HPV-18 confers resistance to TNF-alpha in organotypic cultures of human keratinocytes

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) inhibits normal keratinocytes proliferation. However, many human papillomavirus (HPV)-immortalized or transformed cell lines are resistant to TNF-alpha antiproliferative effect. The present study analyzes the effects of TNF-alpha on organotypic cultures of primary human keratinocytes (PHKs) that express HPV-18 oncogenes. Raft cultures prepared with PHKs acutely transfected with HPV-18 whole genome or infected with recombinant retroviruses containing only E6/E7 or E7 were treated with 2 nM TNF-alpha. While BrdU incorporation into basal/parabasal cells of normal PHKs cultures was markedly inhibited by TNF-alpha cultures transfected with HPV-18 whole genome showed proliferation in all cell strata. Furthermore, BrdU incorporation into cultures expressing E6/E7 or E7 was not significantly reduced, indicating that E7 alone confers partial resistance to TNF-alpha. Besides, TNF-alpha treatment did not alter p16ink4a, p21cip1, p27kip1, or cyclin E levels, but did reduce cyclin A and PCNA levels in sensitive cells.     

Steroid- and retinoid-mediated growth arrest and apoptosis in WEHI-231 cells: role of NF-kappaB, c-Myc and CKI p27(Kip1)

IgM cross-linking induces NF-kappaB inactivation, c-Myc down-regulation, and cyclin kinase inhibitor p27(Kip1) accumulation in WEHI-231 murine B lymphoma cells. p27(Kip1) up-regulation leads to a decreased cyclin-dependent kinase 2 activity, retinoblastoma protein hypophosphorylation, G1 arrest and apoptosis. Similar to membrane (m) IgM cross-linking in B lymphoma cells, steroids and retinoids down-regulate c-Myc (via NF-kappaB inactivation) and induce apoptosis in T cell hybridomas and thymocytes. In this study, we determined if steroids and retinoids have similar effects in WEHI-231 cells. Our results show that steroids and retinoids induce NF-kappaB inactivation, c-Myc down-regulation, p27(Kip1) up-regulation, G1 arrest, and apoptosis. Importantly, these hormones enhance anti-IgM-induced apoptosis in WEHI-231 cells. Similar to mIgM signaling, all these effects are prevented by treatment with CD40 ligand. Caspase inhibition, on the other hand, rescues cells from steroid/retinoid-induced apoptosis, but has no effect on growth arrest, p27(Kip1), and c-Myc. Together, these findings suggest that steroids/retinoids and mIgM cross-linking share a common signal transduction pathway leading to G1 arrest and cell death.     

Differences in transformation activity between HPV-18 and HPV-16 map to the viral LCR-E6-E7 region.

Homologous, subgenomic fragments of the viral LCR and E6/E7 transforming genes of HPV-18 and HPV-16 were amplified from several primary cervical, penile, and vulvar tumors and cloned into a pUC-18-derived vector. When assayed by a quantitative transformation assay using primary human keratinocytes, the subgenomic regions of HPV-16 and HPV-18 exhibited transforming activities similar to that of the full-length, prototype HPV genomes. More importantly, the HPV-18 LCR-E6-E7 region was approximately 10- to 50-fold more active than that of HPV-16. These studies demonstrate (1) that the transforming activity differences previously observed between prototype HPV-16 and HPV-18 map to the LCR-E6-E7 region, and (2) that individual and independent isolates of HPV-16 and HPV-18 exhibit consistent differences in transforming potential, even when isolated from different anatomic sites.     

Synergism between bovine papillomavirus type 4 and the flavonoid quercetin in cell transformation in vitro.

Bovine papillomavirus type 4 (BPV-4) morphologically transforms primary bovine cells in vitro only in the presence of an activated ras gene. The transformed cells are capable of anchorage-independent growth, but are not immortal and are incapable of inducing tumors in nude mice, suggesting that other events are needed to convert the cells to the fully transformed phenotype. We show here that treatment of the cells with a single dose of the flavonoid quercetin leads to full oncogenic transformation of cells transfected with BPV-4 and ras. Quercetin is one of the most potent mutagens found in bracken fern, the environmental cofactor in BPV-4-associated carcinogenesis of the upper alimentary canal of cattle. Our results point to quercetin as the probable in vivo cocarcinogen synergizing with BPV-4 in malignant progression.     

