p21WAF1/CIP1 expression in gestational trophoblastic disease: correlation with clinicopathological parameters, and Ki67 and p53 gene expression.

BACKGROUND: The p21WAF1/CIP1 gene mediates growth arrest by inhibiting G1 cyclin dependent kinases and has been considered as a downstream effector of the tumour suppressor gene p53. AIM: To analyse the role of p21WAF1/CIP1 in gestational trophoblastic disease. METHODS: The immunohistochemical expression of p21WAF1/CIP1 gene was measured in 33 placentas, 28 partial hydatidiform moles, 54 complete hydatidiform moles, and 13 choriocarcinomas in paraffin wax embedded tissue. The results were correlated with p53 (DO7) and Ki67 (MIB1) immunoreactivity as well as clinical progress. RESULTS: p21WAF1/CIP1 immunoreactivity was found predominantly in the nuclei of the syncytiotrophoblasts. p21WAF1/CIP1 protein expression correlated with gestational age in normal placentas (p = 0.0001) but not in hydatidiform moles (p = 0.89). Complete hydatidiform moles and choriocarcinomas had a significantly higher p21WAF1/CIP1 expression compared with normal placentas and partial hydatidiform moles (p < 0.001); there was no difference between placentas and partial hydatidiform moles. No correlation between p21WAF1/CIP1 expression and either the proliferation (Ki67) index (p = 0.34) or p53 protein accumulation (p = 0.68) was demonstrated. There was no significant difference (p > 0.05) in p21WAF1/CIP1 expression between the 17 patients who developed persistent gestational trophoblastic disease and those who did not. CONCLUSIONS: This study suggests that p21WAF1/CIP1 expression in trophoblastic disease may be induced by a p53 independent pathway. The proliferative activity of gestational trophoblastic diseases might not be determined solely by the control of the cell cycle operated by p21WAF1/CIP1. p21WAF1/CIP1 expression is not an accurate prognostic indicator of gestational trophoblastic disease.     

Expression of p21WAF1/CIP1 in fetal and adult tissues: simultaneous analysis with Ki67 and p53.

AIMS: To determine the expression of p21WAF1/CIP1 in relation to the expression of Ki67 and p53 in various normal adult and fetal tissues, and to investigate its distribution throughout the cell cycle. METHODS: The expression of p21WAF1/CIP1 in relation to Ki67 and p53 was analysed in adult and fetal tissues using immunohistochemical techniques. Heat induced epitope retrieval techniques were used to characterise the presence of p21WAF1/CIP1 in different tissues, as well as to detect its distribution throughout the cell cycle. In addition, flow cytometry and western blotting were used to test whether the level of p21WAF1/CIP1 expression varied at different phases of the cell cycle in phytohaemagglutinin (PHA) stimulated lymphocytes. RESULTS: p21WAF1/CIP1 expression varied from one tissue to another, and it was restricted mainly to the squamous and glandular epithelium, where it appeared in association with p53. Human tissues in which p21WAF1/CIP1 was found showed a mutually exclusive topographical sequential expression between p21WAF1/CIP1 and Ki67. This was confirmed by double labelling studies, which showed that p21WAF1/CIP1 positive cells were in the G0 phase. Unlike these findings of a decline in p21WAF1/CIP1 expression after the G0 phase, PHA stimulated lymphocytes showed a level of p21WAF1/CIP1 expression that rose as the cell progressed through the cell cycle. CONCLUSIONS: The analysis of p21WAF1/CIP1 expression in relation to the status of p53 should take into account the existence of variable p21WAF1/CIP1 expression in different tissues. This could provide an explanation for the varying frequency of p53 mutations in tumours of different cellular origin. In tissues characterised by regular p21WAF1/CIP1 expression, it appears in a pattern that is consistent with the proposed role of this inhibitor of cyclin dependent kinases in cell cycle arrest-that of inducing cell differentiation. The conflicting results of in vivo and in vitro studies could support the hypothesis that microenvironmental conditions may influence the location of p21WAF1/CIP1 in different phases of the cell cycle.     

