ABSTRACT
Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV-16)-associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS-ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Oligodeoxyribonucleotides, Antisense/analysis , Oligodeoxyribonucleotides, Antisense/chemistry , Bacterial Proteins/analysis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , DNA Probes, HPV/analysis , DNA Probes, HPV/chemical synthesis , DNA Probes, HPV/chemistry , Flow Cytometry , Green Fluorescent Proteins , Humans , Luminescent Proteins/analysis , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Microscopy, Fluorescence/methods , Molecular Sequence Data , Oligodeoxyribonucleotides, Antisense/chemical synthesis , RNA, Messenger/chemistry , RNA, Viral/antagonists & inhibitors , RNA, Viral/chemistry , Recombinant Proteins/analysisABSTRACT
Human involucrin whose gene transcription is directed by a 2456-nucleotide (nt) 5'-noncoding region is a structural component of the epithelial cornified layer. Transient transfection assays demonstrated that this region is transcriptionally active in multiplying keratinocytes and is enhanced by 2 mM CaCl2 treatment. Calcium-independent transcriptional activity and the interaction with the AP-1 transcriptional factor was located on the proximal part (nt -159 to -1) of the 5'-noncoding region. However, CaCl2 responsiveness was mapped to a distal 1185-nt fragment (nt -2456 to -1272). Moreover, this fragment potentiated the Herpes simplex thymidine kinase promoter in normal keratinocytes and is responsive to calcium treatment in a cell type-specific manner. Interestingly, the absence of a 491-nt fragment located between the two enhancer domains (nt -651 to -160) resulted in transcriptional activation in multiplying keratinocytes. This fragment interacts with AP-1 and the YY1 transcriptional silencer. It is concluded that human involucrin 5'-noncoding region contains at least three regulatory domains, a distal CaCl2-responsive enhancer, a putative transcriptional silencer (that interacts with AP-1 and YY1), and a proximal enhancer/promoter (that interacts with AP-1). Thus, this study demonstrates the presence of particular transcriptional factors can potentially regulate the human involucrin expression.
Subject(s)
Protein Precursors/genetics , Transcription, Genetic , Animals , Base Sequence , Cattle , Cells, Cultured , DNA , DNA Footprinting , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Erythroid-Specific DNA-Binding Factors , HeLa Cells , Humans , Keratinocytes/metabolism , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Transcription Factor AP-1/metabolism , Transcription Factors/metabolism , Transfection , YY1 Transcription FactorABSTRACT
Human papillomavirus (HPV) specifically infect stratified epithelial cells, causing benign and malignant neoplasia. Several elements directing this virus' genetic expression are present in a non-coding region called LCR. HPV infection starts in the basal cells of stratified epithelia, where a particular combination of cellular factors interacting with the LCR starts the transcription of the viral E6 and E7 oncogenes. The E6 and E7 genes alter the cell cycle because they interact and inactivate tumor suppressor proteins: E6 binds and degrades protein p53 and E7 associates with p105RB. E1 and E2 are the next synthesized proteins. E2 blocks the early transcription and permits E1 specific binding to the viral origin of replication located within the LCR, initiating the viral genome replication. Following the course of viral infection, the E2-induced E6 and E7 down-regulation releases p53 and p105RB proteins, and the differentiation process can continue. Then, a putative late promoter can activate the capsid genes L1 and L2. At this step, mature virions can be detected in the upper layers of the epithelium. Disruption in E2 gene transcription is usually associated to genital malignant neoplasia. In the absence of E2, E6 and E7 remain constitutively expressed, sustaining the immortality of the infected cell and blocking the epithelial differentiation program.
Subject(s)
Condylomata Acuminata/genetics , Gene Expression Regulation, Viral , Papillomaviridae/genetics , Down-Regulation , Female , Genes, Viral/genetics , Genital Neoplasms, Female/etiology , Genital Neoplasms, Male/etiology , Humans , Male , Oncogenes/genetics , Transcription, GeneticABSTRACT
In Mexico, about 30% of all malignant tumors in women are uterine cervix carcinomas. It is one of their main causes of death. We have previously shown that in Mexico City, 31% (5/16) of the analyzed tumoral samples contained HPV-16 DNA sequences. We have now extended this observation in Mexico City and included the city of Monterrey and found that the prevalence of HPV-16 is similar in both: 26% (6/23) for Monterrey and 29% (4/14) for Mexico City. HPV-18 was detected in only 10% (1/10) and 7% (1/14) of the tumors in these two populations when assayed with an HPV-18 specific probe. In both cities, the majority of the samples analyzed (including samples from the four stages of severity of the disease) contained integrated papillomavirus DNA sequences. Our results suggest that the mexican population contains a rather low proportion of HPV-16 and HPV-18 sequences in uterine-cervix carcinoma.