Angiotensin II induces p27(Kip1) expression in renal tubules in vivo: role of reactive oxygen species

Previous studies have demonstrated that angiotensin II (ANG II) mediates cell cycle arrest of cultured renal tubular cells by induction of p27(Kip1), an inhibitor of cyclin-dependent kinases. However, it is not known whether ANG II exerts similar effects in vivo. Infusion of ANG II into naive rats for 7 days increased formation of reactive oxygen species in tubular cells of the kidney. Furthermore, ANG II infusion stimulated protein expression of p27(Kip1) as detected by western blotting of tubular lysates and immunohistochemistry. Infusion of ANG II reduced tubular proliferation as detected by proliferating-cell nuclear antigen (PCNA) immunohistochemistry. The increase in p27(Kip1) expression was not due to an increase in mRNA. Immunoprecipitation experiments revealed that the increased p27(Kip1) protein associates with cyclin-dependent kinase 2. Coadministration of the radical scavenger dimethylthiourea abolished this ANG II mediated p27(Kip1) expression without reducing systemic blood pressure. Furthermore, dimethylthiourea infusion attenuates the ANG II mediated G(1)-phase arrest of tubular cells. However, infusion of norepinephrine did not induce reactive oxygen species or p27(Kip1) expression, despite a significant increase in blood pressure. Thus ANG II induces p27(Kip1) expression in renal tubular cells in vivo. This effect is mediated by reactive oxygen species. Since tubular hypertrophy depends on G(1)-phase arrest and may promote subsequent development of interstitial fibrosis, administering oxygen radical scavenger may be a therapeutic tool to counteract ANG II dependent remodeling of renal tubular cells.     

High glucose-induced hypertrophy of mesangial cells requires p27(Kip1), an inhibitor of cyclin-dependent kinases

Hypertrophy of mesangial cells is one of the earliest morphological alterations in the kidney after the onset of diabetes mellitus. We have previously shown that cultured mesangial cells exposed to high ambient glucose arrest in the G1 phase of the cell cycle and that this is associated with an increased expression of inhibitors of the cyclin-dependent kinase (CDK)-inhibitors p21(Cip) and p27(Kip1). To further investigate a potential role of p27Kip1 in the development of glucose-induced hypertrophy, mesangial cells from p27Kip1 wild-type (+/+) and knockout (-/-) mice were established. High glucose medium (450 mg/dl) increased p21(Cip1) protein in p27Kip1+/+ and -/- mesangial cells, and increased p27Kip1 protein levels in p27Kip1+/+ cells. In contrast to high glucose increasing de novo protein synthesis in p27Kip1+/+ cells, high glucose did not increase protein synthesis in p27Kip1-/- cells. High glucose also reduced DNA synthesis and caused cell cycle arrest in p27Kip1+/+ cells. In contrast, despite an increase in transforming growth factor (TGF)-beta mRNA and protein expression, DNA synthesis and cell cycle progression were increased by high glucose in p27Kip1-/- cells. Exogenous TGF-beta comparably induced fibronectin mRNA in p27Kip1+/+ and -/- cells suggesting intact TGF-beta receptor transduction. In addition, high glucose failed to increase the total protein/cell number ratio in p27Kip1-/- cells. However, in the presence of high glucose, reconstituting p27Kip1 expression by transient or stable transfection in p27Kip1-/- cells, using an inducible expression system, increased the de novo protein synthesis and restored G1-phase arrest. These results show that p27Kip1 is required for glucose-induced mesangial cell hypertrophy and cell cycle arrest.     

Oncogenic potential diverge among human papillomavirus type 16 natural variants

We compared E6/E7 protein properties of three different HPV-16 variants: AA, E-P and E-350G. Primary human foreskin keratinocytes (PHFK) were transduced with HPV-16 E6 and E7 and evaluated for proliferation and ability to grow in soft agar. E-P infected keratinocytes presented the lowest efficiency in colony formation. AA and E-350G keratinocytes attained higher capacity for in vitro transformation. We observed similar degradation of TP53 among HPV-16 variants. Furthermore, we accessed the expression profile in early (p5) and late passage (p30) transduced cells of 84 genes commonly involved in carcinogenesis. Most differences could be attributed to HPV-16 E6/E7 expression. In particular, we detected different expression of ITGA2 and CHEK2 in keratinocytes infected with AA and AA/E-350G late passage cells, respectively, and higher expression of MAP2K1 in E-350G transduced keratinocytes. Our results indicate differences among HPV-16 variants that could explain, at least in part, differences in oncogenic potential attributed to these variants.     

