细胞周期调控相关蛋白Cyclin D1、CDK 4和pRb在新疆维吾尔族妇女宫颈癌中的表达及意义

 目的 探讨细胞周期调控相关蛋白Cyclin D1、CDK 4和pRb磷酸化状态在新疆维吾尔族妇女宫颈癌（简称维族）中表达及其意义。方法 应用免疫组织化学方法检测64例维族宫颈癌及43例维族正常宫颈组织中Cyclin D1、CDK 4表达和pRb磷酸化状态。结果 Cyclin D1和CDK4在宫颈癌组中阳性率分别为70%、87%,与正常宫颈组相比,差异有统计学意义（P<0.05）,pRb磷酸化在两组中差异有统计学意义（P<0.01）。结论 Cyclin D1 与CDK4 蛋白的高表达及pRb高磷酸化可能与维族宫颈癌发生发展有关。     

Cyclin D1-CDK4 complex, a possible critical factor for cell proliferation and prognosis in laryngeal squamous cell carcinomas

Cyclin D1 and its catalytic partner CDK4 are known to play important roles in the G1/S checkpoint of the cell cycle. The complex formed by CDK4 and cyclin D1 has been strongly implicated in the control of cell proliferation and prognoses in human malignancies. We investigated the immunohistochemical expression of cyclin D1, CDK4 and proliferating cell nuclear antigen (PCNA) in 102 patients with laryngeal squamous cell carcinoma (LSCC). Cyclin D1 overexpression was observed in 59 cases (57.8%) of LSCC, and was significantly correlated with tumor site, tumor size, lymph node metastasis and advanced stage. CDK4 overexpression was observed in 48 cases (47.1%), and was significantly correlated with tumor size and advanced stage. Cyclin D1 and CDK4 expression was significantly associated with cell proliferation measured by PCNA (r = 0.812, p < 0.0001 and r = 0.725, p < 0.0001, respectively). The Kaplan-Meier analysis showed that cyclin D1 overexpression was significantly associated with disease-free survival and overall survival. CDK4 overexpression was significantly associated with overall survival. When cyclin D1 and CDK4 are combined, the patients with co-overexpression of cyclin D1-CDK4 revealed the poorest overall survival. Additionally, in early-stage (I-II) cases, co-overexpression of cyclin D1-CDK4 was also revealed to possess a significant prognostic role. By multivariate analysis, cyclin D1 overexpression, lymph node metastasis and advanced stage were independent prognostic factors for disease-free survival. Cyclin D1 overexpression, CDK4 overexpression, tumor grade, lymph node metastasis and advanced stage were independent prognostic factors for overall survival. These findings indicate that cyclin D1 and CDK4 overexpression and/or co-overexpression of these proteins may play a pivotal role in the biological behavior of LSCC and may provide a strong prognostic implication.     

Aberrant expression of G(1)/S regulators is a frequent event in sporadic pituitary adenomas

Components of the pRb/p16/cyclin D1/CDK4 pathway are frequent targets in numerous tumour types, including those of pituitary origin. However, previous studies of pituitary tumours have examined individual components of this pathway. Therefore, to determine their overall contribution we have simultaneously examined the immunohistochemical status of pRb, p16 and cyclin D1 and analysed the CDK4 gene for a characterized activating mutation. Of the total pituitary tumour cohort (29 clinically non-functioning adenomas and 16 somatotrophinomas) abnormal expression of either pRb, p16 or cyclin D1 was observed in 36 of 45 (80%) tumours and was significantly (P = 0.005) associated with non-functioning tumours (27/29; 93%) compared with somatotrophinomas (9/16, 56%). Loss of either pRb or p16 expression was mutually exclusive in 23 of 45 (51%) tumours, whilst concomitant loss of pRb and p16 expression was observed in five tumours. Cyclin D1 overexpression was observed in 22 of 45 (49%) tumours, however, there was no significant association between overexpression of cyclin D1 and the expression status of either pRb or p16. In addition, no activating mutations within codon 24 of the CDK4 gene were detected. This study provides evidence for the first time that components of the pRb/p16/cyclin D1/CDK4 pathway, either alone or in combination, are frequently deregulated in human pituitary tumours, suggesting that this pathway may be a useful target in drug or gene therapeutic approaches.     

