Luis Benítez Bribiesca

The E7 protein of human papillomavirus (HPV) type 16 expressed by recombinant vaccinia virus can be used for detection of antibodies in sera from cervical cancer patients

Sera from 128 Mexican cervical cancer patients (age 30-80; mean 53.6) and from 47 healthy women (age 25-69; mean 49.2) were investigated using a newly developed assay for the detection of serum antibodies to the human papillomavirus (HPV) type 16 early protein E7. This test (CIPA), based upon immunoprecipitation followed by Western blot analysis, uses the complete E7 protein expressed in HeLa cells infected with recombinant vaccinia virus. To determine the sensitivity and specificity of this assay, these results were compared with previous results of the same sera tested by enzyme-linked immunosorbent assay (ELISA; using synthetic peptides derived from HPV 16 E7) and radio-immunoprecipitation (RIPA) using in vitro translated HPV 16 E7 protein. CIPA (45% positives) demonstrated a significant increase in detection rate compared to the peptide-ELISA (30% positives; P = 0.014, chi2-test) and only a slight increase compared to RIPA (38% positives; P = 0.204, chi2-test). Based on the testing of sera from patients with HPV 16 DNA positive tumors the specificity and sensitivity of the CIPA were 0.98 and 0.59, respectively.

Regulation of p14ARF expression by HPV-18 E6 variants

A common causative agent for uterine cervical cancer is the human papillomavirus type 18 (HPV‐18) which has three phylogenic variants: Asian‐Amerindian, European, and African. Each variant shows significant molecular differences in the E6 gene. E6 oncoprotein is a negative regulator of tumor suppressor protein p53, hence, this oncoprotein indirectly regulates the expression of tumor‐suppressor p14ARF. p14ARF and p16INK4A genes are overexpressed in—and have been proposed as markers for—HPV‐related cervical cancer. In order to dissect the role of E6 on the regulation of p14ARF expression, separating it from that of other intervening factors, transfection of E6 variants to MCF‐7 cells was performed, assessing cDNA transcript levels by RT‐PCR, whereas p14ARF and p53 expression were evaluated by immunocytochemistry and Western blot. E6 transfected cells differentially expressed transcripts of two molecular forms: E6 and E6*. The ratio of these two forms varied with the transfected E6 variant. With the Asian‐Amerindian variant, the ratio was E6 > E6*, whereas with the European and the African the ratio was E6* > E6. As expected with the E6* construct, E6* transcripts were solely observed. In addition, when E6 > E6* and p53 expression was low, p14ARF was high and when E6* > E6 and p53 expression was high, p14ARF was low. In conclusion, each E6 variant distinctively affects p53 levels and consequently p14ARF expression, finding that could be related with the differences in oncogenic effect of infection with the diverse high‐risk HPV variants. J. Med. Virol. 85:1215–1221, 2013.