人乳头瘤病毒16型E6E7 ORF转化作用的研究

构建了含人乳头瘤病毒１６型（ＨＰＶ１６）－Ｅ６Ｅ７ＯＲＦｓ（ｎｔ８３－８５５）片段和ＨＰＶ１６长控制区（ＬＣＲ）加Ｅ６Ｅ７ＯＲＦｓ片段（ｎｔ７００７－７９０４／０－８７９）的逆转录病毒载体ｐＨ２１和ｐＨ１８质粒，利用Ｌｉｐｏｆｅｃｔｉｎ分别将它们导入病毒包装细胞ｐＡ３１７中，经过筛选获得Ｇ４１８抗性的病毒包装细胞，产生的重组病毒Ｈ２１和Ｈ１８感染的ＮＩＨ３Ｔ３细胞都具有恶性细胞的形态学特征，并能在裸鼠体内形成肿瘤。Ｓｏｕｔｈｅｒｎ杂交结果证明，上述两基因片段都整合到细胞基因组中。本实验结果说明ＨＰＶ１６－Ｅ６Ｅ７基因片段是ＨＰＶ１６转化ＮＩＨ３Ｔ３细胞的关键早期区，其自身ＬＣＲ区在该转化过程中没有显示出重要作用。     

Synergism between bovine papillomavirus type 4 and the flavonoid quercetin in cell transformation in vitro

Bovine papillomavirus type 4 (BPV-4) morphologically transforms primary bovine cells in vitro only in the presence of an activated ras gene. The transformed cells are capable of anchorage-independent growth, but are not immortal and are incapable of inducing tumors in nude mice, suggesting that other events are needed to convert the cells to the fully transformed phenotype. We show here that treatment of the cells with a single dose of the flavonoid quercetin leads to full oncogenic transformation of cells transfected with BPV-4 and ras. Quercetin is one of the most potent mutagens found in bracken fern, the environmental cofactor in BPV-4-associated carcinogenesis of the upper alimentary canal of cattle. Our results point to quercetin as the probable in vivo cocarcinogen synergizing with BPV-4 in malignant progression.     

The human papillomavirus type 16 E5 oncoprotein synergizes with EGF-receptor signaling to enhance cell cycle progression and the down-regulation of p27

E5 oncoprotein activity from high risk human papillomaviruses (HPVs) is associated with growth factor receptor signaling, but the function of this protein is not well understood. In this study, we investigated the role of HPV-16 E5 on the cell cycle progression during EGF-stimulation. Wild-type and NIH 3T3 cells over-expressing human EGF-receptor were transfected with HPV-16 E5 gene and the cell cycle progression was characterized. This analysis showed that the E5-expressing cells increased DNA synthesis (S-phase) by around 40%. Cell cycle protein analysis of E5-expressing cells showed a reduction in the half-life of p27^Kip1 protein as compared to control cells (18.4 vs. 12.7 h), an effect that was enhanced in EGF-stimulated cells (12.8 vs. 3.6 h). Blockage of EGF-receptor activity abrogated E5 signals as well as p27^Kip1 down-regulation. These results suggest that E5 and the EGF-receptor cooperate to enhance cell cycle entry and progression through regulating p27^Kip1 expression at protein level.     

The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 are not essential in T cell anergy

Recent evidence suggests that the cyclin-dependent kinase (Cdk) inhibitors p27Kip1 and p21Cip1 are important factors in T cell anergy, but it has remained unclear whether anergy can be induced in their absence. We therefore induced anergy by stimulation of purified T cells from wild-type, p21Cip1-/-, and p27Kip1-/- mice with anti-CD3 antibodies. Anergic wild-type T cells were arrested in the G1 phase of the cell cycle with a high p27Kip1 protein level and low Cdk2 activity. In p27-/- and p21-/- T cells, the pattern of protein expression was preserved, but Cdk2 activity was increased. To confirm the in vivo relevance of these data, anergy was induced by repeated injection of mice with staphylococcal enterotoxin B (SEB), which leads to partial deletion of the responsive Vbeta8+ T cell population and anergy in the remaining T cells. p21-/- mice and wild-type mice reacted similarly to this treatment. p27-/- mice showed reduced deletion of SEB-responsive T cells, but persisting T cells were anergic. These data indicate that other cell cycle regulators contribute to the cell cycle arrest of anergic T cells, as neither Cdk inhibitor is required for the induction of anergy.     

