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1.  Endogenous Human Papillomavirus E6 and E7 Proteins Differentially Regulate Proliferation, Senescence, and Apoptosis in HeLa Cervical Carcinoma Cells 
Journal of Virology  2003;77(2):1551-1563.
Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins, and repression of HPV gene expression causes the cells to cease proliferation and undergo senescence. However, it is not known whether both HPV proteins are required to maintain the proliferative state of cervical cancer cells, or whether mutations that accumulate during carcinogenesis eliminate the need for one or the other of them. To address these questions, we used the bovine papillomavirus E2 protein to repress the expression of either the E6 protein or the E7 protein encoded by integrated HPV18 DNA in HeLa cervical carcinoma cells. Repression of the E7 protein activated the Rb pathway but not the p53 pathway and triggered senescence, whereas repression of the E6 protein activated the p53 pathway but not the Rb pathway and triggered both senescence and apoptosis. Telomerase activity, cyclin-dependent kinase activity, and expression of c-myc were markedly inhibited by repression of either E6 or E7. These results demonstrate that continuous expression of both the E6 and the E7 protein is required for optimal proliferation of cervical carcinoma cells and that the two viral proteins exert distinct effects on cell survival and proliferation. Therefore, strategies that inhibit the expression or activity of either viral protein are likely to inhibit the growth of HPV-associated cancers.
doi:10.1128/JVI.77.2.1551-1563.2003
PMCID: PMC140828  PMID: 12502868
2.  A Humanized Mouse Model of HPV-Associated Pathology Driven by E7 Expression 
PLoS ONE  2012;7(7):e41743.
Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.
doi:10.1371/journal.pone.0041743
PMCID: PMC3402432  PMID: 22911850
3.  Cell culture model predicts human disease: Altered expression of junction proteins and matrix metalloproteinases in cervical dysplasia 
Background
Cervical cancer is necessarily caused by human papillomaviruses, which encode three oncogenes manifesting their functions by interfering with a number of cellular proteins and pathways: the E5, E6, and E7 proteins. We have earlier found in our microarray studies that the E5 oncogene crucially affects the expression of cellular genes involved in adhesion and motility of epithelial cells.
Methods
In order to biologically validate our previous experimental findings we performed immunohistochemical staining of a representative set of tissue samples from different grades of high-risk human papillomavirus associated cervical disease as well as normal squamous and columnar cervical epithelium. Three-dimensional collagen raft cultures established from E5-expressing and control epithelial cells were also examined. The expression of p16, matrix metalloproteinase (MMP) -7, MMP-16, cytokeratin (CK) 8/18, laminin, E-cadherin and beta-catenin was studied.
Results
In agreement with our previous microarray studies, we found intense staining for E-cadherin and beta-catenin in adherens junctions even in high-grade cervical lesions. Staining for MMP-16 was increased in severe disease as well. No significant change in staining for MMP-7 and cytokeratin 8/18 along with the grade of cervical squamous epithelial disease was observed.
Conclusions
Here we have confirmed, using tissue material from human papillomavirus associated lesions, some of the cellular gene expression modifications that we earlier reported in an experimental system studying specifically the E5 oncogene of papillomaviruses. These findings were partially surprising in the context of cervical carcinogenesis and emphasize that the complexity of carcinogenesis is not yet fully understood. Microarray approaches provide a wide overwiev of gene expression in experimental settings, which may yield biologically valid biomarkers for disease diagnostics, prognosis, and follow-up.
doi:10.1186/1472-6890-12-9
PMCID: PMC3495715  PMID: 22863036
Cadherin; Catenin; CIN; Cytokeratin; E5; HPV; Microarray; MMP
4.  The E7 Oncoprotein of Human Papillomavirus Type 16 Stabilizes p53 through a Mechanism Independent of p19ARF 
Journal of Virology  1999;73(9):7590-7598.
