The use of a human papillomavirus 18 promoter for tissue-specific expression in cervical carcinoma cells.
ABSTRACT The use of tissue-specific promoter elements in the treatment of cervical cancer has been explored in this paper. The P(105) promoter of human papillomavirus 18 (HPV18) was utilised to direct tissue-specific expression in a number of cell types. Expression was examined in three cervical carcinoma cell lines: HeLa (HPV18 positive), SiHa (HPV16 positive), and C33A cells (HPV negative); the epithelial cell line, H1299; and the foetal fibroblast cell line, MRC5, utilising a luciferase expression vector. Expression was highest in the cervical cell lines by a factor of at least 80. The effect of a number of mutations in the P(105) promoter on expression levels was examined. Three deletion constructs of the long control region (LCR) were investigated: an 800 bp fragment (LCR800), a 400 bp fragment (LCR400), and a 200 bp fragment (LCR200), as well as the full length product LCR of HPV18 (LCR1000). The LCR800 construct of the HPV18 P(105) promoter had the highest level of expression in the cervical cell lines and was also highest in the HPV18-positive HeLa cell line. Site-directed mutagenesis was then employed on the LCR800 construct to create four further constructs that each had inactivating mutations in one of the four E2 binding sites (E2BSs). Overall, this study indicated that the LCR800 construct of the HPV18 P(105) promoter could be utilised as a tissuerestricted promoter in cervical cancer cells.
- SourceAvailable from: Moshe Yaniv[show abstract] [hide abstract]
ABSTRACT: The human papillomavirus type 18 (HPV18) long control region (LCR) harbors transcriptional promoter and enhancer elements. Recombinant plasmids bearing all or part of the HPV18 LCR cloned in enhancer or promoter configuration upstream of the chloramphenicol acetyltransferase (CAT) gene were transfected into human fibroblasts and keratinocytes. Although the HPV18 enhancer can function in the absence of E2 gene products in both fibroblasts and keratinocytes, the promoter activity of the HPV18 LCR is detectable in keratinocytes but not in fibroblasts, suggesting that it is tissue specific. This promoter activity was repressed in human keratinocytes not only by the bovine papillomavirus type 1 E2 gene product but also by the homologous HPV18 E2 gene product. The promoter involved in the HPV18 E2 repression is located within a 230-base-pair domain directly upstream of the E6 open reading frame of the HPV18 LCR and is probably the previously identified E6 promoter. Although one cannot rule out the possibility that this repressing effect is mediated by a truncated form of HPV18 E2 protein, as was previously demonstrated for bovine papillomavirus type 1, a more likely explanation would be that the full-length HPV18 E2 protein behaves as a repressor. Indeed, at the same doses at which it inhibits transcription from the homologous HPV18 LCR, the HPV18 E2 gene product activates transcription from constructs bearing E2-binding palindromes cloned in enhancer configuration upstream of a heterologous promoter. The fact that the homologous HPV18 E2 gene product acts as a transcriptional repressor of the HPV18 LCR suggests a possible explanation for the overexpression of E6 and E7 open reading frames in cervical carcinoma cells and in cell lines derived from them.Journal of Virology 11/1989; 63(10):4317-24. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: During the last decade, research progress on cervical cancer has elucidated the role of human papilloma virus (HPV) in the pathogenesis of cervical cancer. Clinical trials on the viral-like particle HPV vaccines have good safety profiles and promising efficacy in preventing genital warts, cervical neoplasia, and cervical cancer. The implementation of the HPV vaccine is a tremendous milestone in our effort toward preventing cervical cancers. However, screening programs will continue to serve as a critical component in prevention due to the limitations of the current vaccines. The greatest impact in cervical cancer incidence worldwide requires improved health care access to underserved areas. Advances are needed to develop single-dose, heat-stable, needle-free, and affordable formulations of the HPV vaccine to overcome the socioeconomic barriers associated with this disease.Journal of Clinical Oncology 08/2007; 25(20):2975-82. · 18.04 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The noncoding region of human papillomavirus type 18 (HPV-18) is shown to contain at least three enhancer elements. Two of these elements are responsive to papillomavirus-encoded trans-acting factors, and the third element functions as a constitutive enhancer, requiring only cellular factors for activity. The first enhancer (IE2) is located proximal to the E6 cap site and is responsive to papillomavirus E2 trans-activator. The second enhancer (IE6) is located approximately 500 base pairs upstream of the E6 cap site and is dependent upon the viral E6 gene product for function. A third enhancer (C) is located between 200 and 400 base pairs upstream of the E6 cap site and possesses a constitutive activity, requiring no HPV-18-encoded factors for function. The constitutive enhancer element exhibits some cell type preference for epithelial cell lines, but also functions in rodent fibroblast lines. Each of these enhancers manifests activity independent of the other elements and may reflect separate transcriptional control elements for different stages of the HPV-18 virus life cycle.Journal of Virology 04/1988; 62(3):665-72. · 5.08 Impact Factor