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1.
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.  相似文献   

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Transforming growth factor-beta (TGF-beta) inhibits cell cycle progression of many types of human cells by arresting them in the G1 phase of the cell cycle. The arrest is mediated through interactions of various cyclin-dependent protein kinases (CDKs) and their inhibitors. We demonstrate that treatment with TGF-beta induces increased levels of WAF1/Cip1/p21, a potent inhibitor of various cyclin-CDK kinase activities, in two colon cancer cell lines (LS1034 and LS513), which are sensitive to TGF-beta-induced growth arrest. The induction in at least one of these cell lines (LS1034,p53-) is p53-independent. No WAF1 induction was observed after TGF-beta treatment in a third cell line (HT-29), which is completely insensitive to the cytoinhibitory effect of TGF-beta. In both LS513 and LS1034, WAF1 induction correlated with reduced cyclin E-associated kinase activity in vitro and suppression of the retinoblastoma susceptibility gene (Rb) protein phosphorylation in vivo. In addition, WAF1 was physically associated with cyclin E in the two sensitive cell lines. These results suggest that WAF1/Cip1/p21 is a mediator of cellular sensitivity to TGF-beta.  相似文献   

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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.  相似文献   

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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.  相似文献   

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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.  相似文献   

8.
Onconase is a 12 kDa protein homologous to pancreatic RNase A isolated from amphibian oocytes which shows cytostatic and cytotoxic activity in vitro, inhibits growth of tumors in mice and is in phase III clinical trials. The present study was aimed to reveal mechanisms by which onconase perturbs the cell cycle progression. Human histiocytic lymphoma U937 cells were treated with onconase and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) p16INK4A, p21WAF1/CIP1 and p27KIP1 (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRb) by a novel method utilizing mAb which specifically detects underphosphorylated pRb in individual cells. Cell incubation with 170 nM onconase for 24 h and longer led to their arrest in G1 which was accompanied by a decrease in expression of cyclin D3, no change in cyclin E, and enhanced expression of all three CKIs. pRb was underphosphorylated in the onconase arrested G1 cells but was phosphorylated in the cells that were still progressing through S and G2/M in the presence of onconase. The cytostatic effect of onconase thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of p27KIP1, p16INK4A and p21WAF1/CIP1, the events which may prevent phosphorylation of pRb during G0/1 and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.  相似文献   

9.
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.  相似文献   

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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.  相似文献   

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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.  相似文献   

16.
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.  相似文献   

17.
Previous studies have revealed that the growth inhibition of A431 cells overexpressing epidermal growth factor (EGF) receptors by a high concentration of EGF is mainly due to the expression of cycline dependent kinase (CDK) inhibitor p21(WAF1/Cip1). However, the signal transduction mechanism from the activated EGF receptor to the induction of p21(WAF1/Cip1) gene is still poorly understood. We investigated which signaling pathway plays an important role in p21(WAF1/Cip1) expression and growth inhibition by using specific inhibitors of the signaling molecules. A broad PKC inhibitor, PKC delta inhibitor, but not the conventional PKC inhibitor suppressed the EGF-induced p21(WAF1/Cip1) expression and the growth inhibition of A431 cells. These inhibitors did not alter either the activation of EGF receptor or the stimulation of MAP kinase at detectable levels. Furthermore, we found that the induction of p21(WAF1/Cip1) at the early phase (within 12 hr after stimulation) by a high concentration of EGF was independent of the MAP kinase activation by using dominant negative Ras. These results suggest that PKC, especially PKC delta plays a crucial role in the EGF-induced p21(WAF1/Cip1) expression, resulting in the growth inhibition of A431 cells.  相似文献   

