Cyclin B1和survivin在非小细胞肺癌中的表达和意义

Objective: We studied the expression of cyclin B1 and survivin in human non-small cell lung cancer (NSCLC), and the relationship between such expression and clinicopathological features of NSCLC. Methods: One hundred cases of tissue specimen including NSCLC, neighboring noncancerous tissue and normal lung tissue were collected at random. These specimens were detected by immunohistochemical methods. Results: The expression of cyclin B1 and survivin showed significant difference (P < 0.01) between NSCLC tissues, proliferating epithelial cells of bronchioles and small bronchi in neighboring noncancerous tissues, and normal lung tissues. Compared with normal lung tissues, there was an overexpression of cyclin B1 and survivin in NSCLC and an enhancing expression of cyclin B1 and survivin in proliferating epithelial cells of bronchioles and small bronchi in neighboring noncancerous tissues. Significantly positive correlation was found between the overexpression of cyclin B1 and that of survivin in 100 NSCLC cases (P < 0.01). The significantly positive correlation was also found between the enhancing expression of cyclin B1 and that of survivin in proliferating epithelial cells of bronchioles and small bronchi in neighboring noncancerous tissues (P < 0.01). No statistical significance was found between the different histological types, the differentiated degree, lymphatic metastasis and the expression of cyclin B1 and survivin (P > 0.05) in NSCLC. Statistical significance was marked between different clinical stages of NSCLC and the expression of cyclin B1 and survivin (P < 0.05). Conclusion: The overexpression of cyclin B1 and survivin was found in NSCLC. The expression of cyclin B1 and survivin might be up-regulated during an early step of tumorigenesis and during the development of NSCLC. The progression of cell cycle could be efficiently connected with the control of apoptosis by the interrelations between the overexpression of cyclin B1 and that of survivin in NSCLC during the G2/M phase. The overexpression of cyclin B1 and survivin might be used as marker in showing the dividing and proliferating ability, and the inhibiting apoptosis ability (lengthening cell lifespan) of NSCLC. Moreover, the overexpression of cyclin B1 and survivin was associated with the clinic stages of NSCLC.     

Effect of ionizing radiation on cell-cycle progression and cyclin B1 expression in human melanoma cells

In the present study we investigated the effect of gamma-irradiation (2.5 and 10 Gy) on cell-cycle progression of a human melanoma cell line, M14, characterized by a moderate radiosensitivity (SF2 = O.5). Flow cytometric analysis showed a dose-dependent S-phase accumulation, which was detectable 8 hr after treatment with 2 and 5 Gy and was still persistent at 12 hr after 10 Gy exposure. Such a delay in S-phase was paralleled or followed by an accumulation of cells in G2M, which was transient at the lowest radiation doses and still persistent at 72 hr after 10 Gy. Such an accumulation was, at least in part, due to a block in G2-M transition, as demonstrated by mitotic index analysis. Bivariate flow cytometric analysis of DNA content and cyclin B1 expression showed that, following 2 and 5 Gy, the fraction of cyclin B1-expressing cells was superimposable upon that of G2M cells. Conversely, in cells treated with 10 Gy, the fraction of cyclin B1-expressing cells was half the G2M cell fraction. Northern-blot analysis indicated that the radiation-induced decrease in cyclin B1 protein expression was accompanied by a reduced cyclin B mRNA level. On the whole, our results indicate a direct inhibitory effect of 10 Gy irradiation on cyclin B1 expression as a possible cause for the persistent G2 block in irradiated M14 cells.     

Clinical significance of cyclin D1 expression in patients with node-positive breast carcinoma treated with adjuvant therapy

BACKGROUND: Experimental and clinical studies suggest that cyclin D1 is involved in transformation and tumour progression. However, there is little and contradictory data on the clinical significance of cyclin D1 in human invasive breast carcinoma. PATIENTS AND METHODS: We investigated whether the determination of cyclin D1 has prognostic value in a series of 180 patients with node-positive breast carcinoma and treated with adjuvant therapy with a median follow-up exceeding 6 years. We assessed cyclin D1 expression using the CDS-6 monoclonal antibody and a highly sensitive immunohistochemical technique. RESULTS: We found that most of the evaluable tumours (117 of 167; 70.1%) presented nuclear cyclin D1 staining and that its expression was significantly associated with both the hormone receptors (P = 0.009 and P = 0.005 for ER and PgR, respectively). Furthermore, 29 (17%) of 167 tumours had a weak (15 cases) or strong (9 cases) cytoplasmic cyclin D1 staining. In a subgroup of cases we also studied the amplification of the cyclin D1 gene and a moderate agreement between cyclin D1 nuclear overexpression assessed immunohistochemically and the gene amplification was found. In univariate analysis, cyclin D1 nuclear positivity was significantly associated with improved 6-year relapse-free survival (RFS) (P = 0.004), but not with overall survival (OS) (P = 0.12). The results of the Cox multivariate analysis (final model) indicate that cyclin D1 expression (P = 0.0049) as well as the number of involved nodes (P < 0.001) and tumour size (P = 0.036) are significant prognostic indicators for RFS. Only the number of involved nodes retained significance (P < 0.001) for OS in our series. The joint assessment of the variables considered in the final model of the multivariate analyses had a moderate prognostic capability as determined using the Harrell c statistic (c = 0.66 and 0.64 for RFS and OS, respectively). CONCLUSIONS: The patients with node-positive breast cancer who have a higher likelihood of gaining benefit from adjuvant therapy are those with tumours with cyclin D1 nuclear expression, small size and less than 3 metastatic nodes. Further studies are needed to verify the prognostic value of cyclin D1 in relation to different adjuvant treatments and to deepen the biological pathways that regulate its activation/ suppression in human breast carcinoma.     