High-risk human papillomaviruses are causally associated with cervical cancer. Two viral oncogenes, E6 and E7, are expressed in most cervical cancers, and these genes cause cancer when expressed in experimental animals. The E6 protein targets the p53 tumor suppressor for degradation, while the E7 protein inactivates the retinoblastoma susceptibility protein (pRb), in part by stimulating its degradation. In contrast, expression of E7 in the absence of E6 leads to stabilization of p53. Here we show that E7 stabilizes p53 in mouse embryo fibroblasts lacking p19ARF. The stable p53 is active as a transcriptional activator, as evidenced by the increased expression of the p53-responsive mdm2 gene. Normally, MDM2 protein inhibits p53 function in an autoregulatory loop. Regulation of p53 by MDM2 is required for murine development as well as for proliferation of cultured human fibroblasts. However, E7-expressing human fibroblasts continue to divide even though E7 abrogates the ability of MDM2 and p53 to bind. Furthermore, E7-expressing cells are not more sensitive to UV light, an agent that has been reported to induce apoptosis mediated by p53. These results indicate that in addition to inhibiting the ability of MDM2 to regulate p53, E7 must block signaling steps downstream of p53 to allow cell division.
PMCID: PMC104286  PMID: 10438849
5.  Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation 
Virology  2011;422(1):114-124.
Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.
doi:10.1016/j.virol.2011.10.012
PMCID: PMC3229657  PMID: 22056390
HPV; HeLa cells; GM1; viral tropism; oncogene addiction; cervical cancer, SV40
6.  The Fanconi Anemia Pathway Limits Human Papillomavirus Replication 
Journal of Virology  2012;86(15):8131-8138.
High-risk human papillomaviruses (HPVs) deregulate epidermal differentiation and cause anogenital and head and neck squamous cell carcinomas (SCCs). The E7 gene is considered the predominant viral oncogene and drives proliferation and genome instability. While the implementation of routine screens has greatly reduced the incidence of cervical cancers which are almost exclusively HPV positive, the proportion of HPV-positive head and neck SCCs is on the rise. High levels of HPV oncogene expression and genome load are linked to disease progression, but genetic risk factors that regulate oncogene abundance and/or genome amplification remain poorly understood. Fanconi anemia (FA) is a genome instability syndrome characterized at least in part by extreme susceptibility to SCCs. FA results from mutations in one of 15 genes in the FA pathway, whose protein products assemble in the nucleus and play important roles in DNA damage repair. We report here that loss of FA pathway components FANCA and FANCD2 stimulates E7 protein accumulation in human keratinocytes and causes increased epithelial proliferation and basal cell layer expansion in the HPV-positive epidermis. Additionally, FANCD2 loss stimulates HPV genome amplification in differentiating cells, demonstrating that the intact FA pathway functions to restrict the HPV life cycle. These findings raise the possibility that FA genes suppress HPV infection and disease and suggest possible mechanism(s) for reported associations of HPV with an FA cohort in Brazil and for allelic variation of FA genes with HPV persistence in the general population.
doi:10.1128/JVI.00408-12
PMCID: PMC3421690  PMID: 22623785
7.  Up-regulation of expression and lack of 5' CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas 
BMC Cancer  2007;7:47.
Background
High risk type human papilloma viruses (HR-HPV) induce carcinomas of the uterine cervix by expressing viral oncogenes E6 and E7. Oncogene E7 of HR-HPV disrupts the pRb/E2F interaction, which negatively regulates the S phase entry. Expression of tumor suppressor p16ink4a drastically increases in majority of HR-HPV associated carcinomas due to removal of pRb repression. The p16ink4a overexpression is an indicator of an aberrant expression of viral oncogenes and may serve as a marker for early diagnostic of cervical cancer. On the other hand, in 25–57% of cervical carcinomas hypermethylation of the p16 INK4a promoter has been demonstrated using a methylation-specific PCR, MSP. To evaluate a potential usage of the p16 INK4a 5' CpG island hypermethylation as an indicator of tumor cell along with p16ink4a overexpression, we analyzed the methylation status of p16 INK4a in cervical carcinomas
Methods
Methylation status of p16 INK4a was analyzed by MSP and by bisulfite-modified DNA sequencing. The expression of p16ink4a was analyzed by RT-PCR and by immunohistochemical technique.
