Human papillomavirus type 16 (HPV16), a causative agent of cervical cancers, encodes the E6 and E7 oncogenes, whose simultaneous expression is pivotal for malignant transformation and maintenance of malignant phenotypes. In the hope of developing a gene-specific therapy for HPV-related cancer, we examined the effects of E6 short-interfering RNA (siRNA) on the expression of these oncogenes and on the cell growth of HPV16-related cervical cancer cells. Using SiHa cervical cancer cells, we demonstrated that E6 siRNA decreased the levels of mRNA encoding E6 as well as that encoding E7 protein and also induced nuclear accumulation of p53, the most important target of E6. E6 siRNA suppressed monolayer and anchorage-independent growth of SiHa cells, which was associated with p21(CIP1/WAF1) induction and hypophosphorylation of retinoblastoma protein. Further, SiHa cells treated with E6 siRNA formed tumors in NOD/SCID mice that were significantly smaller than in those treated with control siRNA. Our results show HPV E6 siRNA as a candidate for gene-specific therapy for HPV-related cervical cancer.
Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16-cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21(WAF1/CIP1), as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.
Vascular endothelial growth factor-C (VEGF-C) has been implicated in lymphangiogenesis, the process of new lymphatics formation. The present study investigated VEGF-C mRNA expression in invasive cervical cancer tissue. Additionally, the association of VEGF-C mRNA with clinicopathological features was examined. VEGF-C mRNA expression was assessed by reverse transcription-polymerase chain reaction using β-action as an internal control. 75 patients presenting with invasive cervical cancer were included in the trial. VEGF-C mRNA expression was markedly higher in tumours in which pelvic lymph node metastasis was diagnosed by magnetic resonance (MR) imaging ( P = 0.002). 53 patients displaying stage Ib–IIb cervical cancer underwent radical hysterectomy and pelvic lymphadenectomy. VEGF-C expression was significantly higher in tumours exhibiting deep stromal invasion, pelvic lymph node metastasis and lymph-vascular space involvement ( P = 0.016, P = 0.006 and P = 0.036, respectively). Multivariate analysis revealed VEGF-C mRNA expression to be the sole independent factor influencing pelvic lymph node metastasis. Subjects demonstrating VEGF-C mRNA expression displayed significantly poorer prognoses than those lacking VEGF-C mRNA expression ( P = 0.049). These findings provide evidence supporting the involvement of VEGF-C expression in the promotion of lymph node metastasis in cervical cancer. Furthermore, examination of VEGF-C expression in biopsy specimens may be beneficial in the prediction of pelvic lymph node metastasis. © 2001 Cancer Research Campaign http://www.bjcancer.com
A rapid quantitative real-time PCR method was employed to quantify the copy number of E2 and E6 genes for analysis of the physical status of human papillomavirus type 16 (HPV-16) DNA. Significant differences with respect to both copy numbers were found when more than 40% of HPV-16 DNA was integrated with disruption of the E2 gene in an experimental model. The physical status of HPV-16 DNA in 50 clinical samples was exclusively episomal in 21 cases (42%), concomitant in 14 cases (28%), and integrated in 15 cases (30%). The prevalence of integrated and/or concomitant forms of HPV-16 DNA increased with progression of cervical disease. Four of 11 cervical intraepithelial neoplasia involved integrated forms of HPV-16 DNA partially or exclusively. This rapid, sensitive technique is useful in the analysis of the physical status of HPV DNA.Certain types of human papillomavirus (HPV) play a pivotal role in the carcinogenesis of cervical cancer; moreover, highlevel expression of HPV E6 and E7 oncoproteins is required for progression of cancer cell replication (7,9,17,21). Three alternative processes could increase the expression of these proteins. A high copy number of HPV DNA would directly contribute to increased expression of E6 and E7 (15). In fact, numerous cervical cancer cell lines harbor hundreds of copies of HPV DNA per cell. Furthermore, the amount of HPV-16 DNA increased by orders of magnitude with increasing disease grade (22). Secondly, mutations affecting YY1 motifs in the long control region can enhance the viral oncogene expression (5,15,16,18). Finally, integration of HPV DNA into the cellular genome also leads to overexpression of E6 and E7 oncoproteins, since either the E1 or E2 open reading frame (ORF), the product of which represses activity from the promoter P97 for the E6 and E7 genes, is preferentially disrupted or deleted following integration (1,4,8,20,23).The present study describes a rapid method of quantitative real-time PCR to quantify copy numbers of E2 and E6 genes for analysis of the physical status of HPV type 16 (HPV-16) DNA. This technique was developed based on the following assumptions: (i) preferential disruption of E2 genes will cause the absence of E2 gene sequences in the PCR product following integration; (ii) copy numbers of both genes should be equivalent in episomal forms; and (iii) E2 gene copy number will be smaller than that for E6 in concomitant forms. MATERIALS AND METHODSSpecimens and DNA extraction. Twenty-two invasive cervical carcinoma specimens analyzed in a prior study were evaluated (25). Additionally, cytobrush specimens were collected from outpatients with a confirmed histological diagnosis of CIN or invasive carcinoma at the Department of Obstetrics and Gynecology, Okayama University Medical School Hospital, Okayama, Japan. All specimens were screened for the presence of HPV DNA as described previously (14). Twenty-eight specimens positive for HPV-16 DNA were included. DNA was extracted from these samples via a routine procedure consisting of proteinase K digest...
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