Enhanced expression of cyclin D1 in senescent human fibroblasts

When human fibroblast, TIG-1, was growth-stimulated with fetal bovine serum, the induction level of cell cycle-dependent genes was generally much lower in senescent cells than in young counterparts. Exceptionally, the expression level of cyclin D1 in senescent cells was constitutively higher than in young cells and further increased after serum stimulation, which was confirmed by Northern and Western blots and immunoprecipitation. This was also true in other human diploid fibroblast lines, TIG-3 and MRC-5. However, cyclin D1-dependent kinase activity was not detected in senescent cells. When sense- or antisense-cyclin D1 cDNA driven by beta-actin promoter was transfected into young TIG-1 cells, the number of appeared colonies from sense-strand transfected cultures was lower than that from antisense-strand-transfected ones. However, clones expressing cyclin D1 at low or undetectable level which were isolated after transfection with antisense-cyclin D1 proliferated up to the same division limit as untransfected and sense-strand transfected cells. Four clones of SV40-transformed TIG-1 expressed cyclin D1 at moderate levels during their extended proliferative lifespan. It appears that, if the extremely overexpressed cyclin D1 could cause an inhibition of cell proliferation at senescent stage, cellular senescence occurs regardless of overexpression of cyclin D1.     

Inhibition of cyclin D expression in human breast carcinoma cells by retinoids in vitro

IPRS is a freely available software system which consists of about 250 library functions in C, and a set of application programs. It is designed to run under UNIX and comes with full source code, system manual pages, and a comprehensive user's and programmer's guide. It is intended for use by researchers in human vision, pattern recognition, image processing, machine vision and machine learning.     

The role of cell cycle regulatory proteins, cyclin D1, cyclin E, and p27 in thyroid carcinogenesis

The cell cycle is controlled in part by cyclin-dependent kinases (CDKs), which are activated by forming complexes with cyclins. CDKs phosphorylate certain substrates to facilitate the proliferating cells through the cell cycle. CDK inhibitors (CDKIs) such as p27 inhibit cyclin-CDK complexes and function as a negative cell cycle regulator. The overexpression of the positive regulators (cyclins) or the underexpression of the negative regulators including p27 has been seen in a variety of neoplasms, but their role and interaction in thyroid carcinogenesis is yet to be established. We studied the expression of cyclins D1 and E, and the CDKI, p27 by immunohistochemistry in 116 cases, including 59 cases of follicular variant of papillary carcinoma (FVPC) and 57 cases of follicular adenoma (FA). The positive staining was divided into four grades: 1+ if less than 10%, 2+ if 11% to 25%, 3+ if 26% to 50%, and 4+ if greater than 50% of the nuclei of tumor cells stained positively. Cyclin D1 expression was seen in 37 (63%) FVPC and 34 (60%) FA. Cyclin E-positive cells were seen in 51 (86%) FVPC and 47 (82%) FA. No significant differences in the grade of cyclins D1 (P = .261) and E (P = .284) staining was seen between FVPC and FA. Of the 59 FVPC, 53 (89%) showed p27-positive cells; of these, 33 were 1+, nine were 2+, seven were 3+ and only four were 4+ positive. Conversely, all 57 FA were p27 positive, 53 were 4+, and four were 3+ positive. This difference in the grade of p27 staining between FVPC and FA was statistically significant (P < .001). This study shows a significant underexpression of p27 in FVPC compared with FA, suggesting that a decrease in p27 expression plays a more important role than overexpression of cyclins D1 and E alone in thyroid carcinogenesis and that p27 immunostaining may be helpful in the diagnosis of FVPC.     

