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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During spermatogenesis, the radiosensitivity of testicular cells changes considerably. To investigate the molecular mechanisms underlying these radiosensitivity differences, p21(Cip1/WAF1) expression was studied before and after irradiation in the adult mouse testis. P21(Cip1/WAF1) is a cyclin-dependent kinase inhibitor (CDI) and has a role in the G1/S checkpoint and differentiation. P21(Cip1/WAF1) expression was observed in the normal testis, using Western blotting analysis. After a dose of 4 Gy, but not after 0.3 Gy, an increase in p21(Cip1/WAF1) expression could be determined in whole testis lysates. To investigate which germ cells are involved in p21(Cip1/WAF1) protein expression, immunohistochemical analysis was performed on irradiated testis. In the normal testis a weak staining for p21(Cip1/WAF1) was found in pachytene spermatocytes in epithelial stage V up to step 5 spermatids. A dose of 4 Gy of X-irradiation resulted in a transient increase of p21(Cip1/WAF1) staining in these cells with a maximum at 6 h post irradiation, despite the fact that the irradiation did not induce an increase in the number of apoptotic spermatocytes. When a dose of 0.3 Gy was given, no increase in p21(Cip1/WAF1) staining was observed. Using the TUNEL technique, a 10-fold increase in apoptotic spermatogonia was found after a dose of 4 Gy. However, no staining for p21(Cip1/WAF1) was observed in spermatogonia, suggesting that these cells do not undergo a p21(Cip1/WAF1)-induced G1 arrest prior to DNA repair or apoptosis. These data imply that p21(Cip1/WAF1) is a factor which could be important during the meiotic prophase in spermatocytes and repair mechanisms in these cells, but not in spermatogonial cell cycle delay or apoptosis induction.
Mol Reprod Dev 1997 Jul
PMID:P21(Cip1/WAF1) expression in the mouse testis before and after X irradiation. 917 Jan 3

Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanism by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin-induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 microM apigenin resulted in dose-dependent cell-cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 microM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 microM apigenin-treated cells. In conclusion, apigenin treatment produced a G1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.
Mol Carcinog 1997 Jun
PMID:Induction of p21/WAF1 and G1 cell-cycle arrest by the chemopreventive agent apigenin. 921 Sep 54

Activins, members of the transforming growth factor-beta family, have been implicated in the regulation of growth and differentiation of various types of cells. We have recently found that activin A induces apoptotic cell death of plasmacytic cells including B cell hybridoma cells and myeloma cells. In the present study, we demonstrated that activin A caused cell-cycle arrest in the G1 phase before appearance of apoptotic cells in mouse B cell hybridoma cells. Phosphorylation of retinoblastoma protein (Rb) and in vitro Rb kinase activity of cyclin-dependent kinase (CDK)4 was inhibited in activin A-treated cells. Analysis of expression of genes regulating Rb phosphorylation revealed that activin A suppressed cyclin D2, the sole D-type cyclin gene expressed in the hybridoma cells, and activated p21CIP1/WAF1 but had no effect on expression of cyclin-dependent kinases (CDK2, CDK4, CDK6) and other CDK inhibitors (p27KIP1, p16INK4a, p15INK4b). Modulation of cyclin D2 and p21CIP1/WAF1 expression resulted in a decrease in level of cyclin D2-CDK4 complex and an increase in level of CDK4 complexed with p21CIP1/WAF1. Moreover, overexpression of cyclin D2 partially abrogated inhibition of Rb phosphorylation and G1 arrest in the hybridoma cells.
Mol Endocrinol 1997 Jul
PMID:Activin A induction of cell-cycle arrest involves modulation of cyclin D2 and p21CIP1/WAF1 in plasmacytic cells. 921 52

The p21(WAF1/CIP1/sdi1) gene product (WAF1) inhibits DNA replication in vitro (J. Chen, P. Jackson, M. Kirschner, and A. Dutta, Nature 374:386-388, 1995; S. Waga, G. Hannon, D. Beach, and B. Stillman, Nature 369:574-578, 1994), but in vivo studies on the antiproliferative activity of WAF1 have not resolved G1-phase arrest from potential inhibition of S-phase progression. Here, we demonstrate that elevated WAF1 expression can retard replicative DNA synthesis in vivo. The WAF1-mediated inhibitory effect could be antagonized by cyclin A, cyclin E, or the simian virus 40 small-t antigen with no decrease in the levels of WAF1 protein in transfected cells. Proliferating-cell nuclear antigen (PCNA) overexpression was neither necessary nor sufficient to antagonize WAF1 action. Expression of the N-terminal domain of WAF1, responsible for cyclin-dependent kinase (CDK) interaction, had the same effect as full-length WAF1, while the PCNA binding C terminus exhibited modest activity. We conclude that S-phase progression in mammalian cells is dependent on continuing cyclin and CDK activity and that WAF1 affects S phase primarily through cyclin- and CDK-dependent pathways.
Mol Cell Biol 1997 Aug
PMID:WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases. 923 44

