Objective. Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL).In patients with primary APS, expression of tissue factor (TF) on the surface of monocytes is increased, which may contribute to thrombosis in these patients. However, the intracellular mechanisms involved in aPL-mediated upregulation of TF on monocytic cells are not understood. This study was undertaken to investigate the intracellular signals induced by aPL that mediate TF activation in monocytes from APS patients.Methods. We analyzed, both in vivo and in vitro, aPL interactions with proteins that have signaling functions, including mitogen-activated protein kinases (MAP kinases) and NF-B/Rel proteins.Results. In vivo studies demonstrated significantly higher levels of both TF messenger RNA and TF protein in monocytes from APS patients compared with controls. At the molecular level, increased proteolysis of I B␣ and activation of NF-B were observed. Constitutive activation of both p38 and ERK-1 MAP kinases was also found. Treatment of normal monocytes with aPL activated ERK-1 and p38 MAP kinases, as well as the I B/NF-B pathway, in a dose-dependent manner. NF-B activation and I B␣ degradation induced by aPL were inhibited by the NF-B inhibitor SN50 and the p38 MAP kinase inhibitor SB203580, thus suggesting crosstalk between these pathways. However, the MEK-1/ERK inhibitor PD98059 did not affect aPLinduced NF-B binding activity. TF expression induced by aPL was significantly inhibited by combined treatment with the 3 inhibitors.Conclusion. Our results suggest that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK-1/ERK proteins, and the p38 MAP kinase-dependent nuclear translocation and activation of NF-B/Rel proteins.

Chronic myelogenous leukemia (CML) is characterized by the expression of the BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show in both BCR-ABL cells (Mo7e-p210 and BaF/3-p210) and primary CML CD34+ cells that STI571 inhibition of BCR-ABL tyrosine kinase activity results in a G 1 cell cycle arrest mediated by the PI3K pathway. This arrest is associated with a nuclear accumulation of p27Kip1 and down-regulation of cyclins D and E. As a result, there is a reduction of the cyclin E/Cdk2 kinase activity and of the retinoblastoma protein phosphorylation. By quantitative reverse transcription-PCR we show that BCR-ABL/PI3K regulates the expression of p27Kip1 at the level of transcription. We further show that BCR-ABL also regulates p27Kip1 protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27Kip1 by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27Kip1 . We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27Kip1 mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27Kip1 contributes to the pathogenesis of CML. In conclusion, these results suggest that BCR-ABL regulates cell cycle in CML cells at least in part by inducing proteasome-mediated degradation of the cell cycle inhibitor p27Kip1 and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias. (Cancer Res 2005; 65(8): 3264-72)

The PARK2 gene, previously identified as a mutated target in patients with autosomal recessive juvenile parkinsonism (ARJP), has recently been found to be a candidate tumor suppressor gene in ovarian, breast, lung and hepatocellular carcinoma that maps to the third common fragile site (CFS) FRA6E. PARK2 is linked to a novel described PACRG gene by a bidirectional promoter containing a defined CpG island in its common promoter region. We have studied the role of promoter hypermethylation in the regulation of PARK2 and PACRG expression in different tumor cell lines and primary patient samples. Abnormal methylation of the common promoter of PARK2 and PACRG was observed in 26% of patients with acute lymphoblastic leukemia and 20% of patients with chronic myelogenous leukemia (CML) in lymphoid blast crisis, but not in ovarian, breast, lung, neuroblastoma, astrocytoma or colon cancer cells. Abnormal methylation resulted in downregulation of PARK2 and PACRG gene expression, while demethylation of ALL cells resulted in demethylation of the promoter and upregulation of PARK2 and PACRG expression. By FISH, we demonstrated that a lack of PARK2 and PACRG expression was due to biallelic hypermethylation and not to deletion of either PARK2 or PACRG in ALL. In conclusion, our results demonstrate for the first time that the candidate tumor suppressor genes PARK2 and PACRG are epigenetically regulated in human leukemia, suggesting that abnormal methylation and regulation of PARK2 and PACRG may play a role in the pathogenesis and development of this hematological neoplasm. ' 2005 Wiley-Liss, Inc.

The role of members of the mitogen-activated protein kinase (MAPK) family on tumor necrosis factor K K (TNF-K K)-mediated down-regulation of col1a1 gene was studied. TNF-K K increased extracellular-regulated kinase and Jun-N-terminal kinase phosphorylation, but these e¡ects were not related to its inhibitory e¡ect on K K1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF-K K, and the speci¢c p38 MAPK inhibitor SB203580 mimicked the e¡ect of TNF-K K on col1a1 mRNA levels. Transforming growth factor L L (TGF-L L) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF-L L. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.