Intracellular expression of a single-chain antibody directed against human papillomavirus type 16 E7 oncoprotein achieves targeted antineoplastic effects

Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. Anti-HPV16 E7 intracellular single-chain antibodies (scFvs) were constructed to down-regulate HPV16 E7 oncoprotein in HPV DNA-containing cell lines. In these studies, we transfected anti-E7 scFvs into the HPV16-positive human cervical carcinoma cell lines CaSki and SiHa and tested them for their ability to inhibit cell proliferation and alter the level of HPV16 E7 oncoprotein. Our results showed that anti-HPV16 E7 scFvs inhibited cell proliferation by >85% in CaSki cells and by 95% in SiHa cells. E7 oncoprotein was down-regulated by anti-HPV16 E7 scFv, and its expression was inversely related to the amount of scFv transfected. However, there were no effects of transfecting scFvs alone in HPV-negative cell lines. These results imply that anti-HPV16 E7 scFvs only have specific anti-HPV16 E7 effects on cell proliferation and on the synthesis of virally encoded proteins in HPV-positive cell lines. Thus, transfection of HPV16 E7-positive tumors with antigen-specific scFvs may be a viable strategy for cervical cancer gene therapy.     

Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein

We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic flux in tumor cells. The glycolytic intermediate fructose 1, 6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.     

Immunoexpression of mutant p53 and human papillomavirus related E6 oncoprotein in anal malignancies

Immunohistochemical expression of mutant p53 protein and human papillomavirus (HPV) 16 and 18 related E6 oncoprotein was studied in 36 biopsy proved anal cancers. Mutant p53 was detected in 61.1% cases. HPV 16 and 18 E6 protein was expressed in 22.2% cases, all of which were squamous cell carcinomas. Coexpression of both mutant p53 and E6 protein was found in only 5 cases (13.8%). In HPV 16/18 positive anal tumors, the degradation of p53 is accelerated by viral E6 oncoprotein. In HPV negative tumors, however, other mutagenic factors probably play a role in carcinogenesis.     

E6 of human papillomavirus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts

Immortalization is the consequence of the inactivation or bypass of two mortality stage mechanisms, M1 and M2, which are controlled by several genes including Rb and p53 in human fibroblasts. Abrogation of the M1 controls can be obtained through the activity of DNA tumor virus genes such as E6 and E7 of human papillomavirus 16 (HPV16). Fibroblasts expressing both E6 (which binds p53) and E7 (which binds Rb) bypass M1 and continue replicating (exhibit an extended lifespan) until an independent mechanism, M2, is activated. The inactivation of the M2 mechanism finally results in cell immortalization. The present study establishes a difference in the tissue-specific mechanisms for the control of the M1 stage of cellular senescence. The expression of HPV16 E6 was sufficient to bypass the M1 stage of cellular senescence and confer an extended lifespan in human mammary epithelial cells but not in fibroblasts. This implies that the M1 mechanism in human mammary epithelial cells does not involve the constitutive activation of Rb function as it does in fibroblasts. In addition, the results confirmed that the expression of HPV16 E6 (or both E6 and E7) did not directly immortalize the human mammary epithelial cells, since the inactivation of a second event, M2, was required to achieve immortalization. These observations are considered in the context of the telomere shortening model of cellular senescence.     

Inactivation of p27Kip1 by the viral E1A oncoprotein in TGFbeta-treated cells

The adenovirus oncoprotein E1A and the simian virus SV40 large T antigen can both reverse the strong growth-inhibitory effect of transforming growth factor(TGF)-beta on mink lung epithelial cells: exposure of TGF-beta causes these cells to arrest late in the G1 phase of the cell cycle (ref. 3). This arrest correlates with an increase in expression of the protein p15Ink4B (ref. 4), inactivation of the cyclin E/A-cdk2 complex by the inhibitory protein p27Kip1 (refs 5-7), and with the accumulation of unphosphorylated retinoblastoma protein. The rescue by E1A of cells from TGF-beta arrest is partly independent of its binding to retinoblastoma protein. Here we show that E1A directly affects the cyclin-dependent kinase inhibitor p27Kip1 in TGF-beta-treated cells by binding to it and blocking its inhibitory effect, thereby restoring the activity of the cyclin-cdk2 kinase complex. In this way, E1A can overcome the effect of TGF-beta and modulate the cell cycle. To our knowledge, E1A provides the first example of a viral oncoprotein that can disable a cellular protein whose function is to inhibit the activity of cyclin-dependent kinases.     

