Expression And Its Significance Of TSG101, P21～(WAF1/CIPI) And P300/CBP In Squamous Cell Carcinoma And Adenocarcinoma Of Lung

Expression and its Significance of TSG101, P21^WAF1/CIPI and P300/CBP in Squamous Cell Carcinoma and Adenocarcinoma of LungIntroductionTumor susceptibility gene 101(TSG101) was identified in a random mutagenesis screen for potential tumor suppressors in NIH 3T3 cells. Human tumor susceptibility gene101 has been mapped to chromosome 11p15.1-15.2, the whole length of cDNA is 1494bp, including six exons and five introns, which encodes .the product with 380 amino acids and 43KD molecular weigh. Stathmin can interact with TSG101'sαcoiled-coil sequence and make it phosphorylation, TSG101 may carry out transcriptional control by phosphorylation. In addition, TSG101 participates in protein degradation mediated by ubiquitin,acting as UBC dominant negative mutant. It has been shown that functional inactivation of TSG101 in human NIH3T3 fibroblasts leads to cellular transformation and the transformed cells can form metastasis, Part of cells'malignant transformation and tumourigenesis aren't reversible by restoration of TSG101, it indicates that inactivation of TSG101 can increase genomic instability and facilitate the development of tumor.Recently, there have been some reports about TSG101 in medullocell leukaemia,lymphoma,prostatic carcinoma,cancer of the cervix, in spite of different findings, it is certain that TSG101 plays an important role in oncogenesis. TSG101 protein binds to the cyclin-dependent kinase(CDK) inhibitor P21^WAF1/CIPI. The increased TSG101's expression dosen't affect stability of the P21^WAF1/CIPI protein in HEK293F cells and differentiating primary keratinocytes, but shows P21^WAF1/CIPI -dependent .Some findings show TSG101 interacts with hormone receptor reactivator P300/CBP both in vivo and vitro, which affects tumor's development in prostate, breast and other tissues. Now there have been few reports about the expression of TSG101 in lung cancer tissues and the relationship with P21^WAF1/CIPI and P300/CBP. So we investigate the expression of TSG101, P21^WAF1/CIPI P300/CBP and the relationship of their expression in squamous cell carcinoma and adenocarcinoma of lung in order to evaluate their possible roles in the oncogenesis and progression of lung cancer.Materials and Methods1. Samples79 lung cancer tissues samples (48 lung squamous carcinoma and 31 lung adenocarcinomas). 48 lung cancer tissue samples among those were obtained from patients who had surgery in the LiaoNing Province Tumor Hospital between 1980 and 2001 .The other 26 fresh lung cancer tissue samples and 26 fresh non-cancerous tissues were obtained from the First Affiliated Hospital of China Medical University from May to July, 2006.2. ReagentsThe anti-human TSG101 mouse monoclonal antibody (sc-7964) was provided by Santa Cruz company (USA). The goat-anti-mouse second antibody (ZB-2305) and DAB agent kit (ZLI-9302) were provided by Beijing zhong shan golden bridge biotechnology co. The anti-human P21^WAF1/CIPI mouse monoclonal antibody fluid (MAB-0235) and the Ultra-sensitive S-P kit (SP-900/9001/9002) were provided by Fujian maixin biotechnology co. Protein marker (#SM0441) was provided by MBI Co. The anti-human P300/CBP mouse monoclonal antibody (cloneNM11)was provided by Neomarkers co.3. Methods(1) ImmunohistochemistryDetect the TSG101,P21^WAF1/CIPI and P300/CBP expression by Immunohistochemistry S-P method. Result determination: The Buffy fine particle appear in intra-cellular. TSG101 Protein is located at alveolar epithelial cell cytoplasm and/or nucleus, P21^WAF1/CIPI protein is located at cancer cell nucleus and tunica mucosa bronchiorum nucleus, P300/CBP is located at cancer cell nucleus and tunica mucosa bronchiorum nucleus, some appear in nucleus- cytoplasm type. Referring to Yao guang yu's score standard, we observe five field of vision each slice in 400 magnification and count 100 cancer cells each field of vision, then we add up to 500 cancer cells.