The functionality of the p53-mediated pathway, activated in response to DNA damage, has been assessed in primary fibroblast cell cultures and Epstein-Barr virus-transformed lymphoblastoid cell lines derived from Nijmegen breakage syndrome (NBS) patients. This autosomal recessive disease is characterized by microcephaly, growth and mental retardation, chromosomal instability, radiosensitivity, and high cancer incidence. The recent mapping of the NBS gene to chromosome 8q21 demonstrates that NBS is genetically distinct from ataxia telangiectasia (AT). Changes in p53 protein levels were significantly reduced and delayed in all the NBS fibroblast cell cultures and lymphoblastoid cell lines examined compared to normal cultures over a 4-h period postirradiation (5 Gy). The transcriptional activation of p21(WAF1/CIP1) mRNA was also lower in 12 NBS fibroblast cultures examined. In agreement with an abrogated p53 function, NBS cells exposed to ionizing radiation show an abnormal cell cycle arrest at G1-S and a prolonged accumulation of cells in the G2 phase. In contrast, exposure to the alkylating agent methyl methanesulfonate results in similar increases of p53 and p21(WAF1/CIP1) mRNA in both cell types. The ATM gene transcript was found to be expressed at similar levels in NBS and normal cells, whereas it was strongly reduced in the AT homozygote cells examined. These results suggest that the ATM gene product cannot substitute for that of the NBS gene in the signaling of cellular damage produced by ionizing radiation and that both are involved in the activation of p53. The suboptimal p53-mediated response could contribute to the high cancer risk and radiosensitivity seen in NBS patients.
Mol Cell Biol 1997 Sep
PMID:Nijmegen breakage syndrome cells fail to induce the p53-mediated DNA damage response following exposure to ionizing radiation. 927 79

The helix-loop-helix transcription factor E2A plays important roles not only in promoting cellular differentiation but also in suppressing cell growth. Id proteins, the inhibitors of E2A, have opposite effects on cell differentiation and growth. To understand the mechanisms by which E2A suppresses cell growth, we examined the role of E2A in regulating the expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1/SD11, which prevents cell cycle progression upon overexpression. By using transient-cotransfection assays of luciferase reporter constructs in HeLa cells, we have found that overexpression of E2A can transcriptionally activate the p21 gene. To identify the sequences that mediate this activation in the promoter of the p21 gene, we carried out mutational analyses. Out of the eight putative E2A-binding sequences (E1 to E8) in the promoter, the E1 to E3 sequences located close to the transcription start site are found to be essential. In addition, loss of the E boxes in the promoter also reduces p21 expression without cotransfection with E2A in HIT pancreatic cells, where the endogenous E2A-like activity is high. Furthermore, we have also shown that overexpression of E2A in 293T cells activates expression of the endogenous p21 gene at both the levels of mRNA and protein. In correlation with the finding that E47 overexpression leads to growth arrest in NIH 3T3 cells, we have shown that Id1 overexpression in NIH 3T3 cells accelerates cell growth and inhibits p21 expression. Taken together, these results provide insight into the mechanisms by which E2A and Id proteins control cell growth.
Mol Cell Biol 1997 Oct
PMID:Regulation of the expression of cyclin-dependent kinase inhibitor p21 by E2A and Id proteins. 931 46