Results
The extensive methylation within p16 INK4a 5' CpG island was not detected either in 13 primary cervical carcinomas or in 5 cancer cell lines by bisulfite-modified DNA sequencing (including those that were positive by MSP in our hands). The number and distribution of rare partially methylated CpG sites did not differ considerably in tumors and adjacent normal tissues. The levels of the p16 INK4a mRNA were increased in carcinomas compared to the normal tissues independently of the number of partially methylated CpGs within 5'CpG island. The transcriptional activation of p16 INK4a was accompanied by p16ink4a cytoplasmic immunoreactivity in the majority of tumor cells and presence of a varied number of the p16 positive nuclei in different tumors.
Conclusion
Hypermethylaion of the p16INK4a 5' CpG island is not a frequent event in HR-HPV-positive cervical carcinomas and cannot be an effective marker of cancer cells with up-regulated expression of p16ink4a. Our data confirm other previous studies claiming specific p16INK4a up-regulation in the majority of cervical carcinomas at both the protein and mRNA levels. Cytoplasmic accumulation of p16ink4a is a feature of cervical carcinomas.
doi:10.1186/1471-2407-7-47
PMCID: PMC1831478  PMID: 17359536
8.  Repression of the Human Papillomavirus E6 Gene Initiates p53-Dependent, Telomerase-Independent Senescence and Apoptosis in HeLa Cervical Carcinoma Cells 
Journal of Virology  2004;78(8):4063-4073.
Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine the role of p53 signaling in senescence or apoptosis after repression of HPV oncogenes, we introduced a dominant-negative allele of p53 into HeLa cells. Dominant-negative p53 prevented senescence and apoptosis when E6 alone was repressed but did not inhibit senescence when both E6 and E7 were repressed. To determine whether reduced telomerase activity was involved in senescence or apoptosis after E6 repression, we generated HeLa cells stably expressing an exogenous hTERT gene, which encodes the catalytic subunit of telomerase. Although these cells contained markedly elevated telomerase activity and elongated telomeres, hTERT expression did not prevent senescence and apoptosis when E6 alone was repressed. These results demonstrate that when the Rb tumor suppressor pathway is inactivated by the E7 protein, E6 repression activates p53 signaling, which in turn is required for growth inhibition, senescence, and apoptosis. Thus, sustained inactivation of the p53 pathway by the E6 protein is required for maintenance of the proliferative phenotype of HeLa cervical carcinoma cells.
doi:10.1128/JVI.78.8.4063-4073.2004
PMCID: PMC374296  PMID: 15047823
9.  Reversible repression of papillomavirus oncogene expression in cervical carcinoma cells: consequences for the phenotype and E6-p53 and E7-pRB interactions. 
Journal of Virology  1994;68(5):2811-2821.
The transforming genes E6 and E7 of high-risk human papillomaviruses are consistently expressed in papillomavirus-associated neoplasms of the anogenital tract. In papillomavirus type 18-associated SW 756 cervical carcinoma cells, transcription of the viral E6-E7 genes is blocked by dexamethasone. Herein we show that dexamethasone-mediated repression of the E6-E7 genes results in loss of the neoplastic phenotype of SW 756 cells. Withdrawal of dexamethasone restores E6-E7 expression and neoplastic growth. Moreover, reconstitution of E6-E7 gene expression by a dexamethasone-inducible expression vector renders the neoplastic phenotype resistant to dexamethasone. These results clearly indicate that the continuous expression of the viral E6-E7 oncogenes is required to maintain the neoplastic growth properties of SW 756 cervical cancer cells. The viral E6 protein destabilizes the p53 tumor suppressor gene product in vitro. Since low levels of p53 have been observed in papillomavirus-transformed keratinocyte cell lines, it was speculated that degradation of p53 by E6 contributes to papillomavirus-associated growth deregulation. Consistent with this hypothesis, we detected a significant increase in p53 levels upon dexamethasone-induced repression of papillomavirus E6-E7 oncogene expression. No p53 increase was observed in dexamethasone-treated cells in which the viral oncogene expression was restored. The viral E7 protein has been shown to complex with the retinoblastoma tumor suppressor gene product (pRB). In some cells, this interaction has been shown shown to release the transcription factor E2F from its complex with pRB, and it has been hypothesized that E7-induced, increased levels of free E2F contribute to the transforming potential of the viral oncogenes. In gel shift experiments, we detected relatively stable complexes of pRB and E2F in all SW 756-derived cells, independent of the level of E7 expression. This suggests that E7-mediated release of E2F from its complex with pRB might not be required to maintain the neoplastic phenotype of human papillomavirus-associated cancer cells, although a possibly relevant partial E7-mediated release of E2F from pRB cannot be excluded.