Cyclin D1 and p21 in ulcerative colitis-related inflammation and epithelial neoplasia: a study of aberrant expression and underlying mechanisms

It is unclear whether and how cyclin D1 and/or p21(WAF1/CIP1) dysregulation contribute to ulcerative colitis (UC)-related inflammation and colorectal carcinogenesis. Cases of quiescent UC (QUC; n = 15), active UC (AUC; n = 23), UC-related dysplasia (n = 35) and UC-related colorectal adenocarcinomas (CRCs; n = 11) were studied with cyclin D1 and p21(WAF1/CIP1) immunohistochemistry. The CRCs were also studied with beta-catenin, bcl2, and p53 immunohistochemistry, p53 and k-ras mutation analyses, and cyclin D1 gene fluorescence in situ hybridization. QUC showed cyclin D1 (negative/weak staining) and p21(WAF1/CIP1) (surface epithelial and upper-third crypt staining) expression similar to that of normal colorectum. Moderate or strong cyclin D1 immunostaining was seen in 9% of AUC cases, 40% of dysplasia cases, and 36% of UC-related CRCs. Although these carcinomas showed neither cyclin D1 gene amplification nor any association between k-ras mutation and cyclin D1 overexpression, the latter was closely related to nuclear beta-catenin expression. Increased lower-third crypt p21(WAF1/CIP1) staining was seen in 57% of AUC cases; decreased upper-third crypt p21(WAF1/CIP1) staining, in 23% of dysplasia cases; and absent or weak p21(WAF1/CIP1) staining, in 55% of UC-related CRCs. The latter change was always associated with p53 mutation but could not be related to p53 or bcl2 expression. In conclusion, AUC shows up-regulated cyclin D1 and p21(WAF1/CIP1) expression. Cyclin D1 up-regulation and p21(WAF1/CIP1) down-regulation occur early in UC-related carcinogenesis. Cyclin D1 up-regulation is less common in UC-related CRCs than in sporadic CRCs, and is related to beta-catenin nuclear signaling. p21(WAF1/CIP1) down-regulation is seen at an equal or higher frequency among UC-related CRCs compared with sporadic CRCs and is attributable to p53 mutation.     

IGFBP7对人乳腺癌MCF-7细胞增殖的影响及其机制

目的: 探究胰岛素样生长因子结合蛋白7(IGFBP7)对人乳腺癌MCF-7细胞增殖的影响及其分子生物学机制。方法: 将质粒pCMV6-IGFBP7转染MCF-7细胞,构建稳定表达IGFBP7的MCF-7细胞系;采用Western blotting检测IGFBP7在MCF-7细胞稳定转染子的表达;采用软琼脂培养克隆形成实验检测IGFBP7对MCF-7细胞克隆形成能力的影响;采用流式细胞术检测IGFBP7对MCF-7细胞周期的影响;采用Western blotting检测IGFBP7对MCF-7细胞细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、细胞周期素D1(cyclin D1)、细胞周期素依赖性激酶4(CDK4)、cyclin E、CDK2、p21^CIP1/WAF1、p27^KIP1、p53、视网膜母细胞瘤蛋白(Rb)和p-Rb蛋白含量的影响。结果: (1)只有稳定转染质粒pCMV6-IGFBP7的MCF-7细胞表达IGFBP7。(2)IGFBP7能够显著降低MCF-7细胞的克隆形成率(P<0.01),阻止细胞从G₁期进入S 期,使其停滞于G₁期(P<0.01)。(3)IGFBP7能够显著抑制ERK1/2的磷酸化(P<0.01)。(4)IGFBP7能够下调cyclin D1和cyclin E蛋白表达(P<0.01),上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达(P<0.01),抑制Rb的磷酸化(P<0.01)。(5)MEK1/2阻断剂PD98059可部分模拟IGFBP7的肿瘤抑制效应。结论: (1) IGFBP7可通过下调cyclin D1和cyclin E蛋白表达,上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达,以及抑制Rb磷酸化发挥抗肿瘤作用;(2) IGFBP7对cyclin D1和p27^KIP1的调节可能与其抑制ERK1/2信号通路有关。     

Oxygen induces S-phase growth arrest and increases p53 and p21(WAF1/CIP1) expression in human bronchial smooth-muscle cells.

