p21Cip1/WAF1 is important for differentiation and survival of U937 cells

Vitamin D3 (VD3) induces monocytic differentiation of U937 cells. Induction of p21Cip1/WAF1 (p21) and subsequent G0/G1 cell-cycle arrest are required in this process. Using a system of inducible expression of ectopic p21, we demonstrated the important role of p21 in the induction of monocytic differentiation in U937 cells. Prior induction of antisense-p21 expression significantly suppressed p21 expression, and resulted in inhibition of VD3-induced U937 differentiation. Moreover, induction of expression of antisense-p21 in VD3-differentiated U937 cells resulted in apoptosis of the cells. This was associated with activation of Cdc2 and caspase-3 like protease. Our results suggest that p21 is required for the initiation of the early steps of differentiation as well as survival of differentiated cells.     

TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1

We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.     

p21WAF1/Cip1 expression is associated with cell differentiation but not with p53 mutations in squamous cell carcinomas of the larynx

The anti-metastatic effect of Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, was investigated in mice bearing B16 melanoma cells. Treatment of BF10 mice implanted with high metastatic B16F10 melanoma cells with a 10 mg/kg dose of Z-100 resulted in the reduction of experimental pulmonary metastasis as compared with that of BF10 mice treated with saline. The number of pulmonary metastatic colonies in BF1 mice (mice implanted with low metastatic B16F1 melanoma cells) was greatly increased after the inoculation of CD4+ CD11b+ CD281+ TCR alphabeta+ type 2 T cells (F10-Th2 cells) derived from BF10 mice, while only a few metastatic colonies were demonstrated in lungs of BF1 mice inoculated with naive CD4+ T cells. However, the numbers of metastatic colonies in BF1 mice were not increased when they were inoculated with the F10-Th2 cell fraction derived from Z-100-treated BF10 mice and the generation of F10-Th2 cells in BF10 mice was effectively suppressed by the Z-100 treatment. These results suggest that Z-100 inhibits pulmonary metastasis of B16 melanoma through the regulation of tumor-associated Th2 cells, which are a key cell in the acceleration of tumor metastasis.     

Growth inhibitory concentrations of EGF induce p21 (WAF1/Cip1) and alter cell cycle control in squamous carcinoma cells

Previous studies have reported inhibition of A431 squamous carcinoma cell growth by nanomolar concentrations of epidermal growth factor (EGF), a potent mitogen for cells of epithelial origin. In this study, we examined potential mechanisms through which inhibition of keratinocyte growth mediated by EGF might occur by analysing components of the cell cycle regulatory machinery in A431, HN6 and HN30 keratinocytes in the presence of growth inhibitory or growth stimulatory doses of EGF. Treatment of cells with 25 pM EGF produced an increase in [3H]thymidine incorporation in A431, HN6 and HN30 cells, with respect to control cultures. Exposure to 2.5 nM EGF reduced [3H]thymidine incorporation in A431 cells and HN6 cells to 11% and 70% of control levels, respectively, whereas HN30 cells continued to proliferate in the presence of EGF. [3H]thymidine incorporation assays carried out over 24 h revealed repression of DNA synthesis in A431 cells after 12 h exposure to 2.5 nM EGF compared to untreated cells. Flow cytometry studies demonstrated accumulation of cells in G0/G1 after addition of 2.5 nM, but not 25 pM EGF. Western blot analysis revealed elevation of p21 (WAF1/CIP1/SDI1) protein levels in A431 and HN6 cells under growth-inhibitory conditions. Stimulatory doses of EGF did not induce p21 in these cells. Northern blot hybridization demonstrated elevated levels of p21 mRNA within 4 h of exposure of A431 cells to 2.5 nM EGF, which remained elevated above basal levels at 24 h. In vitro kinase assays demonstrated temporal differences in CDK2 and CDK6 activities which were related to EGF concentration. Immunocomplex Western blotting demonstrated increased association of p21 with CDK2 and CDK6 in A431 cells treated with 2.5 nm EGF. Furthermore, temporal alterations in the association of PCNA with p21 and with CDK6 were observed. The data indicate that p21 is a likely mediator of EGF-induced growth-inhibition, probably through mechanisms involving sequestration of PCNA and inhibition of CDK activity.     

p21WAF1/CIP1/SDI1 is elevated through a p53-independent pathway by mimosine

Tumor suppressor p53 is a nuclear protein that is induced by DNA damage and is involved in G1 and G2 phase control of the cell cycle. p21WAF1/CIP1/SDI1 (p21), a cyclin-dependent kinase inhibitor, is a downstream target and effector of p53 to induce G1 arrest. Mimosine is a potent reversible late G1 phase blocker of the cell cycle. In this study, we showed that mimosine can increase both p21 mRNA and protein levels, indirectly inhibit cyclin E-associated kinase activity without affecting the cyclin E protein level, block human breast cancer cells (21PT) in the late G1 phase of the cell cycle, and induce a p53-independent p21 pathway in these cells. These results support the possibility of restoring a G1 checkpoint by use of mimosine. They also suggest that the mechanism of the effect of mimosine is complex and may have more than one target in the cell.     