Images
PMCID: PMC236769  PMID: 8151752
10.  Depletion of the cdk Inhibitor p16INK4a Differentially Affects Proliferation of Established Cervical Carcinoma Cells 
Journal of Virology  2014;88(10):5256-5262.
ABSTRACT
Infections with high-risk human papillomaviruses (hrHPV) contribute to cervical carcinoma. The cdk inhibitor and tumor suppressor p16INK4A is consistently upregulated in cervical carcinoma cells for reasons that are poorly understood. We report here that downregulation of p16INK4A gene expression in three different cervical carcinoma cell lines reduced expression of the E7 oncogene, suggesting a positive feedback loop involving E7 and p16INK4A. p16INK4A depletion induced cellular senescence in HeLa but not CaSki and MS-751 cervical carcinoma cells.
IMPORTANCE This study demonstrates that the cdk inhibitor p16INK4A, frequently used as surrogate marker for transforming infections by human papillomaviruses of the high-risk group, is required for high-level expression of the E7 oncoproteins of HPV-16, HPV-18, and HPV-45 in cervical carcinoma cells. It is also demonstrated that depletion of p16INK4A induces senescence in HeLa but not CaSki or MS-751 cervical carcinoma cells.
doi:10.1128/JVI.03817-13
PMCID: PMC4019144  PMID: 24599991
11.  Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein. 
Molecular and Cellular Biology  1993;13(2):953-960.
Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.
Images
PMCID: PMC358979  PMID: 7678696
12.  Critical Roles for Non-pRb Targets of Human Papillomavirus Type 16 E7 in Cervical Carcinogenesis 
Cancer research  2006;66(19):9393-9400.
High-risk human papillomaviruses (HPV) encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. In vivo, HPV-16 E7 has been shown to induce multiple phenotypes in the context of transgenic mice, including cervical cancer. E7 is a multifunctional protein known best for its ability to inactivate the tumor suppressor pRb. To determine the importance of pRb inactivation by E7 in cervical cancer, we pursued studies with genetically engineered mice. E7 expression in estrogen-treated murine cervix induced dysplasia and invasive cancers as reported previously, but targeted Rb inactivation in cervical epithelium was not sufficient to induce any cervical dysplasia or neoplasia. Furthermore, E7 induced cervical cancer formation even when the E7-pRb interaction was disrupted by the use of a knock-in mouse carrying an E7-resistant mutant Rb allele. pRb inactivation was necessary but not sufficient for E7 to overcome differentiation-induced or DNA damage–induced cell cycle arrest, and expression patterns of the E2F-responsive genes Mcm7 and cyclin E indicate that other E2F regulators besides pRb are important targets of E7. Together, these data indicate that non-pRb targets of E7 play critical roles in cervical carcinogenesis.
doi:10.1158/0008-5472.CAN-06-0984
PMCID: PMC2858286  PMID: 17018593
13.  The Human Papillomavirus E6 Oncogene Represses a Cell Adhesion Pathway and Disrupts Focal Adhesion through Degradation of TAp63β upon Transformation 
PLoS Pathogens  2011;7(9):e1002256.
Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical pathway in human carcinogenesis.