18.
The incidence of brain metastases secondary to small cell lung cancer (SCLC) is about 35% and the treatment strategy of brain irradiation with respect to dose and fractionation is controversial. In order to evaluate treatment outcome of brain irradiation in SCLC patients with brain relapse, we retrospectively evaluated all patients treated with brain irradiation in the eastern part of Denmark from 1988 to 1992 (PCI patients excluded). During this 5-year period, 101 evaluable patients were included (44 females, 57 males) (median age 61 years; range, 39-75 years). Forty-four patients, of whom 43 were in extracerebral complete remission (CR), received extended course (EC) brain irradiation (> 45 Gy, treatment schedule > 4 weeks). Fifty-seven patients received short course (SC) brain irradiation (< 30 Gy, treatment schedule < 1 week). Among the SC treated patients, 14 were in CR, 20 had partial remission or stable disease and 23 had progressive extracerebral disease. The median survival (from diagnosis of brain metastases) in the group receiving irradiation with EC (44 patients) was 160 days (range, 74-2021 days), while the 57 patients treated with SC had a median survival of 88 days (range, 20-948 days) (P = 0.00001, Log-Rank analysis). In a subgroup of 14 patients in extracerebral CR, receiving SC irradiation, the median survival was 83 days (range, 15-948 days). When the latter patients were compared to the 43 patients in CR in the group treated with EC, a statistically significant difference was shown (P = 0.034, Log-Rank analysis). Using Cox-hazard regression analysis with backward elimination, liver metastases and poor performance status were adverse prognostic signs, although the only significant parameters of survival were gender (female vs. male, relative risk of dying 1 and 1.52, P = 0.05) and schedule of brain irradiation (extended course vs. short course, relative risk of dying, 0.36 and 1, P < 0.001). Extended course irradiation of brain relapse secondary to SCLC seems in general to be of limited value, although a significant prolonged survival at approximately 7 weeks, was obtained. The prolongation of survival does not seem worthwhile considering the length of treatment time (5-6 weeks) compared to SC treatment (1 week). However, the data do not permit evaluation of the quality of life of the patients. This retrospective evaluation suggests the need for randomized trials with carefully planned quality-of-life assessments.  相似文献   

19.
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.  相似文献   

20.
Loss of attachment to an extracellular matrix substrate arrests the growth of untransformed cells in the G1 phase. This anchorage-dependent cell cycle arrest is linked to increased expression of the p21Cip1 (p21) and p27Kip1 (p27) cyclin-dependent kinase inhibitors. The result is a loss of cdk2-associated kinase activity, especially that of cyclin E-cdk2. The levels of p21 and p27 are also upregulated in unattached transformed cells, but cyclin E-cdk2 activity remains high, and the cells are able to grow in an anchorage-independent manner. Increased expression of cyclin E and cdk2 appears to be partially responsible for the maintenance of cyclin E-cdk2 activity in transformed cells. To explore further the regulation of cyclin E-cdk2 in transformed cells, we have analysed the subcellular distribution of cyclin-cdk complexes and their inhibitors in normal human fibroblasts, their transformed counterparts, and in various human tumor cell lines. In substrate-attached normal fibroblasts, cyclin E and cdk2 were exclusively in the nuclear fraction, associated with one another. When normal fibroblasts were detached and held in suspension, cyclin E-cdk2 complexes remained nuclear, but were now found associated with the p21 and p27 cdk inhibitors and lacked histone H1 phosphorylating activity. In contrast, the transformed fibroblasts and tumor cells, which are anchorage-independent, had more than half of their cyclin E, cdk2, p21 and p27 in the cytoplasmic fraction, both in attached and suspended cultures. The cytoplasmic p21 and p27 were bound to cyclin E-cdk2, as well as to complexes containing cyclin A and cyclin D. The nuclear cyclin E-cdk2 complexes from the transformed cells grown in suspension contained only low levels of p21 and p27 and had histone H1 kinase activity. Thus, at least three mechanisms contribute to keeping cyclin E-cdk2 complexes active in suspended anchorage-independent cells: cyclin E and cdk2 are upregulated, as reported previously, cdk inhibitors are sequestered away from the nucleus by cytoplasmic cyclin-cdk complexes, and the binding of the inhibitors to nuclear cyclin E-cdk2 complexes is impaired.  相似文献   

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