RESUMO
OBJECTIVE: The aim of this study was to evaluate precisely the differences in the spectra of human papillomavirus (HPV) types detected by different generic primer pairs commonly used for detection of this extraordinarily heterogeneous virus. METHODS: Three sets of polymerase chain reaction (PCR) primers for the L1 open reading frame (ORF) and two sets for E6/E7 ORFs were used to detect HPVs in DNAs from 107 cervical tissues, including 77 cervical neoplasias. HPV types were determined by analysis of restriction fragment length polymorphisms (RFLPs) and nucleotide sequencing. RESULTS: A high overall detection rate of HPV in cervical neoplasias (76/77, 98.7%) was achieved by polymerase chain reaction (PCR) amplification with multiple sets of generic primers, while the detection rate for each individual primer pair varied from 48/77 (62%) to 70/77 (91%). Only in 34 of 77 cases (44%) were HPV DNAs positive for all sets of primer pairs. Further determination of HPV types by RFLPs and nucleotide sequencing showed inconsistencies between the PCR primer pairs used. CONCLUSION: Our study revealed that the HPV detection rate is critically affected by the choice of PCR primers, and that appropriate use of combinations of generic PCR primer sets followed by RFLP analyses is both necessary and sufficient for typing most HPVs in cervical lesions. More precise methods such as sequencing would be necessary in only a few cases.
Assuntos
Primers do DNA , DNA Viral/genética , Papillomaviridae/genética , Reação em Cadeia da Polimerase/métodos , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/virologia , Sequência Consenso , Feminino , Humanos , Papillomaviridae/classificação , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Neoplasias do Colo do Útero/patologia , Displasia do Colo do Útero/patologiaRESUMO
Interleukin-6 (IL-6) is a pleiotropic cytokine that is not only a mediator in major immunologic reactions but also a growth factor of keratinocytes. We studied the IL-6 secretion in vitro of 15 human cell lines derived from both squamous cell carcinoma (SCC) and adenocarcinoma of the uterine cervix. Four of the eight well differentiated SCC secreted a large amount (> 1500 pg/48 h/10(6) cells) of IL-6 in nude mice. In contrast, poorly differentiated SCC cell lines and all of the 7 adenocarcinoma cell lines secreted a small amount (< 500 pg/48 h/10(6) cells of IL-6). The expression of IL-6 mRNA of the cell lines correlated well with their IL-6 secretion potential. However, the expression of IL-6 receptor did not correlate with the IL-6 secretory potential. We also studied the IL-6 secretion of freshly isolated normal squamous epithelium and of dysplastic epithelium. In culture, two normal squamous epithelia secreted a large amount (> 2000 pg/48 h/10(6) cells), whereas 8 dysplasia epithelia secreted an extremely small amount (< 10 pg/48 h/10(6) cells). About one-third of patients with SCC had a raised serum IL-6 value. IL-6 production may help to differentiate between SCC and adenocarcinoma of the uterine cervix. IL-6 regulation seems to change in the course of SCC carcinogenesis.
Assuntos
Adenocarcinoma/metabolismo , Carcinoma de Células Escamosas/metabolismo , Interleucina-6/biossíntese , Neoplasias do Colo do Útero/metabolismo , Animais , Antígenos CD/genética , Feminino , Expressão Gênica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA Mensageiro/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina-6 , Células Tumorais CultivadasRESUMO
To investigate the possible role of the multidrug resistance phenotype to chemoresistance in human ovarian carcinoma, we have analyzed human multidrug resistance gene (mdr 1) expression in 8 human ovarian adenocarcinoma cell lines. An increase in P-glycoprotein level specific to multidrug-resistant tumor cells was not apparently associated with the increase in resistance to vincristine (VCR) or doxorubicin (Adriamycin). Mdr 1 transcripts (4.5 kilobases) were observed in the RNA preparation obtained from only one cell line (SHIN-3) that showed the highest resistance to both drugs in vitro and in vivo. No cell lines showed mdr 1 DNA amplification. These results suggest that the insensitivity of human ovarian carcinoma to chemotherapy could be partly explained by the expression of mdr 1.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/genética , Neoplasias Ovarianas/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cistadenocarcinoma/tratamento farmacológico , Cistadenocarcinoma/genética , Cistadenocarcinoma/patologia , Doxorrubicina/uso terapêutico , Endometriose/tratamento farmacológico , Endometriose/genética , Endometriose/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mesonefroma/tratamento farmacológico , Mesonefroma/genética , Mesonefroma/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologia , Vincristina/uso terapêuticoRESUMO
A HPV16DNA integrated in a human cancer cell line of the uterine cervix (QG-U) was isolated and cloned. Three kinds of cells (mouse Balb/3T3, Detroit 551 and human keratinocyte) were co-transfected with HPV16DNA and neo DNA with calcium phosphate co-precipitates. Selection of the Transformants and their characteristics were examined. Transformants of mouse Balb/3T3 cells were able to be obtained about 2 weeks after co-transfection. HPV16DNA was integrated in the transformants. The most characteristic change observed in the transformants was the ability to proliferate in serum-free medium. In contrast, two kinds of human diploid cells (Detroit 551 and human keratinocyte) were not transformed by transfection, and ceased growing at almost the same time as nontransfected cells. These results indicate that HPV-DNA transformants are easily selected in cell lines rodents but selection of transformants from human diploid cells is relatively difficult.