(A) The number of cells stained was graded as follows:≤10%(1), 11%～50%(2), 51%～75%(3),＞75%(4); (B)The staining intensity was scorded as negative (0), weak(1), moderate(2), and strong(3): A×B, according to the score, we divide four grade:: "-"(0,1, 2), "+"(3, 4)"++"(6, 8)"+++"(9, 12).TSG101 staining result determination: scores≥3 is regarded as positive,＜3 is regarded as negative.P21^WAF1/CIPI staining result determination:≥10% that the Buffy fine particle appear in nucleus is regarded as positive.P300/CBP result determination:≥10% that the Buffy fine particle appear in nucleus or cytoplasm is regarded as positive.(2) Western Blot: Detect the expression of TSG101 in lung cancer tissues and noncancerous tissues. Rapidly homogenize tissues(1-2g) in 5 volumes of lysis buffer centriguge the homogenate(10,000rpm, 4℃)for 60 minutes to pellet insoluble material.â‘ Electrophoresis;â‘¡transfer proteins from gel to PVDF membrane;â‘¢blocking;â‘£incubation with primary antibody;⑤enzyme conjugate incubation;â‘¥substrate incubation;⑦DAB staining.4. Statistical analysisAll the datas are analyzed with SPSS for Windows 13.0 software. We useχ² test to analyze the correlation between clinic pathologic parameters of lung cancer and expression of TSG101, P300/CBP and P21^WAF1/CIPI, we use the permutation test for the Spearman correlation coefficient to analyze the correlation between expression of TSG101, P300/CBP and P21^WAF1/CIPI. The result of Western Blot is analyzed by t test. The statistical significance is defined as P＜0.05.Results1. Expression of TSG101 in squamous cell carcinoma of lung, adenocarcinoma of lung and normal tissues.TSG101 is expressed in lung cancer tissues and localized in cytoplasm and/or nucleus.TSG101 is expressed strongly in normal, tissues. Western Blot Method shows the level of TSG101 in 26 tumor tissues is noticeably lower than that in the normal tissues(t=8.578, P=0.000).2. Expression of P21^WAF1/CIPI in squamous cell carcinoma of lung, adenocarcinoma of lung and normal tissues.P21^WAF1/CIPI Protein is located at cancer cell nucleus and tunica mucosa bronchiorum nucleus.3. Expression of P300/CBP in squamous cell carcinoma of lung, adenocarcinoma of lung and normal tissues.P300/CBP is located at cancer cell nucleus and tunica mucosa bronchiorum nucleus, some appear in nucleus- cytoplasm type.4. Clinical significance of TSG101, P21^WAF1/CIPI and P300/CBP.Expression of TSG101, P21^WAF1/CIPI and P300/CBP are found in 47 (59.49%), 48 (60.76%) and 45(56.96%) of 79 lung cancer cases, respectively. Expression of TSG101 is found in 12 (46.15%) of 26 lung cancer cases, which is also analyzed by Western Blot. There is no relationship between TSG101 expression and age, sex, tumor size, histological type and P-TNM stages(P＞0.05); Otherwise, there is closely relationship between TSG101 expression and cellular differentiation, lymph node metastases (P＜0.05). The positive rate of P21^WAF1/CIPI and P300/CBP protein is correlated with the cellular differentiation, lymph node metastases and P-TNM stages (P＜0.05). The expression rate of TSG101, P21^WAF1/CIPI and P300/CBP in well and moderately differentiated cells is significantly higher than that in poorly differentiated cells(P＜0.05, P＜0.01), the expression rate Of TSG101, P21^WAF1/CIPI and P300/CBP in patients without lymphatic metastasis is significantly higher than that in those with lymphatic metastasis (p＜0.01).Additionally, the expression level of TSG101 is positively correlated with P21^WAF1/CIPI (r=0.710) and P300/CBP(r=0.689).DiscussionThere are different opinions about TSG101's role in tumor development. TSG101 is localized to chromosome 11p15.1-15.2, a region which is loss of allei heterozygosity frequently lost in several human tumor types including breast cancer, wilm' s tumor and lung cancer, this region is considered including tumor suppressor. However, Steiner etal proposes contrary opinion. Oh H etal reported that the ability of TSG101 and P21^WAF1/CIPI to suppress differentiation is mediated by specific modulation of gene expression. In fact, both P21^WAF1/CIPI and TSG101 have been implicated in transcription control by interaction with the coactivator P300, and they may be part of same complexe, suppressing tumor cell growth together.In the present study, we find that that TSG101 is expressed in lung cancer, the cytoplasm or nucleus expressing rate is 59.49%, the expression of TSG101 protein in tumor tissues is significantly lower than that in the neighboring noncancerous tissues, as well as Western Blot. There is no relationship between TSG101 expression and age, sex, tumor size, histological