Functional consequences of cyclin D1 overexpression in human mammary luminal epithelial cells

The proliferation of eukaryotic cells is primarily regulated by a decision made during the G1 phase of the cell cycle as to remain in the cycle and divide, or to withdraw from the cycle and adopt a different cell fate. During this time, environmental signals, which regulate the synthesis of the G1 cyclins, are coupled to cell division. In this context, mammalian D-type cyclins have been shown to control progression through the G1 phase of the mammalian cell cycle. Specifically, cyclin D1 has been reported frequently to be amplified, over-tran-scribed and overexpressed in human breast carcinomas. Although the effects of cyclin D1 overexpression have been examined in human breast carcinoma cell lines, the biological consequences of cyclin D1 expression in normal human mammary epithelial cells remain to be elucidated. In this study we have stably over expressed cyclin D1 in human mammary luminal epithelial cells in order to more directly address the role of cyclin D1 in cell cycle control and tumorigenesis of the human breast. Here, we demonstrate that the effect of cyclin D1 overexpression in these cells is to reduce their growth factor dependency, as well as shorten the duration of G1 and correspondingly reduce the mean generation time. Collectively, our data indicate that deregula-tion of cyclin D1 expression in human mammary epithelial cells can provide a growth advantage and hence contribute to the oncogenic potential of these cells. © Rapid Science Ltd.     

Expressions of cyclin E, A, and B1 in Hodgkin and Reed–Sternberg cells: Not suppressed by cyclin-dependent kinase inhibitor p21 expression

p21 Is involved in the control of the mammalian cell cycle through the binding and inhibition of cyclin-dependent kinases. The cyclins are dependent on the phases of the cell cycle, and divided into two classes: mitotic cyclins (A, B1, B2) and G1 cyclins (C, D1, D2, D3, E). The product of the p21 gene is a potent downstream effector of the p53 tumor-suppressor gene function. The Hodgkin and Reed- Sternberg (H & RS) cells in Hodgkin's disease are reported to frequently express p53, p21, and nuclear proliferative activity (Ki-67). To clarify the relationship of p21, p53 and cyclins, we performed the immunohistochemistry of p53, p21, Ki-67, cyclin D1, cyclin E, cyclin A and cyclin B1, using 11 cases with Hodgkin's disease. In addition, we performed p53 gene sequencing of exon 5-8, and in situ hybridization of Epstein-Barr virus (EBV) EBER-1 region, whose products have reported to induce the expression of cyclin D. In this study, in all cases, Ki-67 was expressed in almost all H & RS cells, and p53 and p21 were expressed in H & RS cells. No p53 gene mutations were detected in any case, and p53 protein overexpression did not correlate with p53 gene mutations. The number of p21-positive H & RS cells was significantly related with that of the p53-positive cells. The cyclins E, A, B1 and D1 were also expressed in H & RS cells. Unexpectedly, the expression of the cyclins was not suppressed by p21 and p53 expression. In addition, the existence of EBV was not related to the expression of cyclins. It is considered that H & RS cells are, indeed, in cell cycle and commonly express the cell cyclins, and that the cell cycle of H & RS cells may not be specifically fixed in the G1, S, G2 or M phases.     

Cell cycle-related variation and tissue-restricted expression of human cyclin D1 protein

Recent evidence from genetic studies suggests that abnormalities of some of the members of the cyclin superfamily may be intimately associated with tumourigenesis, most likely through deregulation of the cell cycle control. In an attempt to elucidate the potential role of cyclin D1 (a gene located within the 11q13 amplicon and a candidate BCL-1, PRAD-1 oncogene) in the pathogenesis of human neoplasias, we have developed and characterized a novel monoclonal antibody specifically recognizing cyclin D1 protein in various assays including immunohistochemistry on frozen and paraffin sections. Using the DCS-6 antibody as a tool, we now show a characteristic cell cycle-dependent variation of the cyclin D1 protein in human cultured cells and report on the first immunohistochemical study of this G1 cyclin in a range of normal human tissues and breast carcinomas. Analysis of normal tissues revealed generally low levels of cyclin D1 protein, mainly restricted to the proliferative zones of some epithelial tissues, and the lack of its expression in several human tissues including lymph nodes, spleen, and tonsils. In contrast, pronounced overexpression/nuclear accumulation of cyclin D1 was found in 37 per cent of cases in a series of 35 primary ductal carcinomas of the breast. We conclude that the DCS-6 antibody provides a potentially useful tool for the establishment of simple methods suitable for verifying any diagnostic and/or prognostic value of this novel marker on large series of histological specimens and opens the way for biochemical, immunocytochemical, and immunohistochemical studies of the role played by cyclin D1 aberrations in human oncogenesis.     

