p21 Cip1/WAF1 inhibits cell-cycle progression by binding to G 1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N-and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21 Cip1/WAF1 is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21 Cip1/WAF1 in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21 Cip1/WAF1 , and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21 Cip1/WAF1 demonstrated G 1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21 Cip1/WAF1 , concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21 Cip1/WAF1 forms a complex with the apoptosis signalregulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21 Cip1/WAF1 lacking the nuclear localization signal (ΔNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21 Cip1/WAF1 itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21 Cip1/WAF1 , which are determined by its intracellular distribution and are dependent on the stage of differentiation.

p21Cip1/WAF1 has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21Cip1/WAF1 is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21Cip1/WAF1 lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21Cip1/WAF1 forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21Cip1/WAF1 is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21Cip1/WAF1 may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions.

Improved care of infants born prematurely has increased their survival. However, the incidence of preterm labor has not changed. To understand the processes involved in preterm labor, we used oligonucleotide microarrays to study gene expression in murine and human uterus during pregnancy. The induction of enzymes for prostaglandin synthesis was used as a marker for important changes during pregnancy because prostaglandins strongly contribute to both human and murine labor. We identified 504 genes that changed at least 2-fold between d 13.5 and 19.0 in the gravid mouse uterus. In the pregnant human myometrium, we found 478 genes that changed at least 2-fold in either term or preterm labor compared with preterm nonlabor specimens and 77 genes that significantly varied in both preterm and term labor. Patterns of gene regulation within functional groups comparing human preterm and term labor were similar, although the magnitude of change often varied. Surprisingly, few genes that changed significantly throughout pregnancy were the same in the mouse and human. These data suggest that functional progesterone withdrawal in human myometrium may not be the primary mechanism for labor induction, may implicate similar mechanisms for idiopathic preterm and term labor in humans, and may identify novel targets for further study.

The clonal composition of EBV-infected cells was examined in three cases of EBV-associated

The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation.The hormone 1,25-dihydroxyvitamin D 3 (VD 3 ) can induce differentiation of hematopoietic cell lines such as HL60 and U937 along a macrophage-monocyte pathway. In a search for VD 3 target genes, the cell cycle inhibitor p21 and the homeobox gene product HoxA10 were identified as direct transcriptional targets of the VD 3 receptor (15,19). HoxA10 can directly bind to the p21 promoter, together with its trimeric partners PBX1 and MEIS1, and activate p21 transcription (5). It has been shown that VD 3 -induced monocytic differentiation is associated with the initial nuclear expression and subsequent cytoplasmic translocation of p21 (3). Furthermore, we have demonstrated that peripheral blood monocytes express p21 in the cytoplasm, which appears important for their survival and for specific function. Cytoplasmic p21 expression protects monocytes by preventing the induction of the activated mitogen-activated protein kinase pathway by reactive oxygen species. This protection is accomplished in part by binding to and inhibiting ASK1, which otherwise triggers cell death.Several tumor suppressor genes, including BRCA1, encode nuclear proteins, the functions of which are critically dependent on their correct nuclear localization. BRCA1 is normally located in the nucleus and plays important roles in DNA damage monitoring and repair (20). The mechanisms regulating the nuclear localization of BRCA1 are prerequisite to its tumor suppressor activity, and their dysregulation may lead to cellular transformation. In contrast to normal breast epithelial cells, where BRCA1 is found in the nucleus, in many advanced breast cancer cells BRCA1 is mislocated to the cytoplasmic compartment (6). In an attempt to identify the underlying mechanism for BRCA1 mislocation, Li et al. searched for proteins that interacted with the nuclear localization signal (NLS) of BRCA1 and identified Brap2 (BRCA1-associated protein 2), which is predominantly localized to the cytoplasm (13). Subsequent stud...