Altered expression of cell cycle regulatory proteins in gastrointestinal stromal tumors: markers with potential prognostic implications

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the digestive tract. The prediction of the malignant potential of GISTs is still difficult. Altered cell cycle regulation may underlie the tumorigenesis and/or the progression of human malignancies. Although p53 and Bcl-2 have been extensively investigated in GISTs, little is known about the frequency of expression and possible clinical implications of alterations of other cell cycle regulatory proteins in these neoplasms. We have previously investigated the role of loss of p16(INK4A) by loss of heterozygosity and immunohistochemistry in the progression of GISTs and found that loss of heterozygosity of 9p and loss of p16 expression are confined to malignant GISTs. This has led us to investigate the role of other cell cycle regulatory proteins in these tumors. Twenty-three cases of GIST (9 low malignant potential [LMP], 10 primary malignant, and 4 intra-abdominal recurrences) were examined. All cases were strongly positive for KIT (CD117). Immunohistochemical stains were carried out on tissue microarrays to evaluate the expression of proteins involved in the G(1)-S transition and proteins that regulate apoptosis including Rb, E2F1, cyclin D1, CDK4, CDK6, p27(KIP1), p21(WAF1/CIP1), p53, Mdm2, Bcl-2, and Bax. The positive phenotypes identified were as follows: Rb, 39.1%; E2F1, 69.6%; cyclin D1, 30.4%; CDK4, 100%; CDK6, 30.4%; 39.1%; p27(KIP1), 47.8%; p21(WAF1/CIP1), 39.1%; p53, 43.5%; Mdm2, 17.4%; Bcl-2, 91.3%; and Bax, 100%. Malignant GISTs are more likely to be associated with a positive E2F1 and p53 phenotype and a negative p16 and p27(KIP1) phenotype. It was concluded that aberration of the cell cycle regulators is a frequent finding and may be a contributing factor to the pathogenesis of GISTs. While some alterations are seen in LMP and malignant GISTs and therefore may represent an early event in molecular tumorigenesis of GISTs, other alterations are more common in malignant GISTs than LMP and therefore have potential utility as complementary tools for the prognostication of GISTs.     

Role of differential and cell type-specific expression of cell cycle regulatory proteins in mediating progressive glomerular injury in human IgA nephropathy

The activities of cell cycle regulatory proteins have been reported to be associated with the development of pathological lesions in glomerulonephritis. To assess the cellular mechanisms underlying the mesangial cell proliferation and glomerulosclerosis in progressive human IgA nephropathy (IgAN), we examined the expression of E2F1, Rb, c-Myc, proliferating cell nuclear antigen (PCNA), cyclins (D1, E and A), cyclin-dependent kinase 2 (CDK2) and CDK inhibitors (p21(waf1), p27(kip1), 57(kip2) and p16(ink4a)) by immunohistochemistry in renal biopsy specimens. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was also performed to detect the presence of apoptosis. In total, 51 cases of IgAN were categorized into four subgroups according to histological severity. A dramatic upregulation of E2F1 expression in mesangial cells was identified in proliferating glomeruli, which correlated well with the proliferation index. High endogenous expression of p27(kip1) and p57(kip2) by podocytes in normal glomeruli and glomeruli with minor lesions was observed to decrease in proliferating and sclerosing glomeruli; this pattern displayed a strong inverse correlation with the mean glomerulosclerosis score and the index of glomerular lesion. Increased apoptotic activity was identified in progressive glomerular lesions of advanced IgAN, which correlated with the proliferative activity in these lesions as assessed by total expression levels of PCNA and CDK2 in glomeruli, E2F1 expression levels in the mesangium, cyclin D1 expression levels in endothelium and the c-Myc glomerular staining score. Our results suggest that the onset and magnitude of mesangial cell proliferation and glomerulosclerosis is associated with the upregulation of E2F1 by mesangial cells and the downregulation of p27(kip1) and p57(kip2) by glomerular epithelial cells. The cell type-specific and coordinated regulation of proliferative and proapoptotic activities of cell cycle regulatory proteins may play an important role in mediating progressive glomerular injury in human IgAN.     