Analysis of p21WAF1/CIP1 in primary bladder tumors

The p21WAF1/CIP1 gene is regulated by p53 and encodes a cyclin-dependent kinase (Cdk)-inhibitor involved in senescence and cell quiescence. The role of p21 as a negative regulator of cell proliferation suggests that it may function as a tumor suppressor gene. However, only a few mutations of the p21WAF1/CIP1 gene have been reported to date. In order to assess potential p21WAF1/CIP1 gene alterations in human bladder cancer, we have examined this gene and its encoded product in a well-characterized cohort of 27 primary bladder tumors. Mobility shifts by single-strand conformation polymorphism in the p21WAF1/CIP1 gene were identified in 2 cases. Sequencing analyses revealed that one of these cases had point mutations in the 3' untranslated region, while the other case had a frame shift mutation at positions 322 (C to A) and a deletion of 8 nucleotides (323-->331; CCG-->ACG, codon 81 Arg-->Thr) that produced a stop signal at codon 83 (Gly--Stop). This tumor had a p21-negative phenotype by immunohistochemistry, but did not lose any allele. We further characterized these cases by the study of TP53 mutations using single-strand conformation polymorphism (PCR-SSCP) and sequencing, as well as immunohistochemical assays. Seven mobility shifts were identified and seven cases showed p53 nuclear accumulation. The two cases displaying mutated p21WAF1/CIP1 had wild-type TP53. It is concluded that p21WAF1/CIP1 gene aberrations are infrequent in bladder carcinoma but may be occasionally identified in primary bladder tumors.     

Expression of retinoblastoma gene product and p21 (WAF1/Cip 1) protein in gliomas: correlations with proliferation markers, p53 expression and survival

AIMS/BACKGROUND: The construction and validation of an instrument for the assessment of subjective visual disability in the cataract patient is described. This instrument is specifically designed for measuring the outcome of cataract surgery with respect to visual disability. METHODS: Visually related activities thought to be affected by cataract were considered for the questionnaire. These were reduced by pilot study and principal components analysis to 18 items. A patient's assessment of his/her ability to perform each task was scored on a four point scale. Scores were averaged to create an overall index of visual disability, as well as subscale indices for mobility related disability, distance/lighting/reading related disability, and near and related tasks visual disability. The questionnaire, administered verbally is entitled "The Visual Disability Assessment (VDA)". Reliability testing included test-retest reliability, interobserver reliability (p, the intraclass correlation coefficient), and internal consistency reliability (Cronbach's alpha). Construct validation, the process for proving that a test measures what it is supposed to measure, included consideration of content validity, comparison with the established Activities of Daily Vision Scale (ADVS) and empirical support with factor analysis. RESULTS: For the four indices, interobserver reliability varied from 0.92 to 0.94, test-retest reliability varied from 0.96 to 0.98, and internal consistency reliability varied from 0.80 to 0.93. The VDA compared favourably with the ADVS by correlation, but Bland-Altman analysis demonstrated that the two instruments were not clinically interchangeable. Factor analysis suggests that all test items measure a common theme, and the subgroupings reflect common themes. CONCLUSIONS: The VDA is easy to administer because it has a short test time and scoring is straightforward. It has excellent interobserver, test-retest, and internal consistency reliability, and compares favourably with the ADVS, another test of visual disability. Factor analysis demonstrated that the 18 items measure a related theme, which can be assumed to be visual disability. The VDA is a valid instrument which provides a comprehensive assessment of visual disability in cataract patients and is designed to detect changes within a patient over time.     