Cyclin D1 and cyclin D-dependent kinases enhance oral keratinocyte proliferation but do not block keratinocyte differentiation

Maintenance of oral epithelial homeostasis requires a fine balance between cell proliferation and differentiation. However, the molecular mechanisms that couple these processes, and its deregulation in tumorigenesis are not fully understood. Cyclin D1 and its kinase partners CDK4 and CDK6 play an important role in regulating the G1-S phase of the cell cycle. Deregulation of cyclin D1 is a frequent event in oral squamous cell carcinoma. Here, we examined whether overexpression of cyclin D1, CDK4 and CDK6 can deregulate the link between oral keratinocyte proliferation and differentiation. Our results show that cyclin D1 and its kinase partners CDK4 and CDK6 enhance keratinocyte proliferation, but are not sufficient to block calcium-induced keratinocyte differentiation and suggest that deregulation of these G1-regulatory kinases alone is insufficient to uncouple the link between proliferation and differentiation.     

Frequent disruption of the RB1 pathway in diffuse large B cell lymphoma: prognostic significance of E2F-1 and p16INK4A.

In the present study, we analysed 34 de novo diffuse large B cell lymphoma (DLCL) from a population-based lymphoma registry for alterations of the RB1 pathway at the genetic (RB1 and CDK4) and protein (pRb, cyclin D1, cyclin D3, CDK4, and E2F-1) level. The results were correlated with the data from our previous studies of CDKN2A deletion and hypermethylation, other p53 pathway components, p27Kip1 expression, and proliferation, as well as with clinical outcome, including prognosis. We found aberrant pRb expression in four (12%) of 34 DLCLs. One of these had a point mutation in intron 3 10 bp downstream of exon 3 generating a novel splice signal. Seven tumours (21%) showed cyclin D3 overexpression, including all three thyroid lymphomas (P = 0.006). Cyclin D3 overexpression and p16INK4A/pRb aberrations were mutually exclusive, supporting an oncogenic role for cyclin D3 in DLCL. p16INK4A inactivation, cyclin D3 overexpression, or aberrant pRb expression was identified in 18 of 34 DLCLs (53%). Combining these results with our previous p53 pathway studies showed that 82% of the de novo DLCLs had alterations of these pathways, and that both pathways were altered in 13 cases (38%). Low E2F-1 expression was associated with treatment failure (P = 0.020), and multivariate analysis of overall survival identified both low E2F-1 expression (relative risk = 6.9; P = 0.0037) and p16INK4A inactivation (relative risk = 3.3; P = 0.0247) as independent prognostic markers. These data support a role of E2F-1 as tumour suppressor gene in lymphoma and strongly suggest that the RB1 and p53 pathways are important in the development of de novo DLCL. Furthermore, low E2F-1 expression and p16INK4A inactivation may serve as prognostic markers for patients with this type of lymphoma.     

Overexpression of G1-S cyclins and cyclin-dependent kinases during multistage human pancreatic duct cell carcinogenesis.

PURPOSE: Molecular analysis of pancreatic intraepithelial neoplasia lesions and ductal adenocarcinoma suggested a multistage paradigm for pancreatic duct cell carcinogenesis. This study investigated the molecular basis for the neoplastic duct cells in this pancreatic intraepithelial neoplasia-carcinoma sequence to acquire progressive enhancement of their proliferative potential. EXPERIMENTAL DESIGN: Using tissue microarray blocks containing 15 to 40 pancreatic intraepithelial neoplasia lesions and ductal adenocarcinoma of pancreas, we studied by immunohistochemistry the expression profiles of cyclins and cyclin dependent kinases (CDKs) that regulate the G1-S cell cycle checkpoints. The role of cyclins D3 and D1 in three pancreatic cancer cell lines was investigated using specific short interfering RNA technique. RESULTS: Cyclin D3 overexpression was noted the earliest in pancreatic intraepithelial neoplasia-1A and was prevalent in 90% to 100% of high-grade pancreatic intraepithelial neoplasias and ductal cancer. Cyclin A overexpression was also noted early and reached 50% to 100% of high-grade pancreatic intraepithelial neoplasias and cancer, but the percentage of abnormal duct cells showing overexpression of cyclin A was significantly lower than cyclin D3. Cyclin E overexpression occurred in 20% to 25% of high-grade pancreatic intraepithelial neoplasias and in 75% of ductal carcinoma. Cyclin D1 demonstrated the lowest frequency of overexpression that occurred late. CDK2 and CDK4 overexpression was also noted in early pancreatic intraepithelial neoplasias and progressively increased to reach 60% to 75% in carcinoma. The down-regulation of cyclin D3 mRNA and protein levels using specific short interfering RNA resulted in growth inhibition of pancreatic cancer cell lines. CONCLUSION: The results provide additional insight into the mechanism of G1-S cell cycle checkpoints deregulation during stepwise pancreatic duct cell carcinogenesis, and suggest a p16-independent role for cyclin D3 in deregulating the G1 cell cycle checkpoints during early stages of pancreatic duct cell carcinogenesis.     

