人乳头瘤病毒16型亚基因DNA体外转化功能的细胞学研究

利用HZIP16和HZIP16K(见材料和方法)质粒,将人乳头瘤病毒16型(HPV-16)的全早期区基因及其开放读码框架(ORF)E6-E7分别转入ψ2细胞,所产生的重组病毒能诱导NIH3T3细胞发生转化。转化细胞具有恶性细胞的生物学和形态学特征,可在0.3％软琼脂中形成集落,可使裸鼠致瘤。Southern blot证明,HPV-16 E6-E7 ORFs序列以整合形式存在于转化细胞和裸鼠肿瘤细胞DNA中,表明HPV-16 DNA具有体外诱导NIH3T3细胞恶性转化的作用,E6-E7 ORFs是诱导细胞转化的关键基因。     

Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells.

The close association of human papillomavirus type 16 DNA with a majority of cervical carcinomas implies some role for the virus in this type of cancer. To define the transforming properties of HPV-16 DNA in vitro we have now performed transfection experiments on baby rat kidney cells using HPV-16 DNA in conjunction with an activated ras gene. We have demonstrated that a 6.6-kb DNA fragment, containing the early genes of HPV-16 under the control of Moloney murine leukaemia virus long terminal repeats (MoMuLV-LTRs), cooperates with EJ-ras in transforming these cells. Both DNAs are required and neither alone is effective. The cooperating activity appears to reside in a protein or proteins derived from the E6/E7 region of the HPV-16 genome.     

Analysis of the p53-mediated G1 growth arrest pathway in cells expressing the human papillomavirus type 16 E7 oncoprotein.

Cells expressing human papillomavirus type 16 (HPV-16) E7, similar to those which express HPV-16 E6, are resistant to a p53-mediated G1 growth arrest. We examined the p53-mediated DNA damage response pathway in E7-expressing cells to determine the mechanism by which E7-containing cells continue to cycle. In response to DNA damage, no dramatic difference was detected in G1- or S-phase cyclin or cyclin-dependent kinase (Cdk) levels when E7-expressing cells were compared to the parental cell line, RKO. Furthermore, Cdk2 kinase activity was inhibited in both RKO cells and E7-expressing cells, while Cdk2 remained active in E6-expressing cells. However, the steady-state levels of pRB and p107 protein were substantially lower in E7-expressing cells than in the parental RKO cells or E6-expressing cells. There was no reduction in pRB mRNA levels, but the half-life of pRB in E7-expressing cells was markedly shorter. Infection of primary human foreskin keratinocytes with recombinant retroviruses expressing HPV-16 E7 resulted in a decrease in pRB protein levels, indicating this phenomenon is a consequence of E7 expression, not of immortalization or transformation. These data strongly suggest E7 interferes with the stability of pRB and p107 protein. We propose that the removal of these components of the p53-mediated G1 growth arrest pathway in E7-expressing cells contributes to the ability of E7 to overcome a p53-mediated G1 growth arrest.     

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.     

A role for p53 in maintaining and establishing the quiescence growth arrest in human cells

The p53 tumor suppressor protein induces transient growth arrest or apoptosis in response to genotoxic stress and mediates the irreversible growth arrest of cellular senescence. We present evidence here that p53 also contributes to the reversible, growth factor-dependent arrest of quiescence (G(0)). Microinjection of expression vectors encoding either MDM2 or a pRb-binding mutant of SV40 T antigen, both of which abrogate p53 function, stimulated quiescent normal human fibroblasts to initiate DNA synthesis and were 40-70% as effective as wild-type T antigen. Electrophoretic mobility shift and p53 transactivation assays showed that p53 activity was higher in quiescent and senescent cells compared with proliferating cells. As proliferating cells entered G(0) after growth factor withdrawal, the p53 mRNA level increased, followed by transient accumulation of the protein. Shortly thereafter, the expression (mRNA and protein) of p21, a p53 target gene and effector of cell cycle arrest, increased. Finally, stable expression of the HPV16 E6 oncogene or dominant negative p53 peptide, GSE-22, both of which inhibit p53 function, delayed entry into quiescence following growth factor withdrawal. Our data indicate that p53 is activated during both quiescence and senescence. They further suggest that p53 activity contributes, albeit not exclusively, to the quiescent growth arrest.     

