应用表达芯片筛选小鼠肝癌淋巴道转移相关基因

目的采用表达芯片比较高、低淋巴道转移力小鼠肝癌腹水型细胞株Hca—F和Hca—P的基因表达谱,以筛选出与肿瘤淋巴道转移相关的基因。方法分别提取Hca—F和Hca—P细胞的总RNA,反转录合成生物素标记的cRNA探针,并与Affy—metrix GeneChip MOE430A（包括22690个转录本,对应于约14500个小鼠已知基因和4371个EST）杂交,检测结果利用生物信息学进行分析。结果Hca—F和Hca—P相比,在14500个已知基因中,有901个（6．2％）表达上调幅度≥2倍;在4371个EST中,有129个（3％）上调幅度≥2倍。公布了差异表达≥8倍的37个基因并根据Gene Ontology（GO）分类和TreeView分析,按照其参与的生物学过程和具有的分子功能进行了功能分类。其中有19个基因参与了组织发生、细胞黏附、信号转导、细胞生长、分化及代谢等的生物学过程,29个基因分别具有转运、移动、蛋白激酶、蛋白结合及受体活性等分子功能,7个基因的生物学功能尚不清楚。结论表达芯片检测与肿瘤淋巴道转移模型相结合,为肿瘤转移研究提供新方法、新思路,一些过量表达的基因将为肿瘤转移机制的研究提供新线索。     

Prognostic role of cyclin D1 in lung cancer. Relationship to proliferating cell nuclear antigen

We developed an immunohistochemical assay specific for cyclin D1 and suitable for formalin-fixed and paraffin-embedded sections, to evaluate cyclin D1 expression in a group of 135 surgically resected lung-cancer patients for the purpose of investigating the prognostic role of this protein in lung cancer. In addition, we compared cyclin D1 expression with the expression of proliferating cell nuclear antigen (PCNA), considered to be a reliable index of the proliferation rate. We found cyclin D1 expressed in more than 60% of the neoplastic cells in 26.5% of our specimens. A total of 24.5% of the specimens showed cyclin D1 expression in a percentage of cells ranging from 30 to 60%; 36.7% of the specimens expressed cyclin D1 in less than 30% of the cells; and 12.2% of the specimens expressed cyclin D1 in less than 1% of the evaluated cells. Western blot analyses confirmed the specificity of this assay by correlating statistically in a highly significant fashion with the immunohistochemical results (P = 0.0003). Furthermore, we found a direct relationship between cyclin D1 and PCNA immunodetection (P = 0.0004), which correlated cyclin D1 overexpression with a higher tumor proliferation rate. When we analyzed our data statistically, cyclin D1 expression was found to be a negative prognostic marker (P < 0.00005) whose expression correlates with a shorter patient survival time.     

High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA microarray screening in renal cell carcinoma

Many genes and signaling pathways are involved in renal cell carcinoma (RCC) development. However, genetic tumor markers have not gained use in RCC diagnostics and prognosis prediction. Identification and evaluation of new molecular parameters are of utmost importance in cancer research and cancer treatment. Here we present a novel approach to rapidly identify clinically relevant molecular changes in cancer. To identify genes with relevance to RCC, a cDNA array analysis was first performed on 5184 cDNA clones on a filter to screen for genes with differential expression between the renal cancer cell line CRL-1933 and normal kidney tissue. There were 89 differentially expressed genes in the cancer cell line, one of them coding for vimentin, a cytoplasmic intermediate filament. In a second step, a renal cancer tissue microarray containing 532 RCC specimen was used to determine vimentin expression by immunohistochemistry. Vimentin expression was seen frequently in clear cell (51%) and papillary RCC (61%), but rarely in chromophobe RCC (4%) and oncocytomas (12%). Furthermore, vimentin expression was significantly associated with poor patient prognosis (P < 0.007) independent of grade and stage. These results obtained from minute arrayed tumor samples match well with previous findings on vimentin expression in renal tumors. It is concluded that the combination of tumor arrays and cDNA arrays is a powerful approach to rapidly identify and further evaluate genes that play a role in tumor biology.     

Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry

Proliferating cell nuclear antigen (PCNA)/cyclin is an intranuclear polypeptide antigen that is found in both normal and transformed proliferating cells. We have recently described two mouse monoclonal antibodies reacting with PCNA. In this report we describe the application of these antibodies to the study of proliferating human cells by indirect immunofluorescence microscopy and by flow cytometry. A fixation/permeation procedure was developed in order to obtain satisfactory binding of monoclonal PCNA-specific antibodies to proliferating cells. This method involved fixation with 1% paraformaldehyde followed by methanol treatment. For the staining of cells in suspension with the IgM type monoclonal antibodies lysolecithin was added to the paraformaldehyde solution to achieve a better permeation by the antibody molecules. This procedure gave a good ratio of specific staining relative to the background staining. It also preserved the shape and normal architecture of the cells as judged by visual microscopic observation and by light scatter measurements using a flow cytometer. Furthermore, this fixation technique permits simultaneous labeling of DNA by propidium iodide and PCNA by monoclonal antibodies. PCNA was detected in various types of normal and transformed proliferating cells by indirect immunofluorescence. Quiescent peripheral blood mononuclear cells were PCNA-negative whereas a fraction of lectin-stimulated lymphocytes became PCNA-positive. Similarly, early passages of fetal skin fibroblasts were PCNA-positive but non-proliferating senescent fibroblasts of later passages were PCNA-negative. The association of PCNA-staining by monoclonal antibodies with cell proliferation was confirmed by flow cytometry. Simultaneous labeling of PCNA and DNA showed that the PCNA signal increased during the G1 phase of the cell cycle, reached its maximum in the S-phase, and declined during the G2/M phase. Using cell sorting we demonstrated that mitotic cells had a very low PCNA signal. Thus, monoclonal PCNA-specific antibodies offer a convenient tool for the detection of human cell proliferation by immunofluorescence microscopy and by flow cytometry.     

Proliferative activity of calcifying odontogenic cysts as evaluated by proliferating cell nuclear antigen labeling index

The calcifying odontogenic cyst (COC) presents with diverse hlstologlcal features; thus, several subclasslfl-cations have been proposed. To evaluate the slgnlficance of the various histological features and subtypes of COC from the perspectlve of proliferative activity, the proliferating cell nuclear antigen (PCNA) labellng index (LI; the percentage of positive nuclei) was assessed immunohistochemlcally in 25 cases of COC (21 benign and four malignant). All of the benign cases were of the cystic variety and further subclas-sified into non-proliferative subtype (NPS; four cases); proliferative subtype (PS; eight cases); and COC associated with odontoma (COCaO, nlne cases). The PCNA U of the mallgnant COC (65.2 ± 5.6) was slgnlflcantly higher than that of the benlgn COC (11.6 ± 9.0; P = 0.002). Non-proliferative subtype (6.8 ± 2.8) showed the lowest PCNA LI and PS (17.2 ± 11.2) the highest of among the three subtypes of benign cystic COC (P = 0.028). In nine cases of COCaO, six showed epithelial lining of the non-proliferative type as NPS and the other three had lining wlth proliferative features as PS. The PCNA LI of the latter COCaO group (14.3 ± 6.6) was significantly higher than that of the former (6.1 ± 4.3; P = 0.05), as Seen between PS and NPS. These results demonstrate that PCNA LI is a possible parameter for differentiating mallgnant COC from benign COC and, whatever the subtypes, the proliferative features In the lining are the main factor influencing the prollferatlng actlvity of COC.     

增殖细胞核抗原 siRNA 序列的设计、合成与筛选

目的：设计并筛选能有效抑制人肝癌SMMC-7721细胞、人结肠癌Caco2细胞、人胃癌SCG-7901细胞增殖的增殖细胞核抗原(PCNA) siRNA。方法:总结以往提出的siRNA 设计原则的基础上，设计4 条PCNA siRNA，体外转录合成PCNA siRNA；并作用于3 种肿瘤细胞，WST-8法检测细胞增殖活性，筛选能有效抑制肿瘤细胞增殖的siRNA序列；RT-PCR检测细胞PCNA mRNA水平；免疫细胞化学方法检测 PCNA 蛋白表达水平。结果:4 条PCNA siRNA中，有3 条序列（No.2、No.4、No.3）的siRNA能有效抑制3 种肿瘤细胞的增殖；其中No.2、No.4作用效果最优，以50 nmol/L浓度作用48 h，对3种肿瘤细胞的增殖抑制率均可达到62％以上，No.2、No.4 PCNA siRNA均能在mRNA和蛋白水平显著下调PCNA的表达。结论:PCNA siRNA能在体外有效抑制SMMC-7721细胞、Caco2细胞、SCG-7901细胞的增殖，其适宜作用浓度为50 nmol/L，适宜作用时间为48 h。     

Effects of components derived from diesel exhaust particles on lung physiology related to antigen