Cyclin D1 expression in pre-cancerous and cancerous lesions of the canine mammary gland

Overexpression of cyclin D1, the regulatory subunit of cyclin-dependent kinases (cdk4 and cdk6) involved in cell cycle control, has often been found in breast cancer and other types of human cancer. Increased expression, or stability, of cyclin D1 molecules may cause sufficient cdk4 activation to produce retinoblastoma protein phosphorylation independently of mitogenic signals; this results in commitment of cells to the G1 phase at mitosis. In the present study, cyclin D1 expression was investigated in pre-cancerous and cancerous lesions of the canine mammary gland by a complex experimental approach, which included Western blot and immunohistochemical analysis of cyclin D1 and the related molecular system. Furthermore, to define relationships between cell growth and expression of cyclin D1, proliferative activity was studied by the AgNOR technique. The study provided the following information. Cyclin D1 overexpression was largely independent of the type of proliferative anomaly. Indeed, cyclin D1 was expressed in 60% of the pre-cancerous lesions and in 44% of cancerous lesions. Mitotic activity and cyclin D1 expression were related: mammary lesions that expressed cyclin D1 showed a high proliferative ratio, the opposite being true of cyclin D1-negative cell populations. This study may contribute to the establishment of an animal model for anti-cancer research based on cyclin D1 suppression or cdk inactivation, or both.     

Mammary Epithelial Cell-Cycle Progression via the α2β1 Integrin : Unique and Synergistic Roles of the α2 Cytoplasmic Domain

The alpha(2)beta(1) integrin supports cell-cycle progression of mammary epithelial cells adherent to type I collagen matrices. Integrin collagen receptors containing the alpha(2) cytoplasmic domain stimulated expression of cyclin E and cyclin-dependent kinase (cdk)2, resulting in cyclin E/cdk2 activation in the absence of growth factors other than insulin. Integrin collagen receptors in which the alpha(2) cytoplasmic domain was replaced by the alpha(1) cytoplasmic domain or an alpha(2) subunit cytoplasmic domain truncated after the GFFKR sequence failed to stimulate cyclin E/cdk2 activation or entry into S phase in the absence of growth factors. Although overexpression of cyclins D or E or cdk2 in cells expressing the integrin collagen receptor with the alpha(1)-integrin cytoplasmic domain did not restore G(1) progression when mammary epithelial cells adhered to type I collagen, co-expression of cyclin E and cdk2 did rescue the ability of the transfectants to enter S phase. Activation of cyclin E/cdk2 complex by mammary epithelial cells required synergy between adhesion mediated by an integrin collagen receptor containing the alpha(2)-integrin subunit cytoplasmic domain and the insulin receptor.     

Cyclin D2 is essential for BCR-mediated proliferation and CD5 B cell development

Progression into G(1) in B lymphocytes is regulated by cyclins D2 and D3, components of the cell cycle machinery currently believed to have overlapping and potentially redundant roles in cell cycle control. To study the specific role of cyclin D2 in B lymphocyte proliferation, we examined B cells from cyclin D2(-/-) mice and demonstrate a specific requirement for cyclin D2 in BCR- but not CD40- or lipopolysaccharide-induced proliferation. Furthermore, conventional B cell development proceeds normally in the mutant mice; however, the CD5 B cell compartment is dramatically reduced, suggesting that cyclin D2 is important in CD5 B cell development as well as antigen-dependent B cell clonal expansion.     

Cyclin alterations in giant cell tumor of bone.

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in the cell cycle. Because alterations of several cyclins, especially cyclin D1, have been implicated in the development of many human neoplasms, we examined 32 cases of giant cell tumor of long bones for cyclin D1 gene amplification and protein overexpression using differential polymerase chain reaction and immunohistochemistry, respectively. In addition, the expression of cyclin D3, cyclin B1, and the proliferation-associated antigen Ki-67 (MIB-1) was assessed immunohistochemically. Low-level cyclin D1 gene amplification was detected in 61% of giant cell tumor cases. All tumors showed cyclin D1, cyclin D3, cyclin B1, and Ki-67 (MIB-1) staining; however, the distribution was very characteristic. Cyclin D1 protein expression was seen predominantly in the nuclei of the giant cells, with occasional mononuclear cells staining. There was no correlation between cyclin D1 gene amplification and protein overexpression. Cyclin D3 staining showed a similar distribution, with 88% of cases showing protein overexpression. Cyclin D1 and/or D3 staining in the giant cells was never associated with staining for either cyclin B1 or Ki-67 (MIB-1), as the expression of the latter two proteins was restricted to the mononuclear cells. Cyclin B1 overexpression was seen in 44% of cases. Ki-67 (MIB-1) staining was present in all cases, and between 10 to 50% of the mononuclear cells were positive. These results suggest that alterations in cyclin D1 and/or D3 might play a role in the pathogenesis of giant cell tumor of bone.     