Human papillomavirus oncoproteins E6 and E7 independently abrogate the mitotic spindle checkpoint

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G1/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.     

Elevated wild-type p53 protein levels in human epithelial cell lines immortalized by the human papillomavirus type 16 E7 gene

The role tumor suppressors p53 and retinoblastoma (RB) play in the transformation process has become central to understanding the pathogenesis of DNA tumor viruses. The two oncoproteins of human papillomavirus (HPV)-16, E6 and E7, bind to p53 and RB, respectively, thus inactivating the function of these tumor suppressor genes. Immortalization of primary human foreskin epithelial cells by HPV requires expression of the E7 protein, and the E6 protein greatly enhances the immortalization frequency. Two of three cell lines immortalized by the HPV-16 E7 oncoprotein expressed wild-type p53 and only one of the three cell lines had acquired a p53 mutation and loss of heterozygosity at 17p during the immortalization process. All three E7-immortalized lines contained higher steady-state levels of p53 protein. Mutation of the p53 gene is not required for immortalization in the absence of the HPV-16 E6 inactivation of the p53 protein, and 16E7 expression leads to the stabilization of wild-type p53.     

The bovine papillomavirus E6 protein binds to the LD motif repeats of paxillin and blocks its interaction with vinculin and the focal adhesion kinase

The bovine papillomavirus type 1 (BPV-1) E6 oncoprotein can transform fibroblasts and induce anchorage-independent growth and disassembly of the actin stress fibers. We have previously shown that the E6 protein interacts with the focal adhesion protein, paxillin, suggesting a direct role of E6 in the disruption of the actin cytoskeleton. We have now mapped the E6 binding sites on paxillin to the LD motif repeats region, which has been implicated in mediating paxillin binding to two other focal adhesion proteins, vinculin and the focal adhesion kinase. The five LD motif repeats identified in paxillin do not contribute equally to its interaction with E6. The first LD repeat is most critical for paxillin binding to E6 both in vitro and in vivo. Furthermore, the binding of recombinant wild-type E6 protein to paxillin blocked the interaction of several cellular proteins with paxillin, including vinculin and the focal adhesion kinase. A mutant E6 protein (H105) which does not bind to paxillin had no effect on the binding of these cellular proteins to paxillin. These data suggest that E6 disruption of the actin stress fibers occurs through blocking the interaction of paxillin with its cellular effectors such as vinculin and the focal adhesion kinase.     

Inactivation of p16 in human mammary epithelial cells by CpG island methylation

Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G1 termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.     

Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded. Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cell fractionation experiments indicate that RI332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI332, its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61. From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.     

Up-regulation of hypoxia-inducible factor-1alpha is not sufficient for hypoxic/anoxic p53 induction

Oxygen-deprived regions of a solid tumor can induce tumor suppressor p53 expression and hence select for p53-mutant tumor cells with diminished apoptotic potential. It has been proposed that the hypoxia-inducible factor-1 (HIF-1) alpha subunit binds to p53 and protects it from proteasomal degradation. However, we found that hypoxic conditions that strongly induce HIF-1-dependent endogenous gene expression as well as HIF-1alpha protein neither induce p53-dependent gene expression nor p53 protein. The iron chelator deferoxamine induced both HIF-1alpha and p53, but p53 up-regulation could still be detected in HIF-1alpha-deficient cells, suggesting that mechanisms other than HIF-1alpha activation contribute to oxygen-regulated p53 induction.