Inactivation of both the retinoblastoma tumor suppressor and p21 by the human papillomavirus type 16 E7 oncoprotein is necessary to inhibit cell cycle arrest in human epithelial cells

The human papillomavirus (HPV) type 16 E7 oncoprotein must inactivate the retinoblastoma tumor suppressor (Rb) pathway to bypass G(1) arrest. However, E7 C-terminal mutants that were able to inactivate Rb were unable to bypass DNA damage-induced G(1) arrest and keratinocyte senescence, suggesting that the E7 C terminus may target additional G(1) regulators. The E7 C-terminal mutant proteins E7 CVQ68-70AAA and E7 Delta79-83 (deletion of positions 79 through 83) were further tested in several models of cell cycle arrest associated with elevated levels of p21. C-terminal mutations rendered E7 unable to induce S phase and endoreduplication in differentiated keratinocytes and rendered it less efficient in delaying senescence of human mammary epithelial cells. Interestingly, when cell cycle arrest was induced with a peptide form of p21, the E7 C-terminal mutants were deficient in overcoming arrest, whereas a mutant defective in Rb binding was competent in inhibiting G(1) arrest. These results suggest that the inactivation of both p21 and Rb by E7 contributes to subversion of cell cycle control in normal human epithelia but that neither p21 nor Rb inactivation alone is sufficient.     

The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest.

Functional p53 protein is associated with the ability of cells to arrest in G1 after DNA damage. The E6 protein of cancer-associated human papillomavirus type 16 (HPV-16) binds to p53 and targets its degradation through the ubiquitin pathway. To determine whether the ability of E6 to interact with p53 leads to a disruption of cell cycle control, mutated E6 proteins were tested for p53 binding and p53 degradation targeting in vitro, the ability to reduce intracellular p53 levels in vivo, and the ability to abrogate actinomycin D-induced growth arrest in human keratinocytes. Mutations scattered throughout the amino terminus, either zinc finger or the central region but not the carboxy terminus, severely reduced the ability of E6 to interact with p53. Expression of HPV-16 E6 or mutated E6 proteins that bound and targeted p53 for degradation in vitro sharply reduced the level of intracellular p53 induced by actinomycin D in human keratinocytes. A perfect correlation between the ability of E6 proteins to reduce the level of intracellular p53 and their ability to block actinomycin D-induced cellular growth arrest was observed. These results suggest that interaction with p53 is important for the ability of HPV E6 proteins to circumvent growth arrest.     

Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP-2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome

Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor beta (TGF-beta) signaling by favoring expression of the TGF-beta type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-beta treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-beta by lowering the intracellular amount of TIP-2/GIPC.     

Alternative fates of keratinocytes transduced by human papillomavirus type 18 E7 during squamous differentiation

The human papillomavirus type 18 (HPV-18) E7 protein promotes S-phase reentry in postmitotic, differentiated keratinocytes in squamous epithelium to facilitate vegetative viral DNA amplification. To examine the nature and fate of the differentiated cells that reenter S phase, organotypic cultures of primary human keratinocytes transduced with HPV-18 E7 were pulse-chase-pulse-labeled with (3)H-thymidine ((3)H-TdR) and bromodeoxyuridine (BrdU). The kinetics of the appearance of doubly labeled suprabasal cells demonstrate that E7 expression did not promote prolonged S phase. Rather, there was a considerable lag before a small percentage of the cells reentered another round of S phase. Fluorescence in situ hybridization analysis, indeed, revealed a small fraction of the cells with more than 4n chromosomes in the differentiated strata. Differentiated cells positive for (3)H-TdR, BrdU, or both often had enlarged nuclei or were binucleated. These results suggest that S phase is not followed by cell division, although nuclear division may occur. Interestingly, a significant fraction of differentiated cells that entered S phase subsequently accumulated p27kip1 protein with a kinetics preceding the accumulation of cyclin E. We conclude that E7-transduced, differentiated keratinocytes that enter S phase have two alternative fates: (i) a low percentage of cells undergoes endoreduplication, achieving higher than 4n ploidy, and (ii) a high percentage of cells accumulates the p27kip1, cyclin E, and p21cip1 proteins, resulting in arrest and preventing further S-phase reentry.     

Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV-16 plus EJ-ras.

