ABSTRACT
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 p14(ARF) . p14(ARF) and p16(INK4A) 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 p14(ARF) 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 p14(ARF) 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, p14(ARF) was high and when E6* > E6 and p53 expression was high, p14(ARF) was low. In conclusion, each E6 variant distinctively affects p53 levels and consequently p14(ARF) expression, finding that could be related with the differences in oncogenic effect of infection with the diverse high-risk HPV variants.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Host-Pathogen Interactions , Human papillomavirus 18/physiology , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Tumor Suppressor Protein p14ARF/biosynthesis , Tumor Suppressor Protein p53/biosynthesis , Blotting, Western , Cell Line , Female , Gene Expression Profiling , Humans , Immunohistochemistry , Real-Time Polymerase Chain ReactionABSTRACT
Small RNAs belong to a newly discovered strain of molecules. These molecules are composed of double strand RNA comprised by just about 19-31 nucleotides. They have two main characteristics that make them unique. Firstly, they are noncoding for proteins and second they interfere post-transcriptional with mRNA. This interfering action is the distinguishing hallmark, therefore known as interfering RNA or RNAi. There are three main subclasses of which micro-RNA and siRNA are the most widely studied. Interference RNAs participate in a myriad of cellular functions mainly through modulation of genetic expression. Due to these capabilities it has been used as therapeutic weapon in a number of diseases including cancer. It is known that both miRNA and siRNA participate in carcinogenesis, either inhibiting suppressor genes, or stimulating oncogenes. It has been demonstrated that manipulating small interfering RNAs in cell lines and animal models, the malignant and metastatic phenotype can be reversed. Up to now a few clinical trials using RNAi as a therapeutic agent have demonstrated some success and feasibility. It is forseeable that in the near future cancer treatment with small RNAs will be widely applicable, once the many constrains for its systemic application are surpassed.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Genetic Therapy/methods , MicroRNAs/genetics , Neoplasms/genetics , RNA Interference , RNA, Messenger/genetics , RNA, Small Interfering/therapeutic use , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor/drug effects , Cell Line, Tumor/pathology , Cell Transformation, Neoplastic/genetics , Clinical Trials as Topic , Drug Delivery Systems , Drug Screening Assays, Antitumor , Feasibility Studies , Female , Gene Expression Regulation, Neoplastic/drug effects , Genes, Tumor Suppressor , Genetic Vectors/therapeutic use , Humans , Male , MicroRNAs/biosynthesis , Neoplasms/drug therapy , Neoplasms/therapy , Neoplasms/virology , Oncogenes , RNA, Messenger/antagonists & inhibitors , RNA, Neoplasm/antagonists & inhibitors , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , RNA, Small Interfering/geneticsABSTRACT
The Papanicolaou test (Pap) has been responsible for a significant reduction of cervical cancer-related morbimortality. In order to increase its sensitivity and specificity new markers have been studied and incorporated to cytological and histological methods for diagnosis for cervical cancer, such as p16INK4A that has been considered the immunocytochemical marker of choice for detection of HPV related cancers. We considered that p14ARF could be a complementary marker in order to improve the accuracy of cytological diagnosis because its genetic proximity to p16INK4A. We performed a systematic analysis of several putative cervical cancer markers in order to evaluate their performance in the detection of malignancy, in comparison with p16INK4A and p14ARF, using immunocytochemistry (ICC), immunofluorescence (IF) and Western blot analyses. Most markers were non-specific and could not discriminate HPV infected cancer cell lines from other non HPV malignant. In contrast, nuclear co-expression of p16INK4A and p14ARF was observed only in HPV-transformed cancer cell lines. Notably, in C-33A cervical cancer cells (HPV negative), p14ARF was present in the nucleoli, but p16INK4A was conspicuously absent from the nuclei of these cells. We conclude that both markers; p16INK4A and p14ARF are complementary and should be evaluated jointly in order to improve the accuracy of cytological diagnosis of cervical cancer.