Herpes simplex virus type 1 infection imposes a G(1)/S block in asynchronously growing cells and prevents G(1) entry in quiescent cells

Herpes simplex virus type 1 (HSV-1) infection disrupted cell cycle regulation in at least two ways. First, infection of quiescent human embryonic lung cells simultaneously with readdition of serum caused inhibition of cyclin D/cyclin-dependent kinase (CDK) 4,6-specific and cyclin E/CDK2-specific phosphorylation of the retinoblastoma protein pRb. The inhibition of cyclin D/CDK4,6 kinase activity corresponded to a loss of cyclin D1 protein and a failure of CDK4 and CDK6 to translocate to the nucleus. Failure to detect cyclin E/CDK2 kinase activity was accompanied by a loss of cyclin E protein and a failure of CDK2 to translocate to the nucleus. Levels of pocket protein p130 persisted, whereas p107 did not accumulate. As a result of these effects on cyclin kinase, G(0)-infected cells failed to reenter the cell cycle. The second type of HSV-induced cell cycle dysregulation was observed in asynchronously dividing cell cultures. A rapid inhibition of preexisting cyclin E/CDK2 and cyclin A/CDK2 activities was observed in human embryonic lung cells, as well as two other human cell lines: C33 and U2OS. HSV-1 immediate-early gene expression was necessary for the inhibition of CDK2 kinase activity. Cyclin and CDK subunit protein levels, intracellular localization, and complex stability were unaffected by infection. In addition, levels of cyclin-dependent kinase inhibitors, p27 and p21, were not affected by HSV-1. Previous experiments demonstrated that in asynchronous infected cells, hypophosphorylated pRb and pocket protein-E2F complexes accumulated, and cellular DNA synthesis was rapidly inhibited. Coupled with the present results, this indicates that HSV-1 has evolved mechanisms for preventing cells in G(1) from proceeding through the restriction point and for cells in S from completing a round of DNA replication.     

Expression of cyclins,cyclin-dependent kinases and cyclin-dependent kinase inhibitors in oligodendrogliomas in humans

Cyclins are regulatory proteins of the cell cycle which bind and activate kinases. In gliomas, contrary to many malignancies, cyclin D1 is rarely amplified, but together with other cyclins, it increases with anaplasia. In a series of 23 surgical biopsies of grade II and III oligodendroglioma, cyclin D1, E, A, B1, CDK4-6, CDK2, Cdc2 and p27/Kip.1 have been studied by immunohistochemistry and Western blot. Cyclin D1 and A increased with anaplasia, showing a linear correlation with MIB.1 labeling index and an inverse correlation with p27/Kip.1 expression. Cyclin E and B1 and kinases were almost only expressed in grade III tumors. Normal oligodendrocytes and microglia cells of the cortex and white matter showed a clear positivity for cyclin D1, but not for other cyclins or kinases.     

Inverse correlation between high level expression of cyclin E and proliferation index in transitional cell carcinoma of the bladder.

BACKGROUND/AIMS: Overexpression of the G1 cyclins, D1 and E, and/or downregulation of p27(Kip1) allow uncontrolled tumour cell proliferation. This study investigated the relation between these three cell cycle proteins and tumour proliferation in bladder cancer. METHOD: Nuclear expression of cyclin D1, cyclin E, and p27(Kip1) was determined immunohistochemically in 52 primary transitional cell carcinomas, and the Ki-67 proliferation marker was also assessed. For each protein, the percentage of positive tumour cell nuclei was determined and analysed as a continuous variable. RESULTS: Advancing tumour grade and pathological stage were accompanied by increasing proliferation indices, but decreasing p27(Kip1) and cyclin D1 expression, with no significant change in cyclin E expression. Overall, cyclin D1 and E expression did not correlate with proliferation. However, in cyclin D1 overexpressing tumours (> or = 5% nuclei positive), the level of cyclin D1 expression positively correlated with proliferation. The correlation between cyclin E expression and proliferation changed from positive to negative with increasing levels of cyclin E expression, accompanied by a coordinate increase in p27(Kip1) expression. Overall, there was an inverse association between p27(Kip1) expression and proliferation. However, a subset of tumours displayed high proliferation indices despite high p27(Kip1) expression. The G1 cyclin index (sum of the level of expression of cyclins D1 and E) correlated positively with proliferation in superficial but not muscle invasive tumours. This correlation was stronger when the G1 cyclin index was adjusted for p27(Kip1) expression. CONCLUSION: These findings support a role for these proteins in the proliferation, differentiation, and progression of bladder transitional cell carcinomas.     