The close association between HPV-16 and cervical cancer implies some role for the virus in development of this cancer. Recent studies have shown that the HPV-16 E7 gene encodes the major transforming activity of the virus in baby rat kidney (BRK) cell transformation assays. To investigate the requirement for continued E7 expression in BRK cells transformed by HPV-16 E7 plus EJ-ras, we have developed a system for inducible expression of the E7 gene. The studies reported here show that continued expression of the HPV-16 E7 gene is required for maintenance of the transformed phenotype in these cells. The implications these observations bear on the role of the E7 gene in cervical carcinoma are discussed.     

Growth/differentiation factor-5 induces growth arrest and apoptosis in mouse B lineage cells with modulation by Smad

Bone morphogenetic proteins, including growth/differentiation factor-5 (GDF-5), are multifunctional cytokines. Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta superfamily have focused on Smad proteins. However, scant attention has been given to the mechanism by which GDF-5 exerts its negative growth effect on immunological competent cells. In the present study, we demonstrated that GDF-5 induced cell cycle arrest in the G1 phase before the appearance of apoptosis in mouse B cell hybridoma HS-72 cells, while the ectopic expression of Smad6 and Smad7 in HS-72 cells suppressed the GDF-5-induced G1 cell cycle arrest by abolishing the expression of p21(CIP-1/WAF-1) and hypophosphorylation of retinoblastoma protein. Moreover, we found that Smad6 and Smad7 suppressed GDF-5-induced apoptosis in HS-72 cells. These findings indicated that Smad6 and Smad7 exhibit inhibitory effects toward GDF-5-mediated signaling in B lineage cells.     

The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein.

The human papillomavirus (HPV) E5 proteins are predicted from DNA sequence analysis to be small hydrophobic molecules, and the HPV type 6 (HPV-6) and HPV-11 E5 proteins share several structural similarities with the bovine papillomavirus type 1 (BPV-1) E5 protein. Also similar to the BPV-1 E5 protein, the HPV-6 and HPV-16 E5 proteins exhibit transforming activity when assayed on NIH 3T3 and C127 cells. In this study, we expressed epitope-tagged E5 proteins from both the "low-risk" HPV-6 and the "high-risk" HPV-16 in order to permit their immunologic identification and biochemical characterization. While the HPV-6 and HPV-16 E5 proteins fail to form disulfide-linked dimers and oligomers, they did resemble the BPV-1 E5 protein in their intracellular localization to the Golgi apparatus, endoplasmic reticulum, and nuclear membranes. In addition, the HPV E5 proteins also bound to the 16-kDa pore-forming protein component of the vacuolar ATPase, a known characteristic of the BPV-1 E5 protein. These studies reveal a common intramembrane localization and potential cellular protein target for both the BPV and HPV E5 proteins.     

Sequential activation of cyclin E and cyclin A gene expression by human papillomavirus type 16 E7 through sequences necessary for transformation.

To investigate E7-dependent biochemical changes which are involved in cellular transformation, we analyzed the influence of human papillomavirus type 16 (HPV-16) E7 on the expression of cell cycle regulatory proteins. Expression of E7 in established rodent fibroblasts (NIH 3T3), which was shown to be sufficient for transformation of these cells, leads to constitutive expression of the cyclin E and cyclin A genes in the absence of external growth factors. Surprisingly, expression of the cyclin D1 gene, which encodes a major regulator of G1 progression, is unaltered in E7-transformed cells. In transient transfection experiments, the cyclin A gene promoter is activated by E7 via an E2F binding site. In 14/2 cells, which were used as a model system to analyze the role of HPV-16 E7 in the transformation of primary cells, we observed rapid E7-dependent activation of cyclin E gene expression, which can be uncoupled from activation of the cyclin A gene, since the latter requires additional protein synthesis. E7-driven induction of cyclin E and cyclin A gene expression was accompanied by an increase in the associated kinase activities. Two domains of the E7 oncoprotein, which are designated cd1 and cd2, are essential for transformation of rodent fibroblasts. It is shown here that growth factor-independent expression of the cyclin E gene requires cd2 but not cd1, while activation of cyclin A gene expression requires cd1 function in addition to that of cd2. These data suggest that cyclin A gene expression is controlled by two distinct negative signals, one of which also restricts expression of the cyclin E gene. The ability of E7 to separately override each of these inhibitory signals, via cd1 and cd2, cosegregates with its ability to fully transform rodent fibroblasts. Unlike serum growth factors, E7 induces S-phase entry without activating cyclin D1 gene expression, in keeping with the finding that cyclin D1 function is not required in cells transformed by DNA tumor viruses.