抗CD44单抗A3D8对HL-60细胞Cyclin D1 CDK4 p21 cip1表达的影响*

目的:探讨CD44单克隆抗体A3D8对人急性髓系白血病细胞株HL-60细胞增殖分化影响的分子作用机制.方法:采用FCM、RT-PCR及Western blot方法检测A3D8作用前后HL-60细胞Cyclin D1、CDK4、p21cip1表达的变化.结果:A3D8作用使HL-60细胞发生G0/G1期阻滞.A3D8下调HL-60细胞Cyclin D1及CDK4的表达,上调HL-60细胞p21cip1 mRNA及P21蛋白表达.结论:A3D8抑制HL-60细胞增殖、诱导其分化的分子机制可能与p21cipl表达上调及Cyclin D1、CDK4表达下调有关.     

Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells

One of the characteristic features of human embryonic stem cells (hESCs) is the competence for self-renewal and pluripotency. To date, little is known about cell cycle regulation in these cells and how the cell cycle machinery influences hESCs properties. A common feature of human, murine and primate ESCs is the presence of a short G1 phase, which has been viewed as a time window during which stem cells are exposed to differentiation signals. We used the hESCs differentiation model and comparisons to human embryonic carcinoma (EC) cells to study the key regulators of G1 to S transition in hESCs. Our studies show that hESCs express all G1-specific CYCLINs (D1, D2, D3 and E) and cyclin-dependent kinases (CDK) (CDK2, CDK4 and CDK6) at variable levels. In contrast to murine ESCs, most of the cell cycle regulators in hESCs show cell cycle-dependent expression, thus revealing important differences in the expression of cell cycle regulatory components between these two embryonic cell types. Knockdown of CDK2 using RNA interference resulted in hESCs arrest at G1 phase of the cell cycle and differentiation to extraembryonic lineages. This suggests an important role for CDK2 in cell cycle regulation in hESCs that are likely to bear significant impacts on the maintenance of their pluripotent phenotype.     

Antiproliferative activity of contragestazol (DL111-IT) in murine and human tumor models in vitro and in vivo

Purposes: To evaluate the antiproliferative activity of contragestazol (DL111-IT) in vitro and in vivo and to elucidate potential molecular mechanisms. Methods: Cell killing ability of DL111-IT was measured by MTT/Trypan blue exclusion method and murine and human tumor models; cell cycle was analyzed by flow cytometry; pRb, CDK4 and Cyclin D1 expressions were detected by western blotting. Results: DL111-IT exhibited high efficiency on cell growth inhibition of 12 cancer cell lines, the IC₅₀ values were 4.1–19.7 μg/ml. In Sarcoma-180 (S180) and Hepatoma-22 (H22) tumor bearing mice models, the inhibition rates were 55.9 and 55.6%, respectively, at the doses of DL111-IT 12.5–50.0 mg/kg for 9 days consecutive administration. Human ovarian carcinoma (HO-8910) xenograft study showed that, nine administrations (within 15 days) of DL111-IT (12.5–50.0 mg/kg) significantly inhibited tumor growth with the inhibition rates ranging from 17.0 to 64.3%. DL111-IT induced G1 arrest and overexpression of pRb, CDK4 and Cyclin D1 were observed in HO-8910 cell line, suggesting that cell cycle regulation might contribute to the anticancer property of DL111-IT. Conclusions: DL111-IT could inhibit the proliferation of cancer cells both in vitro and in vivo via a cell cycle regulation pathway.     

