CD8alphaalpha-mediated survival and differentiation of CD8 memory T cell precursors.

Memory T cells are long-lived antigen-experienced T cells that are generally accepted to be direct descendants of proliferating primary effector cells. However, the factors that permit selective survival of these T cells are not well established. We show that homodimeric alpha chains of the CD8 molecule (CD8alphaalpha) are transiently induced on a selected subset of CD8alphabeta+ T cells upon antigenic stimulation. These CD8alphaalpha molecules promote the survival and differentiation of activated lymphocytes into memory CD8 T cells. Thus, memory precursors can be identified among primary effector cells and are selected for survival and differentiation by CD8alphaalpha.

Expression of the MAPK kinases MKK-4 and MKK-7 in rheumatoid arthritis and their role as key regulators of JNK.

OBJECTIVE: The mitogen-activated protein (MAP) kinase JNK is a key regulator of interleukin-1 (IL-1)-induced collagenase gene expression and joint destruction in arthritis. Two upstream kinases, MKK-4 and MKK-7, have been identified as potential activators of JNK. However, the role of MAP kinase kinases (MAPKKs) and their functional organization within fibroblast-like synoviocytes (FLS) have not been defined. We therefore evaluated the interactions between the various MAP kinase components and determined their subcellular localization. METHODS: MKKs were identified by immunohistochemistry of rheumatoid arthritis (RA) and osteoarthritis (OA) synovium. Western blotting was used to determine the expression of FLS. Immunoprecipitation experiments using antibodies specific for MKK-4, MKK-7, and JNK were performed. Phosphospecific antibodies and immunohistochemistry were used to evaluate the activation state of synovial MKK-4 and MKK-7. Confocal microscopy was used to determine the subcellular location of the kinases. RESULTS: Immunohistochemistry studies demonstrated abundant MKK-4 and MKK-7 in RA and OA synovium, but the levels of phosphorylated kinases were significantly higher in RA synovium. MKK-4 and MKK-7 were constitutively expressed by cultured RA and OA FLS, and IL-1 stimulation resulted in rapid phosphorylation of both kinases. JNK was detected in MKK-4 and MKK-7 immunoprecipitates. Furthermore, MKK-4 coprecipitated with MKK-7 and vice versa, indicating that the 3 kinases form a stable complex in FLS. Confocal microscopy confirmed that JNK, MKK-4, and MKK-7 colocalized in the cytoplasm, with JNK migrating to the nucleus after IL-1 stimulation. The signal complex containing MKK-4, MKK-7, and JNK was functionally active and able to phosphorylate c-Jun after IL-1 stimulation of FLS. CONCLUSION: These studies demonstrate that JNK, MKK-4, and MKK-7 form an active signaling complex in FLS. This novel JNK signalsome is activated in response to IL-1 and migrates to the nucleus. The JNK signalsome represents a new target for therapeutic interventions designed to prevent joint destruction.

Tumor promotion by metanil yellow and malachite green during rat hepatocarcinogenesis is associated with dysregulated expression of cell cycle regulatory proteins.

Metanil yellow (MY) and malachite green (MG) are textile dyes, which, despite a ban, are used as food-coloring agents. MY and MG have promoter effects on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine (DEN). Tumor-promoting agents are not mutagenic but may alter the expression of genes whose products are associated with hyper-proliferation, tissue remodeling, and inflammation. Cell cycle controls normally function to ensure the integrity of the genome and arrest of cells at G1/S or G2/M checkpoints until all the prerequisite events are completed. In order to understand the mechanism(s) of tumor promotion by MY and MG, we have studied the levels of PCNA, a marker of cell proliferation and cell cycle regulatory proteins, cyclin D1, and its associated kinase, cdk4, cyclin B1, and associated kinase, cdc2. Immunohistochemical staining showed an elevated level of PCNA in animals administered MY and MG subsequent to DEN treatment. Western and Northern blot hybridization showed an increased expression of both cyclin D1 and its associated kinase cdk4, and cyclin B1 and its associated kinase cdc2, in livers of rats administered MY and MG after administration of DEN as compared to untreated or DEN controls. The increased level of mRNA was due to the increased rate of transcription of these genes as studied by run-on transcription assay. These data obtained by the in vivo model of liver tumor development provide strong evidence for a link between dysregulation of the two critical checkpoints of the cell cycle as one of the possible mechanism(s) during tumor promotion by malachite green and metanil yellow.

Overexpression of cyclin D1 is associated with the decondensation of chromatin during den-induced sequential hepatocarcinogenesis.

D-type cyclins regulate distinct cellular processes such as mitotic cell cycle control, differentiation and transcription. Deregulation of cyclin D1, a component of G1 checkpoint control, can result in enhanced genomic instability, cell transformation, and malignant neoplasia. However, a precise understanding of the molecular and cellular events underlying the regulation of the cyclin D1 gene remains to be elucidated. In this study, we examined the regulation of the cyclin D1 gene during n-nitrosodiethylamine (DEN)-induced sequential liver carcinogenesis. Northern blot studies showed an increase in the level of cyclin D1 mRNA. Southern blot analysis of the DNA restriction fragment showed no alterations and/or amplification in the coding region of the cyclin D1 gene. Bulk chromatin from DEN-treated rat liver is much more sensitive to nuclease digestion than that from normal liver. Increased expression of the cyclin D1 gene is correlated to the upregulation of its transcription, mediated through chromatin decondensation during sequential hepatocarcinogenesis. Thus, the functional inter-relationship between chromatin organization and gene expression appears to be of critical importance for liver tumour development.