Bone morphogenetic protein-2 induces hypophosphorylation of Rb protein and repression of E2F in androgen-treated LNCaP human prostate cancer cells

Bone morphogenetic protein-2 (BMP-2), a multifunctional member of the transforming growth factor (TGF)-beta, superfamily, has powerful osteoinductive effects and causes cell cycle arrest in a variety of transformed cell lines. We have observed BMP-2-induced inhibition of cell proliferation in an androgen-dependent human prostate cancer cell line (LNCaP). To investigate the mechanism of inhibition of androgen-dependent growth by BMP-2, we examined the effect of dihydrotestosterone (DHT) and/or BMP-2 on cell cycle-related proteins in LNCaP cells. BMP-2 decreased the phosphorylation of retinoblastoma (Rb) protein induced by treatment with DHT. DHT-induced expression of cyclin A and cyclin-dependent kinase 2 (CDK2) protein was also inhibited by co-treatment with BMP-2. Furthermore, BMP-2 induced expression of p21(WAF1/CIP1), a CDK inhibitor. These results indicate that changes in expression of these proteins lead to modulation of the phosphorylation state of Rb. DHT-induced E2F-1 protein and mRNA expressions was also inhibited by BMP-2, suggesting that BMP-2 inhibits DHT-induced growth of LNCaP cells through a decrease in E2F protein expression and suppression of E2F activity by hypophosphorylation of Rb.     

Expression of p21Waf1, p27Kip1 and cyclin D1 proteins in breast ductal carcinoma in situ: Relation with clinicopathologic characteristics and with p53 expression and estrogen receptor status

p21Waf1 (p21), p27Kip1 (p27) and cyclin D1 have recently been reported as useful prognostic markers for patients with breast carcinoma. However, studies on these cell cycle regulators in ductal carcinoma in situ (DCIS) have been extremely limited. Therefore, we studied the immunohistochemical expression of p21, p27 and cyclin D1 proteins in 49 DCIS cases and compared the findings with the clinicopathologic parameters (age, tumor size, gross type, histologic type, histologic grade, necrosis and mitotic index), p53 and estrogen receptor (ER) status. A significant correlation was found between positive p21 immunoreactivity (67.3% of the cases) and well-differentiated histologic grade, non-comedo type, ER-positive and p53-negative (p53-) status. DCIS with p21+/p53- is likely to be the non-comedo type. The overexpression of cyclin D1 (59.2% of the cases) correlated positively with the ER expression (P = 0.001). The p27 protein expression (46.9% of the cases) correlated with the cyclin D1 immunopositivity (P = 0.0003) and ER expression (P = 0.005). No significant associations were seen in the p27 or cyclin D1 expression and other clinicopathologic parameters. Our results suggest that p21 might be more related to the useful biologic markers in DCIS than p27 or cyclin D1. The significant positive association between p21, p27 or cyclin D1 and ER status, and close association of p27 and cyclin D1 expression might be implicated in the tumor biology of DCIS.     

Immunoreactivity of p53, Mdm2, p21(WAF1/CIP1) Bcl-2, and Bax in soft tissue sarcomas: correlation with histologic grade.