D-type cyclins in adult human testis and testicular cancer: relation to cell type,proliferation, differentiation,and malignancy

D-type cyclins are proto-oncogenic components of the ‘RB pathway’, a G1/S regulatory mechanism centred around the retinoblastoma tumour suppressor (pRB) implicated in key cellular decisions that control cell proliferation, cell-cycle arrest, quiescence, and differentiation. This study focused on immunohistochemical and immunochemical analysis of human adult testis and 32 testicular tumours to examine the differential expression and abundance of cyclins D1, D2, and D3 in relation to cell type, proliferation, differentiation, and malignancy. In normal testis, the cell type-restricted expression patterns were dominated by high levels of cyclin D3 in quiescent Leydig cells and the lack of any D-type cyclin in the germ cells, the latter possibly representing the only example of normal mammalian cells proliferating in the absence of these cyclins. Most carcinoma-in-situ lesions appeared to gain expression of cyclin D2 but not D1 or D3, while the invasive testicular tumours showed variable positivity for cyclins D2 and D3, but rarely D1. An unexpected correlation with differentiation rather than proliferation was found particularly for cyclin D3 in teratomas, a conceptually significant observation confirmed by massive up-regulation of cyclin D3 in the human teratocarcinoma cell line NTera2/D1 induced to differentiate along the neuronal lineage. These results suggest a possible involvement of cyclin D2 in the early stages of testicular oncogenesis and the striking examples of proliferation-independent expression point to potential dual or multiple roles of the D-type cyclins, particularly of cyclin D3. These findings extend current concepts of the biology of the cyclin D subfamily, as well as of the biology and oncopathology of the human adult testis. Apart from practical implications for the assessment of proliferation and oncogenic aberrations in human tissues and tumours, this study may inspire further research into the emerging role of the cyclin D proteins in the establishment and/or maintenance of the differentiated phenotypes. Copyright © 1999 John Wiley & Sons, Ltd.     

MCPIP1诱导乳腺癌细胞系MDA-MB-231细胞周期停滞

目的研究单核细胞趋化蛋白诱导蛋白1(MCPIP1)在乳腺癌细胞MDA-MB-231中的作用及机制。方法用脂质体转染法转染细胞,在MDA-MB-231中分别过表达,或敲低MCPIP1并筛选稳定表达shRNA的单克隆;MTT法检测细胞增殖;流式细胞计量技术检测细胞周期;实时荧光定量PCR(q-PCR)检测靶基因半衰期;RNA免疫沉淀法(RNA-IP)检测MCPIP1结合的靶基因;荧光素酶报告基因实验检测调节基因3'非编码区(3'UTR)的能力。结果过表达MCPIP1可显著抑制MDA-MB-231细胞增殖(P0.05),敲低MCPIP1显著促进细胞增殖(P0.05);并分别显著增加或降低G1期细胞百分比(P0.01);MCPIP1显著降低细胞周期素依赖性激酶2(CDK2)、细胞周期素依赖性激酶6(CDK6)、cyclin D1和cyclin E1 mRNAs的半衰期(P0.01);MCPIP1结合细胞周期相关基因(P0.05);MCPIP1显著降低含不同3'UTR的报告基因荧光素酶活性(P0.05)。结论MCPIP1在乳腺癌细胞MDA-MB-231中发挥抑癌作用,诱导细胞周期G_1期停滞可能是其发挥抑癌功能的机制之一。     

Role of CDC25 phosphatases in the control of proliferation

Progression in cell cycle is controlled by CDKs (cyclin dependent kinases) and their cyclins regulatory subunits. In mammalian cells, three dual specificity phosphatases called CDC25 activate CDKs/cyclin complexes. The activity of CDC25 is regulated by phosphorylation and dephosphorylation events. CDC25 phosphatases also participate in cell cycle checkpoints activated in response to DNA damage. Two members of this family, CDC25 A and CDC25 B, have oncogenic properties, and their overexpression has been detected in various types of tumors.     