Author Summary
High-risk human papillomavirus infection can cause cancer of the uterine cervix. The viral proteins leading to transformation of the infected keratinocytes are the E6 and E7 oncogenes which interact with and induce degradation of the cell cycle regulators p53 and pRB. In cervical carcinoma cells, repression of E6/E7 stabilizes the p53 transcription factor leading to activation of a large group of cellular p53 target genes. Here we show that repression of E6/E7 also induces transcriptional activation of an additional large set of genes involved in cell adhesion including previously described p63 target genes. Indeed, we further demonstrated that these p63 target genes are activated by TAp63β and not by p53 or by the ΔNp63α or β isoforms, even though these transcription factors are also expressed in these cells. In cervical carcinoma cells, E6 expression therefore leads to TAp63β degradation thereby allowing anchorage independent growth. Our work describes a new E6-dependent transformation pathway in HPV-associated carcinogenesis. TAp63β inhibition may also represent a common pathway to activate anchorage independent growth in cancers.
doi:10.1371/journal.ppat.1002256
PMCID: PMC3182928  PMID: 21980285
14.  Human Papillomavirus E7 Repression in Cervical Carcinoma Cells Initiates a Transcriptional Cascade Driven by the Retinoblastoma Family, Resulting in Senescence▿ †  
Journal of Virology  2006;81(5):2102-2116.
This work demonstrates a central role for the retinoblastoma (Rb) family in driving the transcriptional program of induced and replicative senescence. HeLa cervical carcinoma cells rapidly undergo senescence when the human papillomavirus (HPV) type 18 E7 gene in these cells is repressed by the bovine papillomavirus (BPV) E2 protein. This senescence response requires the endogenous Rb pathway but not the p53 pathway. Microarray analysis 6 days after BPV E2 introduction into HeLa cells identified 224 cellular genes induced by E7 repression and 354 repressed genes. Many repressed genes were involved in cell cycle progression, and numerous induced genes encoded lysosomal proteins. These gene expression changes were blocked by constitutive expression of the wild-type HPV16 E7 or adenovirus E1A gene, but not by E7 or E1A mutants defective for Rb binding. Short hairpin RNAs targeting the Rb family also inhibited these gene expression changes and blocked senescence. Therefore, surprisingly, the transcriptional response to BPV E2 expression was entirely dependent on E7 repression and activation of the Rb family, and the BPV E2 protein did not directly affect the expression of cellular genes. Activation of the Rb family repressed E2F-responsive genes and stimulated transcriptional activators, thereby mobilizing multiple signals, such as repression of B-MYB and DEK, that were independently sufficient to induce senescence. There was extensive overlap between the transcriptional profiles of senescent, late-passage primary human fibroblasts and senescent cervical carcinoma cells, suggesting that this Rb family-mediated transcriptional cascade also plays a central role in replicative senescence.
doi:10.1128/JVI.02348-06
PMCID: PMC1865941  PMID: 17182682
15.  1st international Beatson symposium--cellular, molecular and clinical aspects of squamous cell carcinomas. 
British Journal of Cancer  1994;69(2):406-408.
About 80% of neoplasias are epithelial in origin and, as such, understanding the molecular mechanisms involved in the development of epithelial tumours is vital to the diagnosis, prognosis and treatment of the vast majority of human cancers. Obviously this is no easy task but, as outlined above, great efforts are being made to identify important molecules involved in the progression of normal epithelial cells to carcinoma. The development of techniques to identify new oncogenes is of particular importance, and hopefully the cDNA expression cloning system of Stuart Aaronson will be a useful tool in this respect. The potential of some of these molecules to be used as therapeutic targets will require the development of suitable screening procedures, such as that being established by Chris Marshall for the ras-Map kinase pathway in yeast. It is encouraging that the immune response to virally (HPV) induced cancer is being carefully elucidated and the prospects of vaccine development for the treatment of cervical cancer coming nearer since this particular form of SCC is a major cancer globally. Finally it was fitting to end the meeting on an optimistic note with John Mendelsohn's EGFR monoclonal antibody therapy entering clinical trials, and hopefully this will prove efficacious in the treatment of human SSC.
PMCID: PMC1968689  PMID: 8297743
16.  Targeting the Human Papillomavirus E6 and E7 Oncogenes through Expression of the Bovine Papillomavirus Type 1 E2 Protein Stimulates Cellular Motility▿†  
Journal of Virology  2011;85(20):10487-10498.
Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors.
doi:10.1128/JVI.05126-11
PMCID: PMC3187478  PMID: 21835799
17.  Clonal Selection for Transcriptionally Active Viral Oncogenes during Progression to Cancer 
Journal of Virology  2004;78(20):11172-11186.
Primary keratinocytes immortalized by human papillomaviruses (HPVs), along with HPV-induced cervical carcinoma cell lines, are excellent models for investigating neoplastic progression to cancer. By simultaneously visualizing viral DNA and nascent viral transcripts in interphase nuclei, we demonstrated for the first time a selection for a single dominant papillomavirus transcription center or domain (PVTD) independent of integrated viral DNA copy numbers or loci. The PVTD did not associate with several known subnuclear addresses but was almost always perinucleolar. Silent copies of the viral genome were activated by growth in the DNA methylation inhibitor 5-azacytidine. HPV-immortalized keratinocytes supertransduced with HPV oncogenes and selected for marker gene coexpression underwent crisis, and the surviving cells transcribed only the newly introduced genes. Thus, transcriptional selection in response to environmental changes is a dynamic process to achieve optimal gene expression for cell survival. This phenomenon may be critical in clonal selection during carcinogenesis. Examination of HPV-associated cancers supports this hypothesis.
doi:10.1128/JVI.78.20.11172-11186.2004
PMCID: PMC521852  PMID: 15452237
18.  Aberrant Cell Cycle Regulation in Cervical Carcinoma 
Yonsei Medical Journal  2005;46(5):597-613.
Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16INK4A-cyclin D1-CDK4/6-pRb-E2F, p21WAF1-p27KIP1-cyclinE-CDK2, and p14ARF-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16INK4A, p21WAF1, and p27KIP1, as characteristic features of HPV-infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.
doi:10.3349/ymj.2005.46.5.597
PMCID: PMC2810564  PMID: 16259056
Cell cycle; cervical carcinoma; cyclin
19.  Frequency of down-regulation of individual HLA-A and -B alleles in cervical carcinomas in relation to TAP-1 expression. 
British Journal of Cancer  1995;72(2):405-411.
The development of cervical carcinoma is strongly associated with specific types of human papillomaviruses (HPVs). A role for cellular immunity in cervical disease is supported by the increased occurrence of HPV-associated lesions in immunosuppressed individuals. Upon viral infection or malignant transformation, ensuing alterations in gene expression result in the generation of novel sets of peptides which can form complexes with specific HLA class I heavy chains and beta 2-microglobulin. These are then expressed at the cell surface as potential targets for specific T cells. In this study of 100 carcinomas HLA-A and -B class I expression by the tumour cells was down-regulated at one or more alleles in at least 73% of cervical carcinomas. Interference with the transporter associated with antigen presentation (TAP), which translocates cytosolic peptides from endogenously synthesised proteins (e.g. viral) into the lumen of the endoplasmic reticulum was found in 38% of the HLA class I down-regulated tumours. Loss of expression for common HLA class I alleles ranged from 36% to 71%, and such changes might be expected to influence specific immunogenic peptide presentation and consequent immune recognition. These results underline the importance of single as well as multiple allelic loss in cervical neoplasia and have important implications for attempts to intervene immunologically in cervical cancer.
PMCID: PMC2033975  PMID: 7640226
20.  Papillomavirus-Mediated Neoplastic Progression Is Associated with Reciprocal Changes in Jagged1 and Manic Fringe Expression Linked to Notch Activation†  
Journal of Virology  2004;78(16):8687-8700.