Our previous study has shown that diesel exhaust particles (DEP), main constituents in ambient particulate matters (PM), enhance airway hyperresponsivness in a murine model of allergic asthma (Takano et al., 1998). However, it remains unknown which components in DEP are responsible for the enhancement. The present study investigated the effects of repeated pulmonary exposure to DEP components (extracted organic chemicals in DEP; DEP-OC, carbonaceous nuclei of DEP after extraction; washed DEP) on lung physiology in the presence or absence of antigen. ICR mice were divided into six experimental groups. Vehicle, DEP components, ovalbumin (OVA), or DEP components plus OVA was administered intratrachally for 6 weeks. Twenty-four hr after the last instillation, cholinergic lung reactivity was examined. DEP components alone did not induce any facilitation of lung function as compared to vehicle alone. The values of total respiratory system resistance (R), elastance (E), Newtonian resistance (R(n)), tissue damping (G), and tissue elastance (H) were higher and the value of compliance (C) was lower in the OVA or the DEP component + OVA groups than in the vehicle group. In particular, the hyperreactivity was most prominent in the washed DEP + OVA group. The values in the DEP-OC + OVA group were not significantly different from those in the OVA group. These data suggest that carboneous component in DEP, rather than organic chemical one, can be attributable to the enhancement of lung hyperresponsiveness in allergic asthma.     

Induction of proliferating cell nuclear antigen and Ki-67 expression by cytomegalovirus infection

With the goal of facilitating viral reproduction, cytomegalovirus (CMV) induces changes in the host cell replication machinery. Very little information is available, however, on the effects brought about by CMV on proliferating cell nuclear antigen (PCNA) and Ki-67 expression in infected cells. Fifty-five paraffin-embedded tissue samples (43 gastrointestinal, 10 skin, and 2 kidney biopsies) with both histological and immunohistochemical evidence of CMV infection were investigated for PCNA and Ki-67 expression by the avidin– biotin–peroxidase method. Of the 55 cases studied, 47 were positive for PCNA and 46 for Ki-67. PCNA and Ki-67 immunostaining was more striking in CMV-immunoreactive, inclusion-free cell nuclei, whereas cell nuclei exhibiting well-developed CMV inclusions either showed a weak peripheral signal for both proliferation markers, or were completely negative. Enhanced PCNA and Ki-67 expression appears to be among the changes induced by CMV infection in host cells. Moreover, this induction seems to reach its peak during the earlier phases of CMV infection and abate as the infection proceeds to its inclusion-forming phases, when a sufficiently high viral load would have been attained. © 1998 John Wiley & Sons, Ltd.     

p53 expression in oat and non-oat small cell lung carcinomas: correlations with proliferating cell nuclear antigen.

AIMS--To investigate the immunohistochemical expression of p53 protein in small cell lung carcinoma (SCLC), comparing it with that in non-small cell carcinoma (NSCLC); and to evaluate the correlation between p53 and proliferating cell nuclear antigen (PCNA) expression as well as between p53 and PCNA expression and survival. METHODS--Paraffin wax embedded tissues from 61 cases of primary lung carcinoma were stained for p53 protein and PCNA using the monoclonal antibodies 1801 and PC-10, respectively, in a standard avidin-biotin immunoperoxidase method. RESULTS--Of the 61 lung carcinomas 35 (57%) were positive for p53 (range 1% to 90%). Ninety percent of the non-oat SCLC contained positive cells and the labelling index (LI) was significantly higher than that of the oat SCLC (p < 0.001). SLCC also displayed a higher p53 LI than NSCLC (p < 0.01); no difference was found between squamous carcinoma, adenocarcinoma, and oat cell carcinoma. A p53 LI of greater than 1% tended to be associated with nodal metastases (p < 0.05), and p53 expression in node positive tumours as well as in oat cell carcinomas was indicative of poorer survival (p < 0.01 and p < 0.1, respectively). A p53 Li of greater than 60% was a negative prognostic factor in non-oat SCLC (p < 0.05). PCNA LI was highest in non-oat SCLC and lowest in NSCLC; oat cell carcinomas had a mean LI intermediate between NSCLC and non-oat SCLC (NSCLC v oat cell carcinoma p < 0.05 and oat cell v non-oat cell carcinomas p < 0.01). A PCNA LI was not correlated with nodal metastases or survival, but there was a significant positive correlation between PCNA LI and p53 LI (rs = 0.484, p < 0.001). CONCLUSIONS--p53 and PCNA expression differ substantially among the various types of lung carcinomas. Substantial differences were also found between oat and non-oat types of SCLC, indicating that SCLC is heterogeneous as far as proliferation rate and altered p53 expression is concerned. p53 seems to be of some prognostic value. The relation between PCNA and p53 expression indicates that the PCNA gene is slightly upregulated by p53.     

