We suggest that JNK activation is among the IL-1 elicited responses that injure articular chondrocytes and this activation of JNK is dependent on intracellular oxidant formation (including NO peroxynitrite). In addition, the extraction technique here described is a novel method that permits the quantitation and study of proteins such as JNK involved in the signaling pathways of chondrocytes within osteoarthritic cartilage.
Oncogenic mink cell focus-forming (MCF) viruses, such as MCF 247, show a positive correlation between the ability to replicate efficiently in the thymus and a leukemogenic phenotype. Other MCF viruses, such as MCF 30-2, replicate to high titers in thymocytes and do not accelerate the onset of leukemia. We used these two MCF viruses with different biological phenotypes to distinguish the effect of specific viral genes and genetic determinants on thymotropism and leukemogenicity. Our goal was to identify the viral sequences that distinguish thymotropic, nonleukemogenic viruses such as MCF 30-2 from thymotropic, leukemogenic viruses such as MCF 247. We cloned MCF 30-2, compared the genetic hallmarks of MCF 30-2 with those of MCF 247, constructed a series of recombinants, and tested the ability of the recombinant viruses to replicate in the thymus and to induce leukemia. The results established that (i) MCF 30-2 and MCF 247 differ in the numbers of copies of the enhancer sequences in the long terminal repeats. (ii) The thymotropic phenotype of both viruses is independent of the number of copies of the enhancer sequences. (iii) The oncogenic phenotype of MCF 247 is correlated with the presence in the virus of duplicated enhancer sequences or with the presence of an enhancer with a specific sequence. These results show that the pathogenic phenotypes of MCF viruses are dissociable from the thymotropic phenotype and depend, at least in part, upon the enhancer sequences. On the basis of these results, we suggest that the molecular mechanisms by which the enhancer sequences determine thymotropism are different from those that determine oncogenicity.
Blot hybridization of thymocyte DNA from AKR/J mice was used to detect new proviral junction fragments as markers of clonality at different stages of viral leukemogenesis and to detect DNA rearrangements at the c-myc locus due to proviral insertion. Clonal populations of thymocytes were observed in mink cell focus-forming virus-injected mice as early as 35 days postinjection, at a stage distinguishable from frank leukemia by flow cytometric analysis and transplantation bioassay. Specific proviral integrations in the c-myc locus were detected in 15% of these early clones and in up to 65% of late-developing thymomas and frank leukemias. Thus, in this system c-myc activation appears to be a common mechanism in T-cell leukemogenesis.
Nitric oxide (NO) may play a role in tissue remodeling associated with arthritis. The articular cell sources of human inducible NO synthesis, however, have not been defined. This study demonstrates that human articular chondrocytes in primary or organ culture, but not synovial fibroblasts, produce NO in response to catabolic cytokines such as interleukin-1 (IL-1). As measured by the accumulation of NO2- in culture medium, NO production by IL-1-stimulated chondrocytes was inhibited by the NO synthase inhibitor Ng-monomethyl-L-arginine (NMA) and dependent on the presence of exogenous L-arginine. Other inflammatory cytokines such as tumor necrosis factor, but not transforming growth factor-beta, induced chondrocyte NO synthesis. The stimulation of NO synthesis required both RNA and protein synthesis. Chondrocytes isolated from cartilage derived from osteoarthritic patients also produced large amounts of NO in response to IL-1. In beginning to define potential effects of NO on chondrocyte function, it is shown that IL-1 induced an increase in cyclic guanosine monophosphate (cGMP) which was inhibited by NMA. In summary, these results demonstrate that cytokine-induced production of NO is a response of human articular chondrocytes but not of synovial fibroblasts. A potential role of NO in cytokine-induced tissue remodeling in the joint is provided by the induction of cGMP.
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