Tumor growth depends on 2 distinctive pathways: cell proliferation and apoptosis. The p53 pathway is an important regulator of the cell cycle as it triggers growth arrest or leads to apoptosis in response to cellular stress and therefore is commonly targeted during tumorigenesis. Apoptosis is also controlled by the Bcl-2 family, which includes proapoptotic and antiapoptotic proteins. The aim of this study was to investigate the expression of proteins that are involved in the p53 pathway and apoptosis in different types of soft tissue sarcomas and to correlate the expression of these proteins with the histologic grade of sarcoma cases. One hundred fifty-two cases of different types of soft tissue sarcomas were analyzed. The cases consisted of 54 low-grade, 40 intermediate-grade, and 58 high-grade sarcomas. Immunohistochemical stains for p21(WAF1/CIP1), p53, Mdm2, Bcl-2, and Bax proteins were carried out on tissue microarrays. Nuclear reactivity for p53 was detected in 49 cases (32.2%). Overexpression of Mdm2 was found in 18 cases (11.8%) and p21(WAF1/CIP1) immunostaining was seen in 28 tumors (18.4%). p53 and p21(WAF1/CIP1) expression correlated with the tumor grade (low grade, 5.6% and 3.7%; intermediate grade, 22.5% and 20%; high grade, 63.8% and 31%, respectively). Expression of Bax protein was a common finding in soft tissue sarcoma cases. It was detected in 141 cases (92.8%). Bcl-2 was identified in 59 tumors (38.8%) and was more prevalent in high-grade sarcomas (low grade, 25.9%; intermediate grade, 32.5%; high grade, 55.2%). It was concluded that alterations in the p53 pathway and genes that regulate apoptosis are common events in soft tissue sarcomas. The expression of p53, p21(WAF1/CIP1), and Bcl-2 is closely associated with the histologic grade of the tumor, and therefore these proteins may be used as prognostic markers.     

沉默ClC-3氯通道基因对HeLa细胞周期分布的影响

目的:探讨沉默HeLa细胞的ClC-3氯通道基因后细胞周期分布的变化及其作用机制。方法:依照siRNA设计原则构建沉默ClC-3基因的ClC-3 siRNA并转染HeLa细胞;实验分为空白对照组(control组)、转染试剂对照组(Lipo组)、阴性对照组(negative siRNA组)和ClC-3 siRNA组。采用real-time PCR检测ClC-3 siRNA的沉默效率;流式细胞术检测细胞周期分布情况;Western blot检测ClC-3蛋白及相关细胞周期蛋白(cyclin)D1、细胞周期蛋白依赖激酶(cyclin-dependent kinase,CDK)4、CDK6、P21和P27等表达。结果:CIC-3 siRNA成功沉默HeLa细胞的ClC-3基因。和其它组相比,ClC-3 siRNA组的细胞周期被阻抑在G_0/G_1期。CIC-3 siRNA组的cyclin D1、CDK4和CDK6蛋白表达水平明显下降,P21和P27蛋白表达水平明显上升。结论:沉默HeLa细胞ClC-3氯通道基因可影响cyclin D1、CDK4、CDK6、P21和27蛋白的表达水平胆抑HeLa细胞周期停滞在G_0/G_1期。     

Aberrant cell cycle regulation in cervical carcinoma

Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16(INK4A)-cyclin D1-CDK4/6-pRb-E2F, p21(WAF1)- p27(KIP1)-cyclinE-CDK2, and p14(ARF)-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16(INK4A), p21(WAF1), and p27(KIP1), as characteristic features of HPV- infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.     

Low expression of p27 protein combined with altered p53 and Rb/p16 expression status is associated with increased expression of cyclin A and cyclin B1 in diffuse large B-cell lymphomas.