Infection by high-risk human papillomaviruses (HPV) and persistent expression of viral oncogenes E6 and E7 are causally linked to the development of cervical cancer. These oncogenes are necessary but insufficient for complete transformation of human epithelial cells in vivo. Intracellular Notch1 protein is detected in invasive cervical carcinomas (ICC), and truncated Notch1 alleles complement the function of E6/E7 in the transformation of human epithelial cells. Here we investigate potential mechanisms of Notch activation in a human cervical neoplasia. We have analyzed human cervical lesions and serial passages of an HPV type 16-positive human cervical low-grade lesion-derived cell line, W12, that shows abnormalities resembling those seen in cervical neoplastic progression in vivo. Late-passage, but not early-passage, W12 and progression of the majority of human high-grade cervical lesions to ICC showed upregulation of Notch ligand and Jagged1 and downregulation of Manic Fringe, a negative regulator of Jagged1-Notch1 signaling. Concomitantly, an increase in Notch/CSL (CBF1, Suppressor of Hairless, Lag1)-driven reporter activity and a decrease in Manic Fringe upstream regulatory region (MFng-URR)-driven reporter activity was observed in late-passage versus early passage W12. Analysis of the MFng-URR revealed that Notch signaling represses this gene through Hairy Enhancer of Split 1, a transcriptional target of the Notch pathway. Expression of Manic Fringe by a recombinant adenovirus, dominant-negative Jagged1, or small interfering RNA against Jagged1 inhibits the tumorigenicity of CaSki, an ICC-derived cell line that was previously shown to be susceptible to growth inhibition induced by antisense Notch1. We suggest that activation of Notch in cervical neoplasia is Jagged1 dependent and that its susceptibility to the influence of Manic Fringe is of therapeutic value.
doi:10.1128/JVI.78.16.8687-8700.2004
PMCID: PMC479091  PMID: 15280477
21.  Small-interfering RNA-mediated silencing of the MAPK p42 gene induces dual effects in HeLa cells 
Oncology Letters  2010;1(4):649-655.
The genesis and progression of cervical cancer involve the mutation or deviant expression of numerous genes, including the activation of oncogenes (Ha-ras, C-myc, C-erbB2 and Bcl-2) and inactivation of tumor-suppressor genes (p53 and Rb). Previous studies showed that small-interfering RNAs (siRNAs) targeting the MAPK p42 gene partly inhibit proliferation and increase apoptosis in human cervical carcinoma HeLa cells. Results of a microarray analysis showed that MAPK p42 siRNA inhibited cell growth through the regulation of cell cycle control and apoptosis and induced interferon-like response in HeLa cells. In order to confirm the dual effects of MAPK p42 siRNA, we compared the roles of siRNA and U0126, an inhibitor of MAPK p42, in HeLa cells. Short 21-mer double-stranded/siRNAs were synthesized to target MAPK p42 mRNA in HeLa cells. The siRNAs were transfected into HeLa cells using Lipofectamine. The cells were treated with siRNA or U0126 at different concentrations for a period of 48 h. The biological effect of siRNA and U0126 on HeLa cells was measured by MTT and flow cytometry. MAPK1, NUP188, P38, STAT1, STAT2, PML and OAS1 were analyzed by real-time quantitative PCR. HeLa cell growth was inhibited by siRNA or U0126, and the effect of siRNA inhibition was greater than that of U0126. Cell cycle phases were different for siRNA or U0126, but HeLa cell growth was arrested at the S phase by siRNA and at G1 phase by U0126. A down-regulation in MAPK p42 expression by siRNA and up-regulation by U0126 were noted. The results of real-time quantitative PCR showed that P38 was up-regulated and NUP188 was down-regulated by siRNA in comparison with the control groups, and the results were consistent with those of U0126. Expression levels of STAT1, STAT2, PML and OAS1 induced by siRNA differed from those induced by U0126. siRNA-mediated silencing and deactivation induced by U0126 in MAPK p42 led to growth inhibition in the HeLa cells. The effects of siRNA on HeLa cell growth were different from those of U0126. Dual effects of MAPK p42 siRNA-2 on HeLa cell growth were noted: one consisted of a specific effect induced by siRNA-mediated p42 MAPK silencing and the other exhibited a non-specific interferon-like response.
doi:10.3892/ol_00000114
PMCID: PMC3436355  PMID: 22966358
interferon-like response; targeting response; MAPK p42; RNA interference
22.  Papillomavirus E6 proteins 
Virology  2008;384(2):324-334.