Hyperoxia increases free radical production, leading to DNA damage. Recent studies indicate that oxygen augments the expression of p53 and p21(WAF1/CIP1), and increases apoptotic labeling of airway epithelial cells. Similar changes in regulatory gene products have not been reported in other pulmonary cells, nor have these changes been investigated in conjunction with alterations in cell-cycle distribution. The present study was conducted to determine whether oxygen alters the expression of p53 and p21(WAF1/CIP1) in human bronchial smooth-muscle cells (BSMC). BSMC placed in room air (RA), 40% O(2), or 95% O(2) were examined for 3 d to determine cell number, thymidine incorporation, cell-cycle distribution, and lactate dehydrogenase release. Apoptosis was assessed through the terminal deoxynucleotidyl transferase-deoxyuridine triphosphate end-nick labeling (TUNEL) technique, and p53 and p21(WAF1/CIP1) protein levels were determined through enzyme-linked immunosorbent assay. Exposure of BSMC to 95% O(2) decreased proliferation and DNA synthesis within 24 h, and was accompanied by an increase in S-phase cells (72 h; RA: 12.9 +/- 4.6%, versus 95% O(2): 34.6 +/- 7.0%; P < 0.01). By comparison, exposure to 40% O(2) resulted in decreased proliferation at 48 h without significant alterations in cell-cycle distribution. Both p53 and p21(WAF1/CIP1) levels were increased by 95% O(2), with maximal differences noted at 24 and 48 h, respectively. All atmospheres showed < 8% cell death and few TUNEL-positive cells. Our results indicate that oxygen-mediated alterations in BSMC proliferation are time- and concentration-dependent. Furthermore, high oxygen levels induce S-phase arrest and increased expression of p53 and p21(WAF1/CIP1). Activation of these genes may prevent replication without inducing apoptosis to allow for the repair of oxidative damage.     

Expression and mechanism of mammalian target of rapamycin in age-related renal cell senescence and organ aging

The mammalian target of rapamycin (mTOR) is relevant to cell senescence and organismal aging. This study firstly showed that the level of mTOR expression increased with aging in rat kidneys, rat mesangial cells and WI-38 cells (P < 0.05). The levels of phosphorylated-mTOR (p-mTOR), cyclin D1 and p21^{WAF1/CIP1/SDI1} expression were significantly higher in WI-38 cells treated with l-leucine (an activator of mTOR) (P < 0.05). The positive staining ratio of senescence-associated β-galactosidase, number of cells in G1 phase, and cellular volume were all increased in WI-38 cells treated with l-leucine when the cellular population doubling (PD) number was 34, while the above phenotypes did not appear in control group until its PD number reached 40 (P < 0.05). The levels of p-mTOR, cyclin D1, and p21^{WAF1/CIP1/SDI1} as well as the aging-related phenotypes were all reduced in cells treated with rapamycin (an inhibitor of mTOR) than in control cells (P < 0.05). These results demonstrated that the level of mTOR was increased in kidney with aging, and that mTOR may promote cellular senescence by regulating the cell cycle through p21^{WAF1/CIP1/SDI1}, which might provide a new target for preventing renal aging.     

Medroxyprogesterone acetate inhibits proliferation of colon cancer cell lines by modulating cell cycle-related protein expression

OBJECTIVE: To investigate the effects of medroxyprogesterone acetate on colon cancer cells in vitro. DESIGN: HT29 and HCT116 human colon cancer cell lines were used in this study. Cell growth and WST-1 assays were performed to investigate the antiproliferative effect of medroxyprogesterone acetate. Cell cycle analysis was performed to investigate the effects of medroxyprogesterone acetate on cell cycle distribution. Western blot, immunoprecipitation, and a cyclin-dependent kinase assay were performed to investigate changes in the levels of cell cycle proteins. RESULTS: Medroxyprogesterone acetate inhibited proliferation of the cancer cells by inducing accumulation in the G0/G1 fraction. Medroxyprogesterone acetate decreased expression of cyclin E, increased expression of p21(WAF1/CIP1), and enhanced interaction of p21(WAF1/CIP1) with cyclin-dependent kinase 2, eventually inhibiting its activity. CONCLUSIONS: Medroxyprogesterone acetate exerts its antiproliferative effect by modulating cell cycle-related protein expression and cyclin-dependent kinase 2 activity. These results should help to elucidate the protective effect of medroxyprogesterone acetate on colon cancer risk.     

