Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.

Common fragile sites (CFSs) are seen as chromosomal gaps and breaks brought about by inhibition of replication, and it is thought that they cluster with tumor breakpoints. This study presents a comprehensive analysis using conventional and molecular cytogenetic mapping of CFSs and their expression frequencies in two mouse strains, BALB/c and C57BL/6, and in human probands. Here we show that induced mouse CFSs relate to sites of spontaneous gaps and breaks and that CFS expression levels in chromosome bands are conserved between the two mouse strains and between syntenic mouse and human DNA segments. Furthermore, four additional mouse CFSs were found to be homologous to human CFSs on the molecular cytogenetic level (Fra2D-FRA2G, Fra4C2-FRA9E, Fra6A3

One prominent e ect of IFNs is their cell growthinhibitory activity. The mechanism behind this inhibition of proliferation is still not fully understood. In this study, the e ect of IFN-a treatment on cell cycle progression has been analysed in three lymphoid cell lines, Daudi, U-266 and H9. Examination of the growth-arrested cell populations shows that Daudi cells accumulate in a G0-like state, whereas U-266 cells arrest later in G1. H9 cells are completely resistant to IFN-a's cell growthinhibitory e ects. The G0/G1-phase arrest is preceded by a rapid induction of the cyclin-dependent kinase inhibitors (CKIs), p21 and p15. In parallel, the activities of the G1 Cdks are signi®cantly reduced. In addition to p21/p15 induction, IFN-a regulates the expression of another CKI, p27, presumably by a post-transcriptional mechanism. In the G1 Cdk-complexes, there is ®rst an increased binding of p21 and p15 to their respective kinases. At longer exposure times, when Cdk-bound p15 and p21 decline, p27 starts to accumulate. Furthermore, we found that IFN-a not only suppresses the phosphorylation of pRb, but also alters the phosphorylation and expression of the other pocket proteins p130 and p107. These data suggest that induction of p21/p15 is involved in the primary IFN-a response inhibiting G1 Cdk activity, whereas increased p27 expression is part of a second set of events which keep these Cdks in their inactive form. Moreover, elevated levels of p27 correlated with a dissociation of cyclin E/Cdk2-p130 or p107 complexes to yield cyclin E/Cdk2-p27 complexes. In resistant H9 cells, which possess a homozygous deletion of the p15/p16 genes and lack p21 protein expression, IFN-a causes no detectable changes in p27 expression and, furthermore, no e ects are observed on either pocket proteins in this cell line. Taken together, these data suggest that the early decline in G1 Cdk activity, subsequent changes in phosphorylation of pocket proteins, and G1/G0 arrest following IFN-a treatment, is not primarily due to loss of the G1 kinase components, but result from the inhibitory action of CKIs on these complexes.

Glucocorticoids are diabetogenic hormones because they decrease glucose uptake, increase hepatic glucose production, and inhibit insulin release. To study the long-term effects of increased glucocorticoid sensitivity in ␤-cells, we studied transgenic mice overexpressing the rat glucocorticoid receptor targeted to the ␤-cells using the rat insulin I promoter. Here we report that these mice developed hyperglycemia both in the fed and the overnight-fasted states at 12-15 months of age. Progression from impaired glucose tolerance, previously observed in the same colony at the age of 3 months, to manifest diabetes was not associated with morphological changes or increased apoptosis in the ␤-cells. Instead, our current results suggest that the development of diabetes is due to augmented inhibition of insulin secretion through ␣ 2 -adrenergic receptors (␣ 2 -ARs). Thus, we found a significantly higher density of ␣ 2 -ARs in the islets of transgenic mice compared with controls, based on binding studies with the ␣ 2 -AR agonist UK 14304. Furthermore, incubation of islets with benextramine, a selective antagonist of the ␣ 2 -AR, restored insulin secretion in response to glucose in isolated islets from transgenic mice, whereas it had no effect on control islets. These results indicate that the chronic enhancement of glucocorticoid signaling in pancreatic ␤-cells results in hyperglycemia and impaired glucose tolerance. This effect may involve signaling pathways that participate in the regulation of insulin secretion via the ␣ 2 -AR. Diabetes 53 (Suppl. 1): S51-S59, 2004

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a receptor present on T cells that plays a critical role in the downregulation of antigen-activated immune responses. CTLA-4 interacts with the ligands CD80 and CD86 on antigen-presenting cells (APC), and also directs the assembly of inhibitory signalling complexes that lead to quiescence or anergy. In this study, we show that human monocytes constitutively express CTLA-4. About 3% of monocytes expressed CTLA-4 on the cell surface, whereas the intracellular expression was higher and present in about 20% of the monocytes. The sequences of the cDNAs from human monocytes were identical to the sequences of CTLA-4 from T cells. Expression of CTLA-4 was also confirmed in the activated myelomonocytic cell lines U937 and THP-1. Monocytes, but not T cells, activated by interferon (IFN)-g also secreted soluble CTLA-4 in vitro. The CTLA-4 expression was upregulated upon treatment with phorbol 12-myristate 13-acetate (PMA) and IFN-g. This increased expression could be partially abolished by staurosporine, an inhibitor of protein kinase C (PKC). Ligation of CTLA-4 in the monocyte-like cell-line U937 with antibodies against CTLA-4 partially inhibited the proliferation of cells and the upregulation of cellsurface markers CD86, CD54, HLA-DR and HLA-DQ induced by IFN-g and Staphylococcus aureus, Cowan I strain (SAC). Ligation of CTLA-4 suppressed the PMA-stimulated activation of transcription activator protein 1 (AP-1) and nuclear factor (NF)-kB in the U937 cell line, indicating the involvement of an inhibitory signal transduction. These data provide the first evidence that CTLA-4 is constitutively expressed by monocytes and thus might be important for the regulation of immune mechanisms associated with monocytes.