ABSTRACT
Rheumatoid synovial fibroblasts (RSF) express cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(Cip1) when they are growth-inhibited in vitro. The induction of p16(INK4a) is characteristic of RSF and intra-articular p16(INK4a) gene therapy has been shown to suppress adjuvant arthritis (AA) of rats. The other inducible CDK inhibitor, p21(Cip1), has multiple functions depending on the cell type. They include inhibition of CDK as well as promotion of active CDK complex formation and induction of apoptosis. This study is to discern the biological effects of p21(Cip1) gene transfer into RSF and its therapeutic effects on AA. A recombinant adenovirus containing a human p21(Cip1) gene and control adenoviruses were prepared. RSF infected with these viruses were examined for their cell growth. Apoptotic cell death was evaluated by nuclear staining and DNA fragmentation analysis. In vivo gene therapy of rat AA was carried out by intra-articular injection of the viruses. Severity of the arthritis was clinically scored. The treated joints were examined histologically and proliferating cell nuclear antigens (PCNA) were detected immunohistochemically. The adenoviral p21(Cip1) gene transfer inhibited growth of RSF without inducing apoptosis. p21(Cip1) gene therapy suppressed AA clinically and histologically. The effects were comparable to p16(INK4a) gene therapy. PCNA expression was reduced in the p21(Cip1)-treated joints. The adenoviral gene transfer of p21(Cip1) ameliorated rat AA. The effect was attributable to inhibition of proliferation. Because p21(Cip1) is induced more easily by many chemicals than p16(INK4a), it also appears to be a feasible target in developing anti-rheumatic drugs.
Subject(s)
Arthritis, Experimental/prevention & control , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Cyclins/administration & dosage , Cyclins/genetics , Enzyme Inhibitors/administration & dosage , Joints/enzymology , Adenoviridae/genetics , Animals , Apoptosis/genetics , Arthritis, Experimental/enzymology , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Fibroblasts/enzymology , Fibroblasts/metabolism , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors/administration & dosage , Hindlimb , Humans , Injections, Intra-Articular , Male , Rats , Rats, Inbred Lew , Synovial Membrane/metabolism , Synovial Membrane/pathologyABSTRACT
The transcriptional coactivator p300, a histone acetyltransferase (HAT), plays key roles in the regulation of cell proliferation and differentiation. p300 is targeted by viral oncoproteins, and mutations of p300, accompanied by inactivation of the second allele, have been reported in certain types of cancers originating in the epithelium. Here, we identified a homozygous p300 deletion of exons 15--18 in the SiHa cervical carcinoma cell line, which results in an in-frame deletion that causes specific loss of the bromodomain, a conserved domain implicated in the regulation of HAT activity. Furthermore, we show that the mutation severely impaired its ability to activate the p21(WAF1/CIP1) promoter in transient reporter assay. These results suggest a critical role for the bromodomain in p300 functions as a tumor-suppressor gene.
Subject(s)
Mutation , Nuclear Proteins/genetics , Trans-Activators/genetics , Uterine Cervical Neoplasms/genetics , Base Sequence , Binding Sites/genetics , Blotting, Western , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , DNA, Complementary/genetics , Female , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , K562 Cells , Luciferases/genetics , Luciferases/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Promoter Regions, Genetic/genetics , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Deletion , Trans-Activators/chemistry , Trans-Activators/metabolism , Tumor Cells, Cultured , U937 Cells , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathologyABSTRACT
Using the reverse transcription PCR, we evaluated expression levels of various antigen presentation-related genes, including LMP2, LMP7, MECL-1, PA28alpha, PA28beta, TAP1, TAP2, and tapasin, in two oral squamous cell carcinoma cell lines, HSC5 and HSC7. Expression levels of LMP2, MECL-1, TAP1, and TAP2 transcripts are reduced in both cell lines in comparison with a normal epithelial cell line. Further, HSC5 and HSC7 show diminished expression of LMP7/tapasin, and PA28alpha/beta, respectively. Surface expression of HLA-B alleles is down-regulated in both lines presumably due to low expression of TAP1/2. However, HLA-A2 surface expression is not significantly down-regulated in HSC5 cells, suggesting an involvement of signal-sequence derived peptides in the TAP-independent pathway. The current study would contribute to our understanding of significance of abnormalities in the antigen presentation machinery of oral squamous cell carcinoma, and provide meaningful information in the design of CTL-based tumor vaccines by intra-cellular delivery system.
