo. Our results demonstrate that both E6 and E7 abrogate the inhibition of DNA synthesis in the epidermis after treatment with ionizing radiation. Increases in the levels of p53 and p21 proteins after irradiation were suppressed by E6 but not by E7. Through the study of p53-null mice, we found that radiation-induced growth arrest in the epidermis is mediated through both p53-dependent and p53-independent pathways. The abrogation of radiation responses in both E6 and E7 transgenic mice was more complete than was seen in the p53-null epidermis. We conclude that E6 and E7 each have the capacity to modulate p53-dependent as well as p53-independent cellular responses to radiation. Additionally, we found that the conserved region (CR) 1 and CR2 domains in E7 protein, which are involved in the inactivation of pRb function and required for E7's transforming function, were also required for E7 to modulate DNA damage responses in vivo. Thus pRb and/or pRb-like proteins likely mediate both p53-dependent and p53-independent responses to radiation.

Animals↗
PubMed1998

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes.

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4+ T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/ nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2q) or F1(C57BL/6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further, E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7+ tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in "split" tolerance to that autoantigen.

Animals↗
PubMed1998

Multiple genetic loci modify risk for retinoblastoma in transgenic mice.

PURPOSE: Forty percent of cases of retinoblastoma, a childhood malignancy of the retina, are linked to the inheritance of a mutant allele of the retinoblastoma susceptibility gene Rb1. Tumor penetrance varies among carriers in different family pedigrees, indicating that other genetic factors may modify risk for occurrence of retinoblastoma. This study was undertaken to determine whether multiple genetic loci modify the risk for retinoblastoma in mice. METHODS: A line of alphaAcry-HPV16E6/E7 transgenic mice expressing the human papillomavirus type 16 E6 and E7 oncogenes (HPV-16 E6 and E7) ectopically in the retina was characterized. E6 and E7 proteins bind to and inactivate the cellular tumor suppressor proteins p53 and Rb, respectively. RESULTS: Retinoblastomas developed rarely when the alphaAcry-HPV16E6/E7 transgene was maintained on the FVB background, but tumors arose with high frequency on C57BL/6 X FVB and C3H x FVB F1 hybrid backgrounds. The incidence of retinoblastoma in the LHbeta-TAG transgenic mice, which express simian virus 40 large tumor antigen (SV40 T-ag), was also influenced by the FVB and C57BL/6 backgrounds. Resistance of the alphaAcry-HPV16E6/E7 FVB mice to retinoblastoma mapped in part to the retinal degeneration (rd) locus. However, multiple genetic experiments indicate that resistance to retinoblastoma depends on additional loci in FVB mice. CONCLUSIONS: Multiple cellular genes can modify risk for retinoblastoma in mice.

Animals↗