The expression of the cyclin-dependent kinase inhibitor (CDKI) p27 protein was investigated in relation to (1) the expression of the cell cycle regulators p53, Rb and p16 and (2) the proliferation profile as determined by the expression of Ki67, cyclin A, and cyclin B1 in 80 cases of de novo diffuse large B-cell lymphomas (DLBCL). P27 expression was low/null in large tumor cells in 58/80 cases and intermediate/high in 22/80 cases. Increased expression of p53 protein was observed in 39/80 cases. Decreased expression of Rb and p16 proteins was mutually exclusive and was observed in 5/80 and 14/80 cases, respectively. The analysis of the p27 expression status (low/null versus intermediate/high) with respect to the p53 and/or Rb/p16 expression status showed that low/null p27 expression was significantly correlated with increased p53 expression (P =.018) and showed a strong trend for correlation with concurrent increased p53 expression and decreased Rb or p16 expression (P =.050). These findings suggest a tendency for concurrent alterations of the cell cycle regulators p27, p53, and Rb or p16 in DLBCL, which might result in impaired tumor growth control. Indeed, the analysis of the combined p27/p53/Rb/p16 expression status with respect to the proliferation profile showed that (1) three alterations in the combined p27/p53/Rb/p16 status (i.e., low/null P27 expression, increased expression of p53, and decreased expression of Rb or p16) were significantly correlated with increased expression of cyclin B1 (P =.005) and (2) two or three alterations were significantly correlated with increased expression of cyclin A (P =.014). These findings suggest combined impairment of a complex cell-cycle control network involving the CDK inhibitor p27, the P53 pathway, and the Rb1 pathway, which exerts a cooperative effect resulting in enhanced tumor cell proliferation.     

Alteration of cell cycle in cervical tumor associated with human papillomavirus: cyclin-dependent kinase inhibitors

The ability of viral oncoproteins to subvert cell cycle checkpoints may constitute a mechanism by which viral oncoproteins induce genetic instability. HPV 16 E6 and E7 disrupt cell cycle checkpoints, particularly affecting nearly all cyclin-dependent kinase inhibitors linked to the G1- and G2- checkpoints, in each case by means of a different mechanism. HPV 16 E7 shows homology with the pRb binding sites of cyclin D1, which consequently releases E2F. In addition, E7 directly binds to p21, and releases PCNA and other S-phase promoting genes. In turn, released E2F activates cyclin E, and cyclin E accelerates p27 proteolysis as a function of the antagonistic reaction of its own inhibitor. The induction of p16 expression is assumed to be indirectly associated with E7, which is upregulated only after prolonged inactivation of Rb. HPV 16 E6 decreased the fidelity of multiple checkpoints controlling both entry into and exit from mitosis, with the mechanism of p53 inactivation. In addition, HPV 16 E6 increased the sensitivity to chemically induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function. Alongside the impressive advances made in the understanding of the molecular mechanisms, which HPV disrupts, the validity of these conclusions should be evaluated in the diagnostic and prognostic fields.     

Altered expression of G1 regulatory proteins in human soft tissue sarcomas

CONTEXT: Soft tissue sarcomas constitute a heterogeneous group of tumors for which tumorigenesis is not fully understood. Altered cell-cycle regulation may underlie the development and/or progression of human malignancies. However, data concerning the occurrence of cell-cycle aberrations in soft tissue sarcomas are very limited. OBJECTIVES: To detect the abnormal features of cell-cycle regulatory proteins in soft tissue sarcomas and to determine the potential role of these proteins in clinical behavior. DESIGN: The p53 and Rb-cyclin D pathways were investigated by immunohistochemical studies of p53, mdm2, pRb, p16, cyclin D1, and cdk4 proteins, respectively. RESULTS: Of the 67 sarcomas analyzed, nuclear accumulation of p53 was detected in 25 samples (37%), and overexpression of mdm2 was found in 16 samples (24%). Both p53 and mdm2 expression correlated with tumor grade. Abnormalities involving the Rb-cyclin D pathway were identified in all of the tumors by the altered expression of either pRb (72%) or p16 (94%). Fourteen (21%) and 64 (96%) cases demonstrated cyclin D1 or cdk4 expression, respectively. Overexpression of cyclin D1 showed an association with pRb and p53. There was no correlation between pRb, p16, cyclin D1, or cdk4 and tumor grade or relapse. CONCLUSION: Disturbance in the cell-cycle regulatory system involving the p53 pathway and the Rb-cyclin D pathway is relatively frequent in soft tissue sarcomas and may be a contributing factor in the tumorigenesis of these tumors. The alterations in the Rb-cyclin D pathway probably constitute an early event, whereas the abnormalities in the p53 pathway seem to be involved in tumor progression. It is noteworthy that cyclin D1 may play a key role in linking both pathways.     

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。