布托啡诺调控PBX3基因抑制乳腺癌细胞系MCF7增殖、迁移和侵袭

目的探讨布托啡诺(butorphanol)对乳腺癌细胞增殖、迁移和侵袭的影响以及相关的分子机制。方法用MTT法检测不同浓度布托啡诺对人乳腺癌细胞系MCF7的抑制作用;用Transwell迁移及侵袭实验检测不同浓度布托啡诺对人乳腺癌MCF7细胞迁移及侵袭的影响;RT-qCR与Western blot法分别检测乳腺癌细胞系、正常乳腺上皮细胞以及布托啡诺对MCF7细胞中PBX3 mRNA及蛋白表达的影响;观察转染si-PBX3或si-con后,MCF7细胞增殖、迁移及侵袭能力的变化;PBX3过表达验证布托啡诺对乳腺癌增殖、迁移及侵袭的作用机制。Western blot检测cyclin D1和MMP-2蛋白表达。结果PBX3在乳腺癌细胞系中的表达上调,沉默PBX3表达可明显抑制MCF7细胞增殖、迁移及侵袭,同时抑制cyclin D1和MMP-2的表达;不同浓度的布托啡诺干预能显著抑制MCF7细胞增殖、迁移及侵袭且具有浓度依赖性,还可抑制PBX3、cyclin D1和MMP-2的表达;过表达PBX3可逆转布托啡诺对乳腺癌细胞增殖、迁移及侵袭的抑制作用。结论布托啡诺可通过抑制PBX3降低乳腺癌细胞增殖、迁移及侵袭能力。     

p21Cip1基因对V79-8细胞周期的影响

为了探讨缺乏可测出Ｇ１，Ｇ２期Ｖ７９－８细胞株的细胞周期失调的分子机理，构建了人的周期负调控基因ｐ２１Ｃｉｐ１的可表达重组质粒ｐＸＪＳＣ，转染该细胞系，建立了高表达ｐ２１的Ｖ７９８－ＳＣ细胞以＾３ＨＴｄＲ放射为影法，流式细胞光度术，细胞增殖曲线测定及免疫印迹技术，检测及比较了与细胞周期变化和增殖有关基因的表达，发现该细胞对照细胞Ｖ７９－８－ｎｅｏＧ１期明显延长，Ｇ２期也稍有增加。     

Cyclin D2 overexpression in transgenic mice induces thymic and epidermal hyperplasia whereas cyclin D3 expression results only in epidermal hyperplasia

In a previous report, we described the effects of cyclin D1 expression in epithelial tissues of transgenic mice. To study the involvement of D-type cyclins (D1, D2, and D3) in epithelial growth and differentiation and their putative role as oncogenes in skin, transgenic mice were developed which carry cyclin D2 or D3 genes driven by a keratin 5 promoter. As expected, both transgenic lines showed expression of these proteins in most of the squamous tissues analyzed. Epidermal proliferation increased in transgenic animals and basal cell hyperplasia was observed. All of the animals also had a minor thickening of the epidermis. The pattern of expression of keratin 1 and keratin 5 indicated that epidermal differentiation was not affected. Transgenic K5D2 mice developed mild thymic hyperplasia that reversed at 4 months of age. On the other hand, high expression of cyclin D3 in the thymus did not produce hyperplasia. This model provides in vivo evidence of the action of cyclin D2 and cyclin D3 as mediators of proliferation in squamous epithelial cells. A direct comparison among the three D-type cyclin transgenic mice suggests that cyclin D1 and cyclin D2 have similar roles in epithelial thymus cells. However, overexpression of each D-type cyclin produces a distinct phenotype in thymic epithelial cells.     