层粘蛋白及其受体、增殖细胞核抗原与肝细胞癌肿瘤生物学特性的关系

目的:探讨层粘蛋白(LN)、层粘蛋白受体(LN-R)及增殖细胞核抗原(PCNA)与原发性肝细胞癌(HCC)恶性程度的关系.方法:采用SABC法分别检测42例肝细胞癌及癌旁组织中LN、LN-R及PCNA的表达情况.结果:LN、LN-R及PCNA在肝细胞癌中的表达均显著高于癌旁组织(P＜0.05),且与肝细胞癌Edmondson分级呈正相关;浸润型(IHCC)生长、有转移、肿瘤直径＞3cm者LN、LN-R及PCNA的表达高,而膨胀型(EHCC)生长、无转移、肿瘤直径≤3cm者多表达较低;在高增殖指数组LN、LN-R表达高于低增殖指数组(P＜0.05).结论:细胞内LN、LN-R及PCNA可作为评判肝细胞癌恶性程度及临床预后的良好指标.     

Immunohistochemical detection of proliferating cell nuclear antigen in solid human malignancies

Proliferating cell nuclear antigen (PCNA), also known as cyclin, is a cell cycle-related nuclear protein that is maximally elevated in late G1 and S phases of proliferating cells. In this study, PCNA was identified by paraffin-section immunohistochemistry in 42 of 64 solid human malignancies, and in several benign tissues known to contain proliferating cells. The PCNA-positive nuclei were randomly distributed and ranged from less than 1% (in most cases) to more than 20% of the neoplastic cells. In general, PCNA positivity correlated with mitotic activity and tumor grade. Further study is necessary to evaluate PCNA as a marker of cellular proliferation and a potential prognostic marker in human malignancy.     

Immunoreactivity of proliferating cell nuclear antigen compared with bromodeoxyuridine incorporation in normal and neoplastic rat tissue.

Monoclonal antibodies (MoAbs) against proliferating cell nuclear antigen (PCNA) represent a potentially useful tool for cell kinetic analysis of tumours. Because in paraffin-embedded tissue the relationship between PCNA immunoreactivity and tumour cell proliferation is not well characterized, we have compared PCNA positivity as detected by the PC10 MoAb with the bromodeoxyuridine labelling index (BrdUrd-LI) in two different transplantable hormone-dependent rat mammary tumours. Together, these two tumour models (MCR-83 and EMR-86) cover a wide range of S-phase fractions. Evaluating 31 methacarn-fixed tumours, a strong but non-linear relationship (r = 0.98) was obtained. PCNA-positive fractions were invariably higher than corresponding BrdUrd-LIs and also higher than the estimated growth faction: growth fractions as determined by continuous BrdUrd labelling of the tumour and stromal cell population in EMR-86 carcinomas were 12 and 26 per cent lower than PCNA-positive fractions, implying that a certain fraction of non-cycling cells can also express PCNA. A dramatic disturbance in the relationship of PCNA positivity and the BrdUrd-LI was observed in the EMR-86 model after growth arrest induced by hormonal ablation: PCNA immunoreactivity remained detectable for at least 3 days, whereas the BrdUrd-LI decreased almost immediately. In comparison, PCNA immunoreactivity persisted for a much shorter period in small intestinal cells that had stopped DNA replication when moving from the crypt towards the villus. It is concluded that although differences in PCNA expression exist between various tissues, PCNA as detected by the PC10 MoAb may be used in tumours as an operational marker for the growth fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prognostic value of proliferating cell nuclear antigen in gastric carcinoma.

A new monoclonal antibody to proliferating cell nuclear antigen (PCNA), PC10, which can be used on routinely processed tissue, was applied to 93 cases of gastric carcinoma. Significant intra-tumoural variation in staining occurred. In addition to a PCNA index (percentage of positive cells per 1000 tumour cells), a semiquantitative PCNA grading system was devised, based on estimates of less than or more than 50% of positive tumour cells in whole sections. Neither PCNA index nor PCNA grade showed any correlation with established histological variables, tumour stage, or the presence of lymph node metastases. No significant correlation was observed between PCNA index and S + G2M phase fraction measured by flow cytometric analysis. To analyse survival tumours with PCNA indices above and below the median level (41%) were compared. Those with a higher index tended to have a worse prognosis, but when PCNA grade was considered, it was found to have definite independent prognostic value, tumours of low grade surviving better than those of high grade. The ability of semiquantitative PCNA grading to allow for intra-tumoural variation suggests it may have advantages over absolute counting, which is prone to sampling error when tumour heterogeneity is a major factor. The prognostic value of PC10 staining in gastric carcinoma is therefore promising.     