The papillomaviruses are small DNA viruses that encode approximately eight genes, and require the host cell DNA replication machinery for their viral DNA replication. Thus papillomaviruses have evolved strategies to induce host cell DNA synthesis balanced with strategies to protect the cell from unscheduled replication. While the papillomavirus E1 and E2 genes are directly involved in viral replication by binding to and unwinding the origin of replication, the E6 and E7 proteins have auxillary functions that promote proliferation. As a consequence of disrupting the normal checkpoints that regulate cell cycle entry and progression, the E6 and E7 proteins play a key role in the oncogenic properties of human papillomaviruses with a high risk of causing anogenital cancers (HR HPVs). As a consequence, E6 and E7 of HR HPVs are invariably expressed in cervical cancers. This article will focus on the E6 protein and its numerous activities including inactivating p53, blocking apoptosis, activating telomerase, disrupting cell adhesion, polarity and epithelial differentiation, altering transcription and reducing immune recognition.
doi:10.1016/j.virol.2008.11.017
PMCID: PMC2674106  PMID: 19081593
23.  Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene. 
Journal of Virology  1993;67(7):3720-3729.
Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.
Images
PMCID: PMC237735  PMID: 8389903
24.  Examination of the pRb-Dependent and pRb-Independent Functions of E7 In Vivo 
Journal of Virology  2005;79(17):11392-11402.
High-risk human papillomaviruses encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. Although E7 protein is best known for its ability to inactivate the retinoblastoma tumor suppressor protein, pRb, many other activities for E7 have been proposed in in vitro studies. Herein, we describe studies that allowed us to define unambiguously the pRb-dependent and -independent activities of E7 for the first time in vivo. In these studies, we crossed mice transgenic for human papillomavirus 16 E7 to knock-in mice genetically engineered to express a mutant form of pRb (pRbΔLXCXE) that is selectively defective for binding E7. pRb inactivation was necessary for E7 to induce DNA synthesis and to overcome differentiation-dependent cell cycle withdrawal and DNA damage-induced cell cycle arrest. While most of E7's effects on epidermal differentiation were found to require pRb inactivation, a modest delay in terminal differentiation with resulting hyperplasia was observed in E7 mice on the RbΔLXCXE mutant background. E7-induced p21 upregulation was also pRb dependent, and genetic Rb inactivation was sufficient to reproduce this effect. While E7-mediated p21 induction was partially p53 dependent, neither p53 nor p21 induction by E7 required p19ARF. These data show that E7 upregulates the expression of p53 and p21 via pRb-dependent mechanisms distinct from the proposed p19-Mdm2 pathway. These results extend our appreciation of the importance of pRb as a relevant target for high-risk E7 oncoproteins.
doi:10.1128/JVI.79.17.11392-11402.2005
PMCID: PMC1193607  PMID: 16103190
25.  Construction of a cancer-perturbed protein-protein interaction network for discovery of apoptosis drug targets 
BMC Systems Biology  2008;2:56.
Background
Cancer is caused by genetic abnormalities, such as mutations of oncogenes or tumor suppressor genes, which alter downstream signal transduction pathways and protein-protein interactions. Comparisons of the interactions of proteins in cancerous and normal cells can shed light on the mechanisms of carcinogenesis.
Results
We constructed initial networks of protein-protein interactions involved in the apoptosis of cancerous and normal cells by use of two human yeast two-hybrid data sets and four online databases. Next, we applied a nonlinear stochastic model, maximum likelihood parameter estimation, and Akaike Information Criteria (AIC) to eliminate false-positive protein-protein interactions in our initial protein interaction networks by use of microarray data. Comparisons of the networks of apoptosis in HeLa (human cervical carcinoma) cells and in normal primary lung fibroblasts provided insight into the mechanism of apoptosis and allowed identification of potential drug targets. The potential targets include BCL2, caspase-3 and TP53. Our comparison of cancerous and normal cells also allowed derivation of several party hubs and date hubs in the human protein-protein interaction networks involved in caspase activation.
Conclusion
Our method allows identification of cancer-perturbed protein-protein interactions involved in apoptosis and identification of potential molecular targets for development of anti-cancer drugs.
doi:10.1186/1752-0509-2-56
PMCID: PMC2488323  PMID: 18590547

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