Immunohistochemical markers of cell cycle control applied to ovarian and primary peritoneal surface epithelial neoplasms: p21(WAF1/CIP1) predicts survival and good response to platinin-based chemotherapy.

Immunohistochemistry for p53, p21(WAF1/CIP1), and Ki-67 provides insight into the molecular events controlling the cell cycle. We tested the hypothesis that these cell cycle markers will aid in the clinical evaluation of ovarian and primary peritoneal surface epithelial neoplasms (SENs). Paraffin sections from a retrospective surgical series of 117 SENs were immunostained with anti-p53 (clone DO7, Novacastra Laboratories, UK), anti-p21(WAF1/CIP1) (clone EA10, Oncogene Science, Cambridge, MA), and anti-Ki-67 (clone MIB-1, Immunotech, Westbrook, ME). The Ki-67 proliferation index (Ki-67PI) and immunoreactivity were evaluated. One hundred seventeen SENs reacted as follows: p53 50%+ and p21(WAF1/CIP1) 65%+. Ki-67PI ranged from 4% to 88% (mean/median = 44/46%). p53 reactivity associated with transitional cell histology, decreased p21(WAF1/CIP1) staining, increased Ki-67PI, architectural/nuclear grade, and stage (P < .05, 1 x 10(-7), .01, .05/.0001, .001,). p21(WAF1/CIP1) staining was associated with endometrioid/clear cell histology, decreased Ki-67PI, architectural/nuclear grade, and stage (P < 05/.05, .05, .01/1 x 10(-8), 1 x 10(-5)). Ki-67PI associated with increased architectural/nuclear grade but not mucinous histology (P < 1 x 10(-5)/1 x 10(-6), .01). Sixty-seven patients had disease at last follow-up; 53 were dead of disease at 0 to 67 months (mean/median, 21/18), and 14 were alive with disease at 12 to 224 months (mean/median, 56/40). Fifty patients were disease free at 5 to 214 months (mean/median, 59/41). Predictors of survival include decreased Ki-67PI, stage, architectural/nuclear grade (P < 1 x 10(-6), 1 x 10(-10), 1 x 10(-10)/.005) and p21(WAF1/CIP1) IMS (multivariate P < 1 x 10(-6)). p21(WAF1/CIP1), a potent inhibitor of cyclin-dependent kinases necessary for cell cycle progression, functions as a key checkpoint in cell cycle control. Immunoreactivity for p21(WAF1/CIP1) provides prognostic information independent of other histological and clinical predictors, p53 IMS, and Ki-67PI in this series of 117 PTs with SENs. Our preliminary data suggest an interrelationship between p21(WAF1/CIP1) expression and an effective clinical response to platinin-based chemotherapy, both associated with apoptosis. Further investigation seems warranted.     

p53-Independent Expression of p21CIP1/WAF1 in Plasmacytic Cells during G2 Cell Cycle Arrest Induced by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

The cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans has been shown to induce cell cycle arrest in the G(2)/M phase in HeLa cells. In the present study, the mechanism of CDT-induced cell cycle arrest was investigated by using HS-72 cells, a murine B-cell hybridoma cell line. Using flow cytometric analysis, we found that the recombinant CDT (rCDT) from A. actinomycetemcomitans induced G(2) cell cycle arrest in HS-72 cells and that rCDT upregulated expression of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) and the tumor suppressor protein p53. HS-72 cells transfected with the E6/E7 gene of human papillomavirus type 16, which lacked rCDT-induced accumulation of p53, exhibited expression of p21(CIP1/WAF1) or G(2) cell cycle arrest upon exposure to rCDT. Furthermore, ectopic expression of a dominant negative p53 mutant did not inhibit rCDT-mediated p21(CIP1/WAF1) expression or G(2) cell cycle arrest in HS-72 cells. These results suggest that the CDT from A. actinomycetemcomitans induces p21(CIP1/WAF1) expression and G(2) cell cycle arrest in B-lineage cells by p53-independent pathways. Together with additional observations made with HeLa cells and COS-1 cells cultured with the rCDT from A. actinomycetemcomitans, the results of this study indicate that CDT-induced p53 accumulation may not be required for G(2) cell cycle arrest and that an increased level of p21(CIP1/WAF1) may be important for sustaining G(2) cell cycle arrest in several mammalian cells.     