Expression of proliferating cell nuclear antigen in lung cancer: a systematic study and correlation with DNA ploidy

Heterogeneity of expression of the proliferating cell nuclear antigen (PCNA) was assessed immunohistochemically in 156 tissue samples from 33 surgically resected pulmonary carcinomas using the monoclonal antibody 19A2. The DNA content of each of these samples was measured by flow cytometry. Mean PCNA expression was higher in squamous carcinomas than in adenocarcinomas but there was marked intra-tumour variation in PCNA index in almost all cases. Intra-tumour heterogeneity of DNA content was noted in 11 cases. The PCNA index of these cases (34.1) was higher than that of DNA homogeneous cases (19.4). The wide variation in PCNA expression between different samples within a tumour would indicate that systematic sampling and counting will be necessary in future immunohistochemical studies of cell proliferation in tumour material.     

应用肿瘤基因芯片筛选早期肺鳞癌相关基因

目的： 研究人早期肺鳞癌发生相关基因表达谱, 探讨肺鳞癌发生的分子机制。方法:选取人早期肺鳞癌组织以及相应正常组织，提取RNA, 与含480个与肿瘤相关基因的芯片杂交, 结果经SuperArray Image 软件分析后比较两种组织中的差异表达基因。结果:共筛查出差异表达基因192 条，其中表达上调基因127 条, 下调基因65条; 按照基因功能可分为运输载体、代谢相关基因、细胞信号转导分子、细胞骨架、转录调控因子基因。结论:基因芯片可用于早期肺鳞癌相关基因表达谱的筛查，可为明确早期肺鳞癌发生机制提供重要参考。     

P型铜转运ATP酶和增殖细胞核抗原在大、小鼠肝的表达

目的:研究P型铜转运ATP酶(ATP7B)与增殖细胞核抗原(PCNA)在大鼠和小鼠肝组织中的表达.方法:采用免疫组织化学法检测肝中ATP7B与PCNA的表达.结果:ATP7B在大鼠和小鼠的肝细胞质表达,表达较广泛.肝细胞PCNA的表达强弱不一,强阳性细胞较少,大鼠和小鼠肝的ATP7B和PCNA积分光密度间的差异均无统计学意义;在大鼠或小鼠肝,ATP7B与PCNA的免疫反应阳性物积分光密度间无显著相关性.结论:ATP7B和PCNA在大鼠和小鼠肝组织均有表达,表达的关联性不明显.     

Proliferation in malignant mesothelioma as determined by mitosis counts and immunoreactivity for proliferating cell nuclear antigen (PCNA)

In order to assess its discriminating and prognostic value, we studied immunoreactivity for proliferating nuclear cell antigen (PCNA) in human malignant mesothelioma (31 cases) and in human non-neoplastic mesothelium (33 cases with reactive mesothelium and 20 cases of normal mesothellum) using the murine monoclonal antibody PC 10. We also compared it with mitosis counts expressed as the mitotic volume index (MV index). There were differences between malignant mesothelioma, reactive mesothelium, and normal mesothelium for percentage of PCNA immunoreactive cells (mean ± SD; 27 ± 9, 9·5 ± 5·1, and 3·6 ± 1·6, respectively) and for their MV index (20·3 ± 4·5, 9·4 ± 2·1, and 3·6 ± 0·6, respectively). The median actuarial survival was 10·1 months for patients with less than 25 per cent PCNA immunoreactive cells, 9·4 months for patients with less than 20 mitoses per mm² of tumoural tissue, 5·9 months for patients with more than 25 per cent PCNA immunoreactive cells, and 5·3 months for patients with more than 20 mitoses per mm² of tumoural tissue. Our results suggest that PCNA immunoreactivity is useful in discriminating between neoplastic and non-neoplastic mesothelium and that it may have prognostic value in malignant mesothelioma.     

Human gene for proliferating cell nuclear antigen has pseudogenes and localizes to chromosome 20

We have isolated from a human genomic library a pseudogene of the proliferating cell nuclear antigen (PCNA)gene. Its sequence shows a 78% similarity with the human PCNA/cDNA. The PCNAgene is located on human chromosome 20, while the pseudogene maps to chromosome region XpterXq13. An additional locus detected by the full-length PCNA cDNA, but not by intron probes, segregates concordantly with chromosome region 6p126pter and probably represents a second pseudogene.