Expression and gene amplification of primary (A, B1, D1, D3, and E) and secondary (C and H) cyclins in colon adenocarcinomas and correlation with patient outcome

BACKGROUND/AIMS: Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3, and E) are crucial for cell cycle progression. Secondary cyclins (C and H) have putative indirect effects on cell cycle progression and have not previously been evaluated in colon cancer. This study examined cyclin protein expression and gene amplification in colon adenocarcinoma and the correlation with patient outcome. METHODS: Immunohistochemistry and real time quantitative polymerase chain reaction were used to determine cyclin expression and gene amplification in 219 tumours. The results were compared with clinical variables and patient outcomes. RESULTS: Cyclin H was overexpressed in all tumours, cyclin C in 88%, cyclin B1 in 58%, cyclin A in 83%, cyclin D3 in 36%, cyclin E in 25%, and cyclin D1 in 11% of the tumours. Extra gene copies of cyclin A were seen in 6.2% of the tumours, cyclin B1 in 9%, cyclin C in 26.9%, cyclin D1 in 55%, cyclin D3 in 20.5%, cyclin E in 19.1%, and cyclin H in 5.1%. A significant correlation between protein overexpression and gene amplification was seen for cyclin C only. High expression of cyclin A was independently associated with improved survival. Amplification of cyclin C was independently associated with an unfavourable prognosis. CONCLUSIONS: Amplification of the cyclin C gene was related to an unfavourable prognosis and high protein expression of cyclin A was associated with a better outcome in colon adenocarcinoma.     

Expression of cyclin D1 and cyclin E in human gastric carcinoma and its clinicopathologic significance.

Cyclin D1 and cyclin E are the mammalian G1 cydins that are both required and rate limiting for entry into S phase. Alterations in cell cycle regulators and subsequent deregulation of the cell cycle are frequently involved in tumorigenesis and/or tumor progression. We investigated the expression of cyclin D1 and cyclin E protein in 84 gastric carcinoma by immunohistochemical staining and also the relevance of each cyclin expression to the clinical outcomes. Overexpression of cyclin D1 and cyclin E was noted in 21 of 84 (25.0%) and 34 of 84 (40.5%) gastric cancer tissues, respectively. There was a significant correlation between overexpression of cyclin E and lymph node metastasis (p=0.003), recurrence (p=0.043), disease free survival (p=0.0378) and overall survival (p=0.0319), but no correlation was noted between overexpression of cyclin D1 and other clinicopathologic variables. These findings suggest that overexpression of cyclin E and cyclin D1 is a frequent finding in gastric cancer and immunohistochemical analysis for cell cycle regulators, especially cyclin E might be a useful prognostic indicator in gastric cancer.     

Living with or without cyclins and cyclin-dependent kinases

Entry into, progression through, and exit from the G1 phase of the mammalian cell cycle in response to extracellular mitogenic cues are presumed to be governed by cyclin-dependent kinases (Cdks) regulated by the D-type and E-type cyclins. Studies performed over more than a decade have supported the view that these holoenzymes are important, if not required, for these processes. However, recent experiments in which the genes encoding all three D-type cyclins, the two E-type cyclins, cyclin D-dependent Cdk4 and Cdk6, or cyclin E-dependent Cdk2 have been disrupted in the mouse germ line have revealed that much of fetal development occurs normally in their absence. Thus, none of these genes is strictly essential for cell cycle progression. To what extent is the prevailing dogma incorrect, and how can the recent findings be reconciled with past work?     

mTOR激酶抑制剂CC-223抑制乳腺癌细胞增殖的实验研究

目的:探讨哺乳动物雷帕霉素靶蛋白(mTOR)激酶抑制剂CC-223对乳腺癌细胞增殖的抑制作用及其相关分子机制。方法:CCK-8法检测CC-223对人乳腺癌细胞MCF-7和MDA-MB-231细胞活力的抑制作用;流式细胞术分析CC-223对乳腺癌细胞周期的影响;Western blot实验检测CC-223对细胞周期调控相关蛋白及细胞增殖相关蛋白c-Myc和存活蛋白(survivin)表达的影响。结果:CCK-8结果表明,CC-223能够显著抑制MCF-7细胞和MDA-MB-231细胞活力(P<0.05);流式细胞术实验结果显示,CC-223能够诱导MCF-7细胞发生G 1期和G 2/M期阻滞(P<0.05);较低浓度的CC-223诱导MDA-MB-231细胞周期阻滞于G 2/M期(P<0.05),而处于G 1期的细胞数量无显著差异。CC-223处理乳腺癌细胞24 h后,细胞周期蛋白B1、细胞周期蛋白D1表达和细胞分裂周期蛋白2(Cdc2)磷酸化水平显著降低(P<0.05)。Western blot结果表明,MCF-7和MDA-MB-231细胞经CC-223作用后,c-Myc和survivin的表达水平显著下调(P<0.05)。结论:CC-223能够抑制乳腺癌细胞活力,阻滞细胞周期进程,同时下调乳腺癌细胞中c-Myc与survivin的蛋白表达。