p21WAF1/CIP1 expression in colorectal carcinoma correlates with advanced disease stage and p53 mutations

Defects in the mechanisms controlling the cell cycle are crucial in cell transformation and/or tumour progression. p21^WAF1/CIP1 is an inhibitor of cyclin-dependent kinases, induced by p53-dependent and p53-independent pathways, which can block progression through the cell cycle. p21^WAF1/CIP1 expression has been investigated immunohistochemically in a series of 191 patients with colorectal cancer of known p53 status. The purpose of the study was two-fold: to assess the relationship between p21^WAF1/CIP1 immunoreactivity and p53 alterations, and to evaluate the prognostic significance of p21^WAF1/CIP1 expression. In 96 carcinomas (51 per cent), p21^WAF1/CIP1 was expressed in over 10 per cent of tumour cells, whereas in 26, p21^WAF1/CIP1 was detected in under 10 per cent of neoplastic cells; 69 tumours lacked p21^WAF1/CIP1 expression. Immunoreactivity was more frequent in tumours of the right colon (p < 0·003) and was inversely correlated with tumour stage (p < 0·03), p53 gene mutations (p < 0·0007), p53 protein accumulation (p < 0·019), and Bcl-2 expression (p < 0·0005). In univariate analysis, down-regulation of p21^WAF1/CIP1 expression was associated with poor overall (p = 0·0022) and disease-free survival (p = 0·0009). Multivariate analysis, however, did not confirm any independent prognostic significance of p21^WAF1/CIP1 expression. The results indicate that p21^WAF1/CIP1 is associated with abnormal accumulation of p53 protein and the occurrence of p53 gene mutations in colorectal cancer and that lack of p21^WAF1/CIP1 expression is correlated with reduced patient survival in univariate analysis. These data underline the crucial pathogenetic role of the p53–p21^WAF1/CIP1 pathway in carcinomas of the large bowel. Copyright © 1999 John Wiley & Sons, Ltd.     

Cyclin A expression in superficial spreading malignant melanomas correlates with clinical outcome.

The present study analysed by immunohistochemistry the protein level of cyclin A and Ki-67 in a panel of paraffin-embedded tissue obtained from 172 primary (110 superficial and 62 nodular) and 73 metastatic melanomas, and ten benign naevi. Since cyclin A exists in the same quaternary complex in the S-phase of the cell cycle as the cdk inhibitor p21WAF1/CIP1, the levels of the two proteins were compared. Cyclin A and Ki-67 were heterogeneously expressed in the malignant tumours, whereas in benign naevi, only rare positive cells were detected. In superficial spreading melanomas, the cyclin A level was related to tumour thickness, with less expression in thinner lesions (p<0.00001), and to Ki-67 (p<0.00001) and p21WAF1/CIP1 (p=0.01) scores. Multivariate analysis showed that in addition to the depth of the primary tumour, the protein level of cyclin A was an independent indicator of relapse-free period (thickness, p<0.00001; cyclin A, p=0.0003). In contrast, in nodular melanoma, the cyclin A level was associated with Ki-67 expression, but neither cyclin A nor Ki-67 was related to tumour thickness (cyclin A, p=0.06; Ki-67, p=0.61) and neither had any impact on relapse-free (cyclin A, p=0.64; Ki-67, p=0.32) or overall (cyclinA, p=0.94; Ki-67, p=0.45) survival. In conclusion, the results indicate that cyclin A is a strong prognostic factor for patients with superficial spreading melanomas. In nodular melanomas, the proliferation rate seems to have little impact on disease progression.     