Cloning and sequence analysis of two cDNAs encoding cyclin A and cyclin B in the zebra mussel Dreissena polymorpha

Cyclins are key components in the progression of both mitotic and meiotic cell cycle control. Full-length cDNA clones encoding cyclin A and cyclin B were isolated from a zebra mussel testis cDNA library. The clones contained open reading frames of 419 and 434 amino acids, had similarity to cyclins A and B from other species, but also some unique features in their sequences. Cyclin A and B mRNA was expressed in testis, ovary, gill, mantle, muscle, and eggs, as shown by specific polymerase chain reaction.     

Immunohistochemical detection and gene amplification of cyclin D1 in mammary infiltrating ductal carcinoma

The deregulation of cyclin D1 (BCL-1, PRAD1, CCND1) protein, normally synthesized in the G1 phase of the cell cycle, has been implicated in the pathogenesis of some malignant neoplasms, including invasive mammary carcinomas. We used rabbit polyclonal antibody 19 to detect cyclin D1 in 55 infiltrating ductal carcinomas and compared the findings to six important clinicopathologic parameters and cyclin D1 gene amplification. Nuclear immunoreactivity of variable intensity for cyclin D1 was present in 35% of the neoplasms, whereas immunoreactivity of normal mammary epithelial nuclei was absent. No significant correlations were observed between immunoreactivity and patient age, axillary lymph node status, estrogen receptors, progesterone receptors, histologic grade, or any of its three components. There was a correlation between cyclin D1 immunostaining and tumor size (P = 0.013). Fourteen of 15 tumors 2 cm or less were negative, whereas 7 of 12 neoplasms larger than 4 cm were immunopositive. Fifteen percent of the invasive carcinomas had cyclin D1 gene amplification. Of these eight tumors, six showed cyclin D1 immunoreactivity (P = 0.017). In this study, cyclin D1 was detected immunohistochemically in approximately one-third of infiltrating ductal carcinomas; approximately one-third of these had detectable cyclin D1 gene amplification. These results further implicate cyclin D1 in breast tumorigenesis and are additional evidence for the role of cell cycle regulatory proteins in invasive mammary carcinoma.     

A link between cyclin A expression and adhesion-dependent cell cycle progression

Cell adhesion has an essential role in regulating proliferation during the G1 phase of the cell cycle, and loss of this adhesion requirement is a classic feature of oncogenic transformation. The appearance of cyclin A messenger RNA and protein in late G1 was dependent on cell adhesion in both NRK and NIH 3T3 fibroblasts. In contrast, the expression of Cdc2, Cdk2, cyclin D1, and cyclin E was independent of adhesion in both cell lines. Transfection of NRK cells with a cyclin A complementary DNA resulted in adhesion-independent accumulation of cyclin A protein and cyclin A-associated kinase activity. These transfected cells also entered S phase and complete multiple rounds of cell division in the absence of cell adhesion. Thus, cyclin A is a target of the adhesion-dependent signals that control cell proliferation.     

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.     

Absence of cyclin D1 protein expression in splenic marginal zone lymphoma

We studied the endoscopic removal of tracheobronchial foreign bodies from 13 patients. Eight patients were treated under local anesthesia and 5 under general anesthesia. Complications included mild bleeding during endoscopic treatment of 3 patients. One patient was placed under general anesthesia because the patient was irritable and because hemorrhage made observation difficult. Laryngeal masks were used in 4 cases, and resulted in no complications. The follow-up courses ranged from 4 months to 12 years and were uneventful for all patients. Use of a fiberoptic bronchoscope in combination with laryngeal masks facilitated the management and removal of tracheobronchial foreign bodies by providing a secure airway and by minimizing the effect on the cardiorespiratory system.     

Clinical relevance of cyclin D1 protein overexpression in laryngeal squamous cell carcinoma

PURPOSE: To investigate the prognostic relevance of cyclin D1 gene overexpression in laryngeal squamous cell carcinomas (LSCCs). PATIENTS AND METHODS: The overexpression of cyclin D1 was analyzed in 149 LSCC patients with a median follow-up duration of 60 months using the DCS6 monoclonal antibody; only cases that overexpressed cyclin D1 in more than 5% of neoplastic cells were considered positive. RESULTS: Forty-eight cases (32.2%) were immunoreactive to the DCS6 antibody. Cyclin D1 overexpression was significantly associated with tobacco smoking and alcohol consumption, tumor extension, advanced clinical stage, and the presence of lymph node metastases. Univariate analysis showed that a shorter disease-free and overall survival were significantly associated with supraglottic site, tumor extension, advanced clinical stage, and cyclin D1 overexpression. At multivariate analysis, tumor extension and cyclin D1 overexpression were significantly associated with tumor recurrence, whereas tumor extension, supraglottic site and, at a borderline level of statistical significance, cyclin D1 overexpression, were associated with reduced overall survival. CONCLUSION: The overexpression of cyclin D1 in LSCC is associated with unfavorable clinicopathologic features and represents an independent significant predictor of laryngeal carcinoma prognosis, particularly for disease-free survival. This indicates that cyclin D1 evaluation may be a further useful element for selecting subgroups of patients who should be treated with more aggressive therapies.     