Low ratio hybridization subtraction technique was previously used in this laboratory to enrich and isolate a number of low abundance UV-inducible hamster transcripts (Fornace, A. J., Jr., Alamo, I. J., and Hollander, M. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8800-8804) that led to the identification and cloning of five important hamster and human GADD genes (Fornace, A. J., Jr., Nebert, D. W., Hollander, M. C., Luethy, J. D., Papathanasiou, M., Fargnoli, J., and Holbrook, N. J. (1989) Mol. Cell. Biol. 9, 4196-4203). In this study we have characterized the remaining DNA damage-inducible (DDI) transcripts. Of the 24 DDI clones, 3 clones (A13, A20, and A113) representing different regions of the same hamster cDNA exhibited near perfect homology to human p21(WAF1/CIP1) cDNA. The DDI clones A26, A88, and A99 displayed very high sequence homologies with the human proliferating nuclear antigen, rat translation initiation factor-5 (eIF-5), and human thrombomodulin, respectively, whereas clones A29 and A121 matched with express sequence tagged sequences of unknown identity. The DDI clones A18, 106, and A107 were different isolates of the same hamster cDNA (hereafter referred to as A18) and displayed high sequence homology with the members in the heterogeneous ribonucleoprotein (hnRNP) family. Using the hamster A18 partial-length cDNA as a probe, we screened human fibroblast cDNA library and isolated the corresponding full-length human cDNA. The deduced amino acid sequence revealed that the putative protein contains all the canonical features of a novel glycine-rich hnRNP. The A18 mRNA levels were specifically increased in response to DNA damage induced by UV irradiation or UV mimetic agents. Thus the putative A18 hnRNP is the first hnRNP whose mRNA is specifically regulated in response to UV-induced DNA damage; accordingly, it may play some role in repair of UV-type DNA damage.
 ...
PMID:Identification of several human homologs of hamster DNA damage-inducible transcripts. Cloning and characterization of a novel UV-inducible cDNA that codes for a putative RNA-binding protein. 933 57

A tetracycline-regulated system was used to characterize the effects of c-Rel on cell proliferation. The expression of c-Rel in HeLa cells led to growth arrest at the G1/S-phase transition, which correlated with its nuclear localization and the induction of endogenous IkappaB alpha expression. These changes were accompanied by a decrease in E2F DNA binding and the accumulation of the hypophosphorylated form of Rb. In vitro kinase assays showed a reduction in Cdk2 kinase activity that correlated with elevated levels of p21WAF1 Cdk inhibitor and p53 tumor suppressor protein. While the steady-state levels of WAF1 transcripts were increased, pulse-chase analysis revealed a sharp increase in p53 protein stability. Importantly, the deletion of the C-terminal transactivation domains of c-Rel abolished these effects. Together, these studies demonstrate that c-Rel can affect cell cycle control and suggest the involvement of the p21WAF1 and p53 cell cycle regulators.
Mol Cell Biol 1997 Nov
PMID:c-Rel arrests the proliferation of HeLa cells and affects critical regulators of the G1/S-phase transition. 934 16

Trichothiodistrophy (TTD), xeroderma pigmentosum (XP), and Cockayne's syndrome (CS) are three distinct human diseases with sensitivity to ultraviolet (UV) radiation affected by mutations in genes involved in nucleotide excision repair (NER). Among the many responses of human cells to UV irradiation, both nuclear accumulation of p53, a tumor suppressor protein, and alterations in cell-cycle checkpoints play crucial roles. The purpose of this study was to define the signals transmitted after UV-C-induced DNA damage, which activates p53 accumulation in TTD/XP-D fibroblasts, and compare this with XP-D cell lines that carry different mutations in the same gene, XPD. Our results showed that p53 was rapidly induced in the nuclei of TTD/XP-D and XP-D fibroblasts in a dose-dependent manner after UV-C irradiation, as seen in XP-A and CS-A fibroblasts, much lower doses being required for the protein accumulation than in normal human fibroblasts, XP variant cells, and XP-C cells. The kinetics of accumulation of p53 and two effector proteins involved in cell-cycle arrest, WAF1 and GADD45, were also directly related to the repair potential of the cells, as in normal human fibroblasts their levels declined after 24 h, the time required for repair of UV-induced lesions, whereas NER-deficient TTD/XP-D cells showed p53, WAF1, and GADD45 accumulation for over 72 h after irradiation. Our results indicate that p53 accumulation followed by transcriptional activation of genes implicated in growth arrest is triggered in TTD/XP-D cells by the persistence of cyclobutane pyrimidine dimers, which are known to block transcription, on the transcribed strands of active genes.
Mol Carcinog 1997 Dec
PMID:Prolonged p53 protein accumulation in trichothiodystrophy fibroblasts dependent on unrepaired pyrimidine dimers on the transcribed strands of cellular genes. 943 78

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the "young" morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.
Mol Cell Biol 1998 Mar
PMID:Reinitiation of DNA synthesis and cell division in senescent human fibroblasts by microinjection of anti-p53 antibodies. 948 78


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