Papel cooperativo de c-MYC e E6/E7 de duas variantes moleculares do Papilomavírus Humano tipo 16 na proliferação e transformação in vitro de queratinócitos humanos primários

As funções das oncoproteínas E6 e E7 do Papilomavírus Humano tipo 16 (HPV-16) na progressão de células epiteliais imortalizadas para tumores invasivos não são totalmente compreendidas. Aqui, estabelecemos uma nova ligação entre E6 e E7 de duas variantes moleculares de HPV-16 (AA e E-350G) e c-MYC, em relação à cooperação na promoção da transformação maligna de queratinócitos humano primários de prepúcio de recém-nascido (PHK). Nosso objetivo foi estudar os efeitos sinérgicos de E6/E7 e c-MYC na proliferação e no potencial de transformação in vitro de PHKs. Avaliou-se a proliferação celular através da expressão proteica do Antígeno Nuclear de Células Proliferantes (PCNA). Também avaliamos a capacidade de transformação, in vitro, dos PHKs através de dois ensaios complementares. Observamos que E-350G-c-MYC PHKs exibiram um aumento discreto na expressão de PCNA e formaram significativamente mais colônias tanto nos ensaios de soft-ágar quanto nos ensaios em placas de cultura de baixa adesão. No geral, concluímos que a variante E-350G co-transfectada com c-MYC pode promover a transformação celular maligna com eficiência maior do que a variante AA-c-MYC. As propriedades oncogênicas exibidas pela variante E-350G permitem entender em maior detalhe os mecanismos que podem levar à neoplasia cervical humana, dada a maior frequência de sua ocorrência na progressão de lesões precursoras de alto grau para carcinomas invasivos.The roles of E6 and E7 oncoproteins of Human Papillomavirus type 16 (HPV-16) in the progression of immortalized epithelial cells to invasive tumors are not fully understood. Here, we establish a novel link between E6 and E7 of two molecular variants of HPV-16 (AA and E-350G), and c-MYC, regarding the cooperation in promoting malignant transformation of primary human foreskin keratinocytes (PHK). We aimed to study the synergistic effects of E6/E7 and c-MYC upon proliferation, and the in vitro transformation potential of PHK. We evaluated cellular proliferation through the expression of the Proliferating Cell Nuclear Antigen (PCNA) protein and colony formation abilities using soft agar and low attachment plates. We observed that E-350G-c-MYC PHKs exhibited discrete higher PCNA levels and formed significantly more colonies in both soft-agar and when growth in low-adhesion culture plates. Overall, we concluded that the E-350G variant co-transfected with c-MYC might promote malignant cellular transformation with a better efficiency than the AA-c-MYC counterpart. The enhanced oncogenic properties exhibited by the E-350G-c-MYC variant offer insights into mechanisms that may operate in human cervical neoplasia, given the higher frequency of its occurrence in the progression of high-grade precursor lesions to invasive carcinomas

Use of the Normalized Absorbance Ratio as an Internal Standardization Approach To Minimize Measurement Error in Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Papillomavirus Infection▿

The serological detection of antibodies against human papillomavirus (HPV) antigens is a useful tool to determine exposure to genital HPV infection and in predicting the risk of infection persistence and associated lesions. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for seroepidemiological studies of HPV infection but are not standardized. Intra- and interassay performance variation is difficult to control, especially in cohort studies that require the testing of specimens over extended periods. We propose the use of normalized absorbance ratios (NARs) as a standardization procedure to control for such variations and minimize measurement error. We compared NAR and ELISA optical density (OD) values for the strength of the correlation between serological results for paired visits 4 months apart and HPV-16 DNA positivity in cervical specimens from a cohort investigation of 2,048 women tested with an ELISA using HPV-16 virus-like particles. NARs were calculated by dividing the mean blank-subtracted (net) ODs by the equivalent values of a control serum pool included in the same plate in triplicate, using different dilutions. Stronger correlations were observed with NAR values than with net ODs at every dilution, with an overall reduction in nonexplained regression variability of 39%. Using logistic regression, the ranges of odds ratios of HPV-16 DNA positivity contrasting upper and lower quintiles at different dilutions and their averages were 4.73 to 5.47 for NARs and 2.78 to 3.28 for net ODs, with corresponding significant improvements in seroreactivity-risk trends across quintiles when NARs were used. The NAR standardization is a simple procedure to reduce measurement error in seroepidemiological studies of HPV infection

E6/E7 Functional Differences among Two Natural Human Papillomavirus 18 Variants in Human Keratinocytes

It is suggested that HPV-18 variants from the A lineage have higher oncogenic potential compared to B variants. Some studies show uneven distribution of HPV-18 variants in cervical adenocarcinomas and squamous cell carcinomas. Regarding HPV-18 variants’ functions, the few studies reported focus on E6, and none were performed using natural host cells. Here, we immortalized primary human keratinocytes (PHKs) with E6/E7 of HPV-18 A1 and B1 sublineages and functionally characterized these cells. PHK18A1 reached immortalization significantly faster than PHK18B1 and formed a higher number of colonies in monolayer and 3D cultures. Moreover, PHK18A1 showed greater invasion ability and higher resistance to apoptosis induced by actinomycin-D. Nevertheless, no differences were observed regarding morphology, proliferation after immortalization, migration, or epithelial development in raft cultures. Noteworthy, our study highlights qualitative differences among HPV-18 A1 and B1 immortalized PHKs: in contrast to PHK18A1, which formed more compact colonies and spheroids of firmly grouped cells and tended to invade and migrate as clustered cells, morphologically, PHK18B1 colonies and spheroids were looser, and migration and invasion of single cells were observed. Although these observations may be relevant for the association of these variants with cervical cancer of different histological subtypes, further studies are warranted to elucidate the mechanisms behind these findings

Prevalence of high risk HPV DNA in esophagus is high in Brazil but not related to esophageal squamous cell carcinoma

Background. The first publication that associated Human Papillomavirus (HPV) infection and esophageal cancer was published in 1982. However, data are still contradictory and require further investigation. The aim of this study was to identify high risk HPV DNA in esophageal tissue of patients with and without esophageal squamous cell carcinoma (ESCC) and correlate HPV presence with classical risk factors. Methods. Invited patients signed the informed consent form, and interviews were conducted in order to obtain information about sociodemographic and lifestyle behavior. During endoscopy, esophageal biopsies were collected from case and controls. Multiplex polymerase chain reaction genotyping was conducted on endoscopic biopsies to identify HPV types and HPV-16 was further evaluated by specific PCR real time. Results. Among 87 cases, 12 (13.8%) had tumors harboring high risk HPV DNA and among 87 controls, 12 (13.8%) had high risk HPV DNA (OR:1.025 [CI:0.405:2.592]). Variables regarding consumption of alcohol and use of tobacco continued to characterize risk factors even after adjustments by presence or absence of high risk HPV. Conclusion. HPV was demonstrated to be frequently and similarly associated to normal and malignant esophageal tissues, but not as an independent risk factor to esophageal cancer. Impact. To contribute to the Brazilian population data on this subject, which is still contradictory