Inhibition of γ-secretase by retinoic acid chalcone (RAC) induces G2/M arrest and triggers apoptosis in renal cell carcinoma

The present study was devised to investigate the effect of RAC on inhibition of cell proliferation and apoptosis of renal carcinoma cells. MTT assay and flow cytometry analysis were used to determine cell proliferation and apoptosis along with cell cycle examination. Western blot analysis and immunohistochemistry were used for the detection of expression levels of Notch1 and Jagged1 in renal cell carcinoma (RCC) and normal kidney tissues. The results revealed a significant inhibition of cell proliferation, G2/M phase cell cycle arrest and cell apoptosis at 30 μM concentration of RAC after 72 h. In ACHN and 769-P cells, the population in G2/M phase was increased to 45.27, and 54.23% respectively on treatment with 30 μM RAC for 72 h. In 769-P and ACHN renal carcinoma cells treatment with 30 μM RAC caused 69.71 and 59.27% of the cells to undergo apoptosis compared to 5.23 and 4.93% respectively in control cells. The positive staining rates of Notch1 and Jagged1 in renal carcinoma tissues were 95.3 and 93.0% compared to normal kidney tissues 36.4 and 42.4% respectively. Treatment of renal carcinoma tissues caused a significant decrease in staining rates of Notch1 and Jagged1 after 96 h. Thus RAC can be a potent agent in the treatment of renal cell carcinoma.     

Early alteration of cell-cycle-regulated gene expression in colorectal neoplasia.

Aberrant crypt foci with dysplasia are thought to be the first detectable lesions of colorectal neoplasia. Because cell cycle disruption appears crucial for tumorigenesis, we analyzed the immunohistochemical expression patterns of the prototype cyclin-dependent kinase inhibitor p21 WAF1/CIP1 and the proliferation marker Ki67 in the early stages of colorectal tumorigenesis. In colorectal epithelium, p21 WAF1/CIP1 expression was undetectable in the lower third of the crypts, where Ki67 was expressed, but then sharply increased as cells passed out of the proliferating zone and migrated toward the humen. Hyperplastic polyps retained this normal compartmentalized pattern. In contrast, markedly decreased p21 WAF1/CIP1 immunostaining was observed in dysplastic aberrant crypt foci as well as in small adenomas. Moreover, the compartmentalization of Ki67 and p21 WAF1/CIP1 was lost, as Ki67 expression extended into the small p21-expressing zone at the top of the crypts. These data suggest that the dysregulated expression of cell-cycle-controlling genes and the consequent release from normal cell cycle controls may represent an essential early step in colorectal neoplasia.     

Activation of M-phase-specific histone H1 kinase by modification of the phosphorylation of its p34cdc2 and cyclin components

An M-phase-specific histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types undergoing the G2/M transition in the cell division cycle. We have used p13suc1-Sepharose affinity chromatography to purify H1K to near homogeneity from matured starfish oocytes. A yield of 67% was obtained. Active H1K behaves as a 90- to 100-kD protein and appears to be constituted of equimolar amounts of cyclin and p34cdc2. The p34cdc2 subunit becomes tyrosine-dephosphorylated as the H1K is activated during entry of the oocytes into M phase, whereas the cyclin subunit is reciprocally phosphorylated. Acid phosphatase treatment of inactive p34cdc2/cyclin complex induces p34cdc2 dephosphorylation and three- to eightfold stimulation of the enzyme activity. These results suggest that active M-phase-specific H1K is constituted of both dephosphorylated p34cdc2 and phosphorylated cyclin.     

Cell-cycle inhibition by TNP-470 in an in vivo model of hepatocarcinoma is mediated by a p53 and p21WAF1/CIP1 mechanism

The objective of this study was to determine in a rat model of hepatocellular carcinoma (HCC) the effects of the antiangiogenic agent TNP-470 on cell proliferation and effectors of the apoptotic pathway, including p53, p21WAF1/CIP1, cyclin D, and cyclin E. Tumor was induced in male Wistar rats by diethylnitrosamine and promoted by two-thirds hepatectomy plus acetaminofluorene administration. Experiments were carried out at 28 weeks after initiating the treatment. TNP-470 was administered at 30 mg/kg, 3 times per week from 20 to 28 weeks. Serum levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor beta (HGFbeta) liver expression were increased by hepatocarcinogenesis (+38% and +183%, respectively), and treatment with TNP-470 was able to prevent the increase in these angiogenic factors induced by HCC. HCC coursed with reduced expression of p21WAF1/CIP1 and p53 (-63% and -60%, respectively). Hepatic expression of cyclin D and cyclin E were significantly increased in rats with HCC (+108% and +115%, respectively). In animals with experimental carcinogenesis, a significant increase in the expression of Cdk4 and CdK2 was also observed (+119% and +187%, respectively). These effects were prevented by TNP-470 administration. In conclusion, cell-cycle inhibition by TNP-470 is mediated at least in part by an activation of p21WAF1/CIP1 because of a p53-dependent mechanism, with reduction of the cyclin D-Cdk4 and cyclin E-Cdk 2 expression. These cytostatic effects should be considered when assessing the efficacy of TNP-470 for anti-angiogenic therapy. These findings may prove useful for the development of therapies for the treatment of human HCC.     