1,25（OH）2D3联合顺铂对原代肺腺癌细胞周期及调控因子的影响

目的:探讨1,25（OH）2D3联合顺铂对原代肺腺癌细胞周期及周期相关调控因子的影响。方法:体外培养手术切除的肺腺癌组织细胞,经药物作用后CCK-8法测定细胞抑制率,流式细胞仪检测细胞周期,RT-PCR检测细胞周期调控因子Cyclin D1、CDK4转录水平。结果:不同浓度的1,25(OH)2D3、顺铂单药作用于肺腺癌细胞均有明显的抑制作用,两药联合表现为协同作用,与单药相比,差异有统计学意义(P＜0.05)。细胞周期分析显示,经药物联合处理后的肺癌细胞,G0/G1期细胞数增多,Ｓ期和G2/M期细胞减少。细胞周期调控因子Cyclin D1、CDK4转录水平均有所降低,差异有统计学意义(P＜0.05)。结论:1,25（OH）2D3联合铂类抗癌药物抑制原代肺腺癌细胞的增殖能力、诱导细胞周期阻滞,与下调Cyclin D1和CDK4 的表达有关。     

细胞周期蛋白cyclin D1、CDK4在食管癌中的表达及其意义

目的：获得食管癌发生过程中调节Ｇ１细胞周期各种因子的作用。方法：采用抗ｃｙｃｌｉｎＤ１和ＣＤＫ４的单克隆抗体对１０例下沉食管和５０例食管鳞状上皮癌标本进行免疫组织化学染色。结果：ｃｙｃｌｉｎＤ１和ＣＤＫ４在正常食管上皮呈现较低水平的表达,在食管鳞状上皮癌中则过表达。２７／５０食管鳞状上皮癌ｙｃｙｃｌｉｎＤ１染色阳性,其中８例强阳性,１２例只表达ＣＤＫ４,１１例只表达ｃｙｃｌｉｎＤ１,１４例既表达ｃ     

Alterations of pRb1-cyclin D1-cdk4/6-p16(INK4A) pathway in endometrial carcinogenesis

The retinoblastoma protein pathway (pRb1-cyclin D1-cdk4/6-p16(INK4A)) participates in the regulation of the cellular processes at the transition of G1/S phases of the cell-cycle. Derailments of this pathway, caused either by lack of pRb1 or p16(INK4A) expression or overexpression of cyclin D1 and/or cdk4/6, are implicated in the deregulation of the cell-cycle machinery, resulting in uncontrolled cell proliferation, tumor heterogeneity, invasion and metastasis. Several studies conducted so far have assessed the deregulation of the pRb1-pathway components in various human tumors and cell-lines, provided these pathway alterations play an obligatory role in tumorigenesis. This review briefly summarizes the current information on the pRb1-cyclin D1-cdk4/6-p16(INK4A) alterations in sporadic uterine cancer, placing emphasis on the influence on the dualistic model of endometrial carcinogenesis.     

Concomitant Presence of p16/Cyclin-dependent Kinase 4 and Cyclin D/Cyclin-dependent Kinase 4 omplexes in LNCaP Prostatic Cancer Cell Line

The cyclin D/cyclin-dependent kinase (CDK)/CDK-inhihitory proteins/retinoblastoma protein (pRb) pathway is hypothesized to control the G1-S check point. The role of this pathway is reported to be different depending on the status of pRb. In the present study, we examined nine human urological tumor cell lines. Cells lacking functional pRb expressed p16, instead of forming cyclin D/ CDK4 complex. In the LNCaP prostatic cancer cell line, however, both p16/CDK4 and cyclin D/ CDK4 complexes were present independently, probably because of partial loss of pRb. In view of the concomitant presence of the incompatible complexes, LNCaP should provide us with a valuable model for the study of this pathway in cancer cells.