Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

Department of Genetics, Yale School of Medicine, New Haven, CT 06520-8005, USA.
Virology (Impact Factor: 3.32). 11/2011; 422(1):114-24. DOI: 10.1016/j.virol.2011.10.012
Source: PubMed


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 that is 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.

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Available from: Daniel Dimaio, Apr 14, 2014
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    • "As a result, HPV-infected cells with integrated HPV DNA acquire extended lifespan, retain the capacity to proliferate, and accumulate mutations attributable to the actions of E6 and E7 proteins (Moody and Laimins, 2010). The E6 and E7 oncogenes are continuously expressed in human cancer cells and are required for proliferation and survival of the cells (Goodwin and DiMaio, 2000; Magaldi et al., 2012). "
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    ABSTRACT: High-risk human papillomaviruses (HPVs) cause a variety of malignancies of the mucosal epithelium. However, the local immune evasion strategies used by HPV-transformed cells remain unclear. Here, we examined the effect of HPV-positive cancer cells on human peripheral blood monocytes, which are precursors of Langerhans cells, key antigen-presenting cells in the squamous epithelium. HPV-positive cervical cancer cells and HPV-E6 expressing cells inhibited monocyte differentiation to Langerhans cells in a contact-dependent manner. Unlike Langerhans cells, monocytes that differentiated in the presence of HPV16 E6-expressing cells exhibited high levels of endocytic activity. Our results suggest that cells infected by high-risk HPV evade immune surveillance by blocking the differentiation of monocytes into competent antigen presenting cells.
    Virology 07/2013; 444(1-2). DOI:10.1016/j.virol.2013.06.020 · 3.32 Impact Factor
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    • "In addition to being a transcriptional regulator of HPV 16 E6 and E7 in early stages of the viral lifecycle, the E2 protein has potent antitumor activity in HPV 16-associated carcinogenesis [7]. HPV 16 E2 expression affects important cellular processes such as cellular proliferation or death, and loss of E2 gene integrity plays a role in the outcome and local control of cervical carcinomas [8,9]. "
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    ABSTRACT: Background Human papillomavirus type 16 (HPV 16) E2 protein is a multifunctional DNA-binding protein. HPV 16 E2 regulates many biological responses, including DNA replication, gene expression, and apoptosis. The purpose of this study was to investigate the relationship among the receptor for globular heads of the human C1q (gC1qR) gene expression, HPV 16 E2 transfection and apoptosis regulation in human cervical squamous carcinoma cells (C33a and SiHa). Methods gC1qR expression was examined in C33a and SiHa cells using real-time PCR and Western blot analysis. Apoptosis of C33a and SiHa cells was assessed by flow cytometry. C33a and SiHa cell viability, migration and proliferation were detected using the water-soluble tetrazolium salt (WST-1) assay, a transwell assay and 3H-thymidine incorporation into DNA (3H-TdR), respectively. Results C33a and SiHa cells that were transfected with a vector encoding HPV 16 E2 displayed significantly increased gC1qR gene expression and p38 mitogen-activated protein kinase (p38 MAPK)/ c-jun N-terminal kinase (JNK) activation as well as up-regulation of cellular apoptosis, which was abrogated by the addition of gC1qR small interfering RNA (siRNA). Furthermore, the changes in C33a and SiHa cell viability, migration and proliferation that were observed upon HPV 16 E2 transfection were abrogated by SB203580 (a p38 MAPK inhibitor) or SP600125 (a JNK inhibitor) treatment. Conclusion These data support a mechanism whereby HPV 16 E2 induces apoptosis by silencing the gC1qR gene or inhibiting p38 MAPK/JNK signalling in cervical squamous cell carcinoma.
    Journal of Translational Medicine 05/2013; 11(1):118. DOI:10.1186/1479-5876-11-118 · 3.93 Impact Factor
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    • "Suitable candidates include genes encoded by cancerassociated viruses, as these are not present in uninfected normal cells. An important example is high-risk human papillomavirus (HR-HPV), a necessary cause of cervical carcinoma (Walboomers et al, 1999), as maintenance of HPV oncogene expression is essential for the survival of HPV-positive cervical cancer cells in vitro (Magaldi et al, 2012). For RNAi to be therapeutically successful, siRNAs need to be developed and systematically assessed, ensuring they have minimal harmful, unintended 'off-target' effects (OTEs) (Birmingham et al, 2007). "
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    ABSTRACT: Background: When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3′ UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells. Methods: We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to ‘bystander' OTEs following transfection in vivo. Results: We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0–5.9% of differentially expressed genes overlapped between the two cell types. Conclusion: These data do not support the assumption that actual OTEs correlate well with predicted OTEs.
    British Journal of Cancer 01/2013; 108(2). DOI:10.1038/bjc.2012.564 · 4.84 Impact Factor
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