Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1

BACKGROUND: Intestinal mucosal turnover is a process of proliferation, differentiation, and apoptosis; the mechanisms remain largely undefined. The purpose of our study was to (1) assess the relationship between apoptosis and enterocyte differentiation and (2) determine whether the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1, or the apoptosis inhibitors, Bcl-2 and Bcl-XL, may be involved. METHODS: Gut-derived Caco-2 cells were treated with sodium butyrate. Apoptosis was assessed by Hoechst stain, DNA laddering, and annexin V assay; differentiation was determined by alkaline phosphatase and sucrase activity. RNA and protein were analyzed for expression of p21Waf1/Cip1, p27Kip1, and members of the Bcl-2 family. RESULTS: Treatment of Caco-2 cells with sodium butyrate resulted in the concomitant induction of both differentiation (increased alkaline phosphatase and sucrase activity) and apoptosis. Increased levels of p21Waf1/Cip1 and p27Kip1 mRNA and protein were detected at 24 hours, occurring before apoptosis or differentiation; decreased mRNA levels of Bcl-2 and Bcl-XL were noted at 24 hours. CONCLUSIONS: Differentiation and apoptosis occurred simultaneously in Caco-2 cells, suggesting that apoptosis may be linked to enterocyte differentiation. The induction of p21Waf1/Cip1 and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL further suggest a link between the cell-cycle mechanisms regulating enterocyte differentiation and apoptosis.     

Comparative effects of overexpression of p27Kip1 and p21Cip1/Waf1 on growth and differentiation in human colon carcinoma cells

Recent studies have shown that decreased expression of p27Kip1 is associated with high grade tumors and an unfavorable prognosis in several types of human cancer. To clarify the role of p27Kip1 in colon cancer, we have overexpressed this protein in the HT29 colon cancer cell line. The derivatives displayed an increase in the p27Kip1 protein in cyclin E/CDK2 immunoprecipitates and a decrease in cyclin E-associated kinase activity when compared to vector control clones, providing evidence that the overexpressed protein was functional. Clones with a high level of p27Kip1 displayed partial growth inhibition in monolayer culture and a decrease in plating efficiency, even though they expressed increased levels of the cyclin D1 protein. Using alkaline phosphatase expression as a marker, we found that the p27Kip1 overexpressor clones displayed a 2-3-fold increase in sensitivity to induction of differentiation by 2 mM sodium butyrate. In contrast to these results, derivatives of HT29 cells that stably overexpressed p21Cip1/Waf1 displayed decreased sensitivity to the induction of differentiation. These findings may explain why decreased levels of p27Kip1 in certain human cancers is associated with high grade (poorly differentiated) tumors, and suggest that strategies that increase the level of p27Kip1 may be useful in cancer therapy.     

Altered expression of the p53-regulated proteins, p21Waf1/Cip1, MDM2, and Bax in ultraviolet-irradiated human skin

The gemistocytic astrocytoma is a histological variant of diffuse astrocytomas and is characterised by the presence of large, GFAP-expressing neoplastic astrocytes (gemistocytes) and a tendency towards rapid progression to glioblastoma. In this study, we analyzed 28 gemistocytic astrocytomas (mean fraction of gemistocytes, 35.0+/-9.9%) for mutations in the p53 and PTEN (MMAC1) tumour suppressor genes. Single strand conformation polymorphism (SSCP), followed by direct DNA sequencing of p53 exons 5-8, revealed a mutation in 23 of 28 (82%) cases. Regional analysis of four tumours revealed identical p53 mutations in gemistocytic and fibrillary tumour areas. In contrast, none of 15 gemistocytic astrocytomas (WHO Grade II) and only two of 11 (18%) anaplastic gemistocytic astrocytomas (WHO Grade III) contained a PTEN mutation. Of these, one was a 1 bp deletion in codon 345 and the other a 1 bp insertion in intron 4. Differential PCR did not reveal homozygous PTEN deletion in any of the tumours analysed. These results indicate that p53 mutations are a genetic hallmark of gemistocytic astrocytomas, whilst PTEN mutations are absent in low-grade and rare in anaplastic gemistocytic astrocytomas.     

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.     

Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1

Small GTPases act as molecular switches in intracellular signal-transduction pathways. In the case of the Ras family of GTPases, one of their most important roles is as regulators of cell proliferation, and the mitogenic response to a variety of growth factors and oncogenes can be blocked by inhibiting Ras function. But in certain situations, activation of Ras signalling pathways arrests the cell cycle rather than causing cell proliferation. Extracellular signals may trigger different cellular responses by activating Ras-dependent signalling pathways to varying degrees. Other signalling pathways could also influence the consequences of Ras signalling. Here we show that when signalling through the Ras-related GTPase Rho is inhibited, constitutively active Ras induces the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 and entry into the DNA-synthesis phase of the cell cycle is blocked. When Rho is active, induction of p21Waf1/Cip1 by Ras is suppressed and Ras induces DNA synthesis. Cells that lack p21Waf1/Cip1 do not require Rho signalling for the induction of DNA synthesis by activated Ras, indicating that, once Ras has become activated, the primary requirement for Rho signalling is the suppression of p21Waf1/Cip1 induction.     

Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase delta and replication factor C

BACKGROUND: PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary. RESULTS: Loop-region mutants in which human PCNA sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA regions were prepared. Analysis of their functions, along with previously prepared alanine scanning mutants, demonstrated that some loops interact with DNA polymerase delta (pol delta) and replication factor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be located within a distinct structure of the PCNA monomer, overlap with RFC- and pol delta-interaction sites. Competition between p21 and pol delta or RFC for binding to PCNA results in efficient inhibition of its stimulation of pol delta DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC. CONCLUSIONS: Semi-saturated amounts of p21 selectively block formation of the active pol delta complex but not the RFC-PCNA complex at 3'-ends of DNA primers. This differential effect may explain the specific inhibition of DNA replication by p21.     

Association between human cancer and two polymorphisms occurring together in the p21Waf1/Cip1 cyclin-dependent kinase inhibitor gene

BACKGROUND: The cyclin-dependent kinase inhibitor gene p21Waf1/Cip1 plays a role in signaling cellular growth arrest. In response to DNA damage, p21 is induced by the p53 gene, thereby playing a direct role in mediating p53-induced G1 arrest. Alterations in this gene may adversely affect regulation of cellular proliferation and increase susceptibility for cancer. Two polymorphisms have previously been characterized in the p21 gene: a C-->A transversion at codon 31 (ser-->arg) and a C-->T transition 20 nucleotides downstream from the 3' end of exon 3. METHODS: The codon 31 polymorphism in exon 2 of the p21 gene was identified by restriction digestion (Alw26I) of products amplified by polymerase chain reaction (PCR). The polymorphism downstream from exon 3 of the p21 gene was identified by single strand conformation polymorphism (SSCP) analysis of PCR amplified products and was confirmed by PstI enzyme restriction digestion. DNA variant alleles were confirmed by direct DNA sequencing. The entire coding region and the promoter region (p53 binding domain) of the p21 gene were screened for mutations by SSCP analysis or DNA sequencing. RESULTS: The two polymorphisms were found in 18 of 96 tumor samples lacking p53 alterations (18.8%). Nine of 54 prostate adenocarcinoma samples (16.7%) contained both p21 variants, whereas 9 of 42 squamous cell carcinomas of the head and neck (21.4%) displayed both polymorphisms. Of the 110 controls examined, 10 (9.1%) had both alterations. Both p21 polymorphisms occurred together in all samples examined and there was no indication of mutation in the coding region of the p21 gene or in the p53 binding domain of the promoter region. CONCLUSIONS: These data suggest that p21 gene variants may play a role in increased susceptibility for the development of some types of cancer. In the current study, the authors demonstrated that the occurrence of these two polymorphisms is increased in prostate adenocarcinoma and squamous cell carcinoma of the head and neck. The polymorphic sites may be directly responsible for this apparent increased susceptibility or they may be linked to regulatory region alterations.     

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.     

The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases

The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.     

p21(Waf1/Cip1) inhibition of cyclin E/Cdk2 activity prevents endoreduplication after mitotic spindle disruption

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G1. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21(Waf1/Cip1) enter S phase with a >/=4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G1/S and G2/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21(+/+) and p21(-/-) cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21(-/-) cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G1-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21(+/+) cells paralleled the onset of endoreduplication in HCT116 p21(-/-) cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.     

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.     

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.     

p53-independent down-regulation of cyclin D1 and p21Waf1 in the process of immortalization of human esophageal epithelial cells

The temporal correlation between adenosine outflow and changes in field excitatory post synaptic potentials (fEPSP) occurring during ischemia-like conditions was investigated in rat hippocampal slices. Five-minute long ischemia-like conditions resulted in a 100% depression of fEPSP amplitude, followed by a complete recovery after 6-7 min of reperfusion. By reducing the duration of the ischemic insult to 2 min, fEPSP was depressed by 50%. During both 5 and 2 min of ischemia-like conditions, a significant increase in adenosine outflow was detected. During reperfusion, when fEPSP amplitude recovered completely, the adenosine level in the extracellular fluid returned to basal values. The strict relationship between the increase in adenosine outflow and fEPSP inhibition supports the hypothesis that adenosine is largely responsible for the synaptic transmission depression during cerebral ischemia.     

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 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.