Expression of a novel form of p21Cip1/Waf1 in UV-irradiated and transformed cells

The tumor suppressor p53 and its target the CDK inhibitor p21 (Cip1/Waf1) are key components of the cellular response to DNA damage. Insight into how p21 is regulated in normal cells, and how it may be deregulated in tumor cells is important for the understanding of tumorigenesis. p21 was induced in normal human diploid fibroblasts after UV irradiation-induced DNA damage, but, at a high dose of UV irradiation, a faster mobility form of p21 on SDS-PAGE (designated p21delta) was expressed. Surprisingly, in a variety of growing transformed cell lines, the level of p21 was low but p21delta was prominent. We found that p21delta appeared to be derived through a loss of around 10 amino acids from the C-terminus of p21, which theoretically would remove the PCNA binding domain, a second cyclin binding domain and the nuclear localization signal sequence. Several characteristics distinguish p21 from p21delta. Both the full length p21 and p21delta could be stabilized by a proteasome inhibitor, but only the full length p21 was associated with Cdk2 and PCNA. Consistent with this, gel filtration chromatography revealed that all the full length p21 in the cell was complexed to other proteins, whereas a significant portion of p21delta was in monomeric form. Moreover, p21 was mainly localized to the nucleus, but p21delta was mainly localized to the cytoplasm. We propose that the decrease in p21 and increase in p21delta could contribute to the deregulation of the cell cycle, and could be a mechanism involved in cellular transformation.     

Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition

Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/Waf1/Sdi1). We provide evidence that in addition to exerting negative control of the G1/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21-/- mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G2 that may contribute to the implementation of late cell cycle checkpoint controls.     

Enhanced expression of Ki-67, topoisomerase IIalpha, PCNA, p53 and p21WAF1/Cip1 reflecting proliferation and repair activity in UV-irradiated melanocytic nevi

To investigate the effect of ultraviolet (UV) irradiation on the expression of cell cycle-associated proteins, melanocytic nevi from healthy volunteers were partially covered, irradiated with a defined UV dose, and excised 1 week thereafter. The irradiated and the protected parts were examined separately by conventional microscopy and immunohistochemistry using the antibodies Ki-S11 (Ki-67), Ki-S7 (topoisomerase IIalpha), PC10 (proliferating cell nuclear antigen [PCNA]), DO-7 (p53), 6B6 (p21WAF1/Cip1), and the melanocytic marker HMB-45. DNA nick-end labeling was used as a marker of apoptosis. Irradiation resulted in morphological changes and increased HMB-45 reactivity. Proliferation, as assessed by Ki-67 and topoisomerase IIalpha expression, was also clearly enhanced in the UV-exposed areas. This was confirmed by the appearance of occasional mitotic figures. PCNA expression levels markedly exceeded those of the proliferation markers and did not correlate with the latter in most cases. p21 immunolabeling indices were also consistently augmented after UV exposure; hence it is likely that growth-inhibitory mechanisms partly compensate for the proliferative impulse, and the disproportional rise in PCNA expression probably reflects DNA repair activity. Enhanced p53 immunostaining in four cases suggests that the induction of p21 after irradiation may be p53 mediated, whereas no concomitant apoptotic events were observed. We conclude that UV light can stimulate the proliferative activity of melanocytes in melanocytic nevi, but that simultaneously cell cycle inhibitors are activated to permit DNA repair.     

G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cip1/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells

UCN-01 (7-hydroxyl-staurosporine) was originally isolated as a Ca2+- and phospholipid-dependent protein kinase C selective inhibitor and now is being developed as an anticancer agent. Results from our and other laboratories have suggested that UCN-01 induces preferential G1-phase accumulation in several human tumor cell lines tested. To elucidate this mechanism, we examined the effects of UCN-01 on several cell cycle-regulatory proteins critical for G1-S-phase transition in p53-mutated human epidermoid carcinoma A431 cells. After 24 h exposure at around 50% growth-inhibitory concentrations (IC50s), 260 and 520 nM, UCN-01 induced the accumulation of pRb (the dephosphorylated retinoblastoma protein form). The protein expression of cyclin A but not cyclin E was markedly reduced and that of cyclin D1 was partially reduced under the same condition. UCN-01 also showed the concentration-dependent inhibitions of the activity of cyclin-dependent kinase 2 (CDK2) using histone H1 and pRb as substrates in vitro (IC50, 530 and 640 nM, respectively). In addition, CDK2 activities of the cells pretreated with UCN-01 for 24 h at 260 and 520 nM were markedly inhibited, giving IC50s of far less than 260 nM. When the same cell lysates were analyzed by Western blotting for CDK2, the lower band (e.g., active and phosphorylated CDK2) was remarkably reduced, in accordance with the reduced activity. Furthermore, UCN-01 induced the expression of the CDK inhibitor p21 protein and its complex formation with CDK2 after 24 h exposure at 260 and 520 nM, whereas the expression level was very low or undetectable in untreated or DNA-damaged cells. The increase of p21 mRNA levels was also induced under the same condition. UCN-01 further increased luciferase activities in A431 cells transiently transfected with p21 promoter-luciferase reporter plasmid after 24 h exposure at 260 and 520 nM. UCN-01 also increased the expression of the CDK inhibitor p27 protein after 24 h exposure at 260 and 520 nM. These results suggest that G1-phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitors p21 and p27.