Characterization of the in vitro reconstituted cyclin A or B1-dependent cdk2 and cdc2 kinase activities

Human cyclins A and B1 were assembled with the cdk2 or cdc2 protein to reconstitute their respective kinase activities in vitro. Both cyclins complemented either cdk2 or cdc2, yielding kinase activities that supported the phosphorylation of histone H1. Activation of cdk2-catalyzed H1 kinase activity by cyclin A required a 10-min preincubation of the two components, whereas cdc2 kinase supported phosphate incorporation without a detectable time lag upon the addition of cyclin B1, suggesting a slower association rate of cdk2 with cyclin A compared with cdc2 and cyclin B1. Both cdk2 and cyclin A, as well as cdc2 and cyclin B1, formed stable complexes in the absence of ATP and substrate that could be isolated after glycerol gradient centrifugation. Incubation of the isolated complexes with ATP and histone H1 supported the phosphorylation of the substrate. Cyclin A-activated cdk2 or cdc2 phosphorylated p107, a pRB-related cellular protein, 10 times more effectively than the cyclin B1-complexed kinases. This was most likely due to a direct association of cyclin A with p107 (Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85-87; Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87-90). The reconstituted cdc2-cyclin B1 complex incorporated 4-5-fold more phosphate into the p34 subunit of the three-subunit (p70, p34, and p14) human single-stranded DNA-binding protein (also called RP-A), a DNA replication and DNA repair factor, than cdc2-cyclin A. No detectable phosphorylation of the p34 protein was observed with cdk2 complexed with either cyclin B1 or A. These data indicate that both cyclins as well as the catalytic subunits are important factors in controlling the rate of phosphorylation of a given substrate. The cyclin-activated cdc2 family kinases may target their cellular substrates through cyclin-mediated protein-protein interactions.     

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.     

Expression patterns of cyclin D1 and related proteins regulating G1-S phase transition in uveal melanoma and retinoblastoma

BACKGROUND/AIMS: A checkpoint mechanism in late G1, whose regulation via loss of retinoblastoma protein (pRB) or p16, or overexpression of cyclin D1 or cyclin dependent kinase 4 (CDK4), has been proposed to constitute a common pathway to malignancy. The aims of this study were (a) to compare markers of cell cycle G1-S phase transition in an intraocular tumour with known pRB deficiency (retinoblastoma) and compare it with one with an apparently functional pRB (uveal melanoma); (b) to determine if one of these markers may have a role in the pathogenesis of uveal melanoma; and (c) to determine if there is a difference in cell cycle marker expression following treatment of uveal melanoma and retinoblastoma. METHODS: 90 eyes were enucleated from 89 patients for retinoblastoma (n = 24) or for choroidal or ciliary body melanoma (n = 66). Conventional paraffin sections were assessed for cell type and degree of differentiation. Additional slides were investigated applying standard immunohistochemical methods with antibodies specific for cyclin D1 protein, pRB, p53, p21, p16, BCL-2, and MIB-1. RESULTS: Cyclin D1 protein and pRB were negative in retinoblastoma using the applied antibodies. In contrast, cyclin D1 protein expression was observed in 65% of uveal melanomas; a positive correlation between cyclin D1 cell positivity and tumour cell type, location, growth fraction, as well as with pRB positivity was observed. p53, p21, and p16 could be demonstrated in both tumours. An inverse relation between p53 and p21 expression was demonstrated in most choroidal melanomas and in some retinoblastomas. Apart from a decrease in the growth fractions of the tumours as determined by MIB-1, a significant difference in the expression of G1-S phase transition markers in vital areas of uveal melanoma and retinoblastoma following treatment with radiotherapy and/or chemotherapy was not observed. CONCLUSION: Retinoblastomas and uveal melanomas, two tumours of differing pRB status, differ also in their immunohistochemical pattern for markers of the G1-S phase transition of the cell cycle. The results of the present study support the concept of (a) an autoregulatory loop between pRB and cyclin D1 in tumours with a functional pRB and the disruption of this loop in the presence of pRB mutation, as well as (b) a checkpoint mechanism in late G1, whose regulation via loss of p16 or pRB, or overexpression of cyclin D1 constitutes a common pathway to malignancy. Further, the results raise the possibility of cyclin D1 overexpression having a role in the pathogenesis of uveal melanoma.