HCV core/gC1qR interaction arrests T cell cycle progression through stabilization of the cell cycle inhibitor p27Kip1

Hepatitis C virus (HCV) is efficient in the establishment of persistent infection. We have previously shown that HCV core protein inhibits T cell proliferation through its interaction with the complement receptor, gC1qR. Here we show that HCV core-induced inhibition of T cell proliferation involves a G(0)/G(1) cell cycle arrest, which is reversible upon addition of anti-gC1qR antibody. Correspondingly, the expression of cyclin-dependent kinases (Cdk) 2/4 and cyclin E/D, as well as subsequent phosphorylation of retinoblastoma (pRb), is reduced in core-treated T cells in response to mitogenic stimulation. Remarkably, degradation of p27(Kip1), a negative regulator of both Cdk4/cyclin D and Cdk2/cyclin E complexes, is significantly diminished in T cells treated with HCV core upon mitogenic stimulation. These data indicate that the stability of p27(Kip1) by HCV core is associated with blocking activated T cells for the G(1) to S phase transition and inhibiting T cell proliferation.     

Chromosome aberrations in cattle with chronic enzootic haematuria

Chromosomal aberrations were investigated in 56 cattle with chronic enzootic haematuria (CEH) raised on pastures giving access to bracken fern. Of these animals, 27 were slaughtered and showed neoplastic lesions of the urinary bladder. Tumour tissue from 11 of the 27 cattle contained bovine papillomavirus type 2 (BPV-2) DNA. Increased numbers of chromosomal aberrations were seen in all animals with CEH, as compared with 30 control cattle that had had no access to bracken fern. The highest clastogenic effect was observed in cattle with urinary bladder cancer and evidence of BPV-2 DNA, suggesting that BPV-2 and bracken fern act synergistically in the production of chromosomal instability. In 19 of 20 animals with CEH, two bracken fern toxic compounds (quercitin and ptaquiloside) were demonstrated in urine, serum and milk.     

Increased P27KIP1 protein expression in the dentate gyrus of chronically stressed rats indicates G1 arrest involvement

Various chronic stress paradigms decrease new cell proliferation in the hippocampal dentate gyrus, yet the exact underlying mechanism is still unclear. In the first gap (G1) phase of the cell cycle, both stimulatory and inhibitory signals derived from the extracellular environment converge. Corticosteroids, which increase during stress and are well-known anti-mitotics, cause cells in vitro to arrest in the G1 phase. Following 3 weeks of unpredictable stress, we therefore expected a change in protein expression of various important G1 cell cycle regulators in the adult rat subgranular zone. Using quantitative immunocytochemistry, we show that particularly cyclin-dependent kinase inhibitor p27Kip1 expression is significantly increased. In addition, 3 weeks of recovery after stress normalized the numbers of p27Kip1-expressing cells, consistent with the recovered adult cell proliferation in these animals. P27Kip1-positive cells do not overlap with GFAP-staining and only to a limited extent with Ki-67-expressing cells. Numbers of cyclin E- and cyclin D1-expressing cells did not change after chronic stress. These results indicate that chronic stress causes cycling cells in the adult hippocampus to arrest in G1, thereby providing more mechanistic insight in the stress-induced decrease in cell proliferation.     

p27(Kip1) is important in modulating pulmonary artery smooth muscle cell proliferation.

Vascular remodeling due to pulmonary arterial smooth muscle cell (PASMC) proliferation is central to the development of pulmonary hypertension. Cell proliferation requires the coordinated interaction of cyclins and cyclin-dependent kinases (cdk) to drive cells through the cell cycle. Cdk inhibitors can bind cyclin-cdk complexes and cause G(1) arrest. To determine the importance of the cdk inhibitor p27(Kip1) in PASMC proliferation we studied [(3)H]thymidine incorporation, changes in cell cycle, cell proliferation, and protein expression of p27(Kip1) following serum stimulation in early passage rat PASMC. p27(Kip1) expression decreased to 40% of baseline after serum stimulation, which was associated with an increase in both [(3)H]thymidine incorporation and the percent of cells in S phase. p27(Kip1) binding to cyclin E decreased at 24 h, and this correlated with an increase in phosphorylation of retinoblastoma both in vivo and in vitro. Overexpression of p27(Kip1) decreased [(3)H]thymidine incorporation and reduced cell counts at 5 d compared with controls. PASMC obtained from p27(Kip1-/-) mice showed a 2-fold increase in [(3)H]thymidine incorporation (at 24 h) and cell proliferation compared with p27(Kip1+/+) PASMC when cultured in 10% fetal bovine serum (FBS). These results suggest an important role for p27(Kip1) in regulating PASMC mitogenesis and proliferation.