膀胱移行细胞癌细胞周期调节相关基因表达与肿瘤生物学行为的关系

目的：探讨细胞周期调节相关基因p16INK4,p21WAF／CIP1,p53mlt在膀胱移行细胞癌中的表达与肿瘤增殖能力,病理分级及临床分期的关系。方法：应用免疫组织化学技术分析77例膀胱移行细胞癌组织p16INK4,p21WAF/CIP1,p53mlt基因的表达和增殖细胞核抗原（PCNA）表达情况,并与病理分级及临床分期之间进行综合分析。结果：p16INK4,p21WAF/CIP1,p53mlt在膀胱移行细胞癌中的表达及PCNA增殖指数与肿瘤的病理分级有关,与临床分期无关。p16,p21,p53阳性组与阴性组分别比较,其PCNA增殖数之间有差异性。多因素分析发现,p16INK4和p21WAF／CIP1阴性及p53mlt阳性组的PCNA值明显高于p16INK4和p21WAF／CIP1阳性及p53mlt阳性组,两者比较不同病理分级的阳性表达构成比亦有显著性差异。结论:联合检测p16INK4,p21WAF/CIP1,p53mlt基因的表达情况能充分反映膀胱移行细胞癌的增殖能力及生物学行为,对膀胱移行细胞癌患者的预后判断及治疗有指导意义。     

Overexpression of p21WAF1/CIP1 and MDM2 characterizes serous borderline ovarian tumors

Ovarian epithelial tumors are classically divided into benign, malignant, and borderline or of low malignant potential. It is controversial whether this last group of tumors should be considered benign or malignant. Expression of cell cycle markers has recently been linked to tumor behavior and response to treatment. It has been shown that one of the pathways through which the p53 gene controls the cell cycle is by transactivating p21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor. By inhibiting cdks, p21WAF1/CIP1 blocks the G-1 to S-phase transition in the cell cycle. p53 can be regulated by MDM2 (murine double minute-2) through direct inactivation or promotion of its cytoplasmic degradation. In an attempt to investigate the cell cycle checkpoint mechanisms of these tumors, we studied the expression of p53, Ki-67, MDM2, and p21WAF1/CIP1 by immunohistochemistry. We analyzed the expression of these proteins in 19 cystadenomas (8 serous and 11 mucinous), 40 borderline tumors (31 serous and 9 mucinous), and 18 serous carcinomas of the ovary. p21WAF1/CIP1 was expressed in 7 of 19 (37%) benign cystadenomas, 32 of 40 (80%) borderline tumors (93.5% of serous and 33% of mucinous), and in 9 of 18 (50%) serous carcinomas. Ki-67 was only weakly expressed in 8 of 19 (42%) benign cystadenomas, all borderline tumors showed Ki-67 staining in less than 50% of the cells, and 55% of serous carcinomas stained in more than 50% of tumor cells. p53 was absent in all but 1 of the cystadenomas, was expressed in 9 of 40 (22.5%) borderline tumors (25.8% of serous and 11% of mucinous), and in 10 of 18 (55%) carcinomas. All 11 implants of serous borderline tumors expressed p21WAF1/CIP1. Most serous borderline tumors expressed higher levels of MDM2 compared with the benign cystadenomas and carcinomas. Four of the serous borderline implants (40%) expressed MDM2. Coexpression of p21WAF1/CIP1 and MDM2 characterizes serous borderline tumors of the ovary and their implants, which suggests that these cell cycle control proteins are important in these tumors and may be related to tumor progression. Low expression of p53 protein in serous borderline tumors might be in part mediated by MDM2. This suggests that the p53 pathway is intact in most of these tumors, in contrast with carcinomas, in which high expression of p53 has been related to mutations of this gene.