type and P-TNM stages (P＞0.05); Otherwise, there is closely relationship between TSG101 expression and cellular differentiation, lymph node metastases (P＜0.05). The expression rate of TSG101 in well and moderately differentiated cells is significantly higher than that in poorly differentiated cells (P＜0.05, P＜0.01 ) , the expression rate of TSG101 in patients without lymphatic metastasis is significantly higher than that in those with lymphatic metastasis (p＜0.01). Yun oh etal reported that TSG101 was not mutated in lung cancer, the wide-type transcript was expressed in normal lung tissues and primary non-small cell lung cancer, some non-small cell lung cancer and small cell lung cancer existed a number of splicing transcripts, the expression of short splicing transcripts in cancer tissues was higher than that in normal tissues and hemacyte, TSG101 did not alter in the process of tumor development, but the spicing of its transcript showed abnormality, the full-length transcripts decrease, so the expression of TSG101 becomes low. Our findings indicate: the low expression of TSG101 in lung cancer may exist more splicing transcripts than that in noncancerous tissues, so expression of TSG101 protein in tumor tissues is significantly lower than that in the neighboring noncancerous tissue, with the tumor grade rising, the expression of TSG101 becomes low. Feng etal reported here that TSG101 protein outside of a narrow range can lead to abnormal cell growth. TSG101 protein is maintained at an almost constant steady-state level in cultured murine and human cells. We suppose that the low expression of TSG101 in lung cancer be dose-dependent, owing to the low expression of TSG101, the ability that prevents tumor development decreases and leads to lung cancer. The findings that between TSG101 aberrant splicing and tumor stage show: there is close relationship between TSG101 aberrant splicing expression and tumor development. Our findings show: there is no relationship between TSG101 expression and P-TNM staged (P＞0.05). Due to the insufficient samples, we should enlarge the samples to confirm.Expression of P21^WAF1/CIPI is found in 48 (60.76%) of 79 lung cancer cases. There is relationship between P21^WAF1/CIPI expression and lung cancer cell differentiation, we find that The expression rate of TSG101 in well and moderately differentiated cells is significantly higher than that in poorly differentiated cells (P＜0.05, P＜0.01 ) , the expression rate of P21^WAF1/CIPI in patients with lymphatic metastasis is significantly lower than that in those without lymphatic metastasis (p＜0.01). As Akitak has reported. We find that P21^WAF1/CIPI expressed in 42 of 48 (87.50%) with expressed TSG101, the expression level of TSG101 is positively correlated with P21^WAF1/CIPI (r=0.710). Hyesun oh eal have reported TSG101 can regulate P21^WAF1/CIP arrested at G1 stage, so the stability of P21^WAF1/CIP protein by TSG101 is accompanied by only limited interference with P21^WAF1/CIP ubiquitination. Our findings show that: TSG101 binds to cyclin- dependent kinase CDK inhibitor P21^WAF1/CIP during the lung cancer development. TSG101 is critical regulating factor in regulating P21^WAF1/CIP protein stability. P21^WAF1/CIPI acts as cell cycle inhibitor interacting with CyclinE/cdk2 complex, implying the function that TSG101 acts as cell cycle inhibitor and potential tumor suppressor, they coordinate with each other in the process of lung cancer development.Expression of P300/CBP is found in 45 (56.96%) of 79 lung cancer cases. With the tumor grade rising, the expression of P300/CBP becomes low, the expression rate of P300/CBP in patients with lymphatic metastasis is significantly lower than that in those without lymphatic metastasis (p＜0.01).â… ,â…¡stage expression of P300/CBP is significantly higher than that ofâ…¢,â…£stage. We find that P300/CBP expressed in 40 of 45(88.89%) with expressed TSG101, the expression level of TSG101 is positively correlated with P300/CBP(r=0.689). We presume that the C-terminal half of TSG101, which binds to P300,repress transcription. TSG101 affects transcriptional activation by nuclear receptors through coil-coiled region in the process of lung cancer development, which prevents tumor cell proliferation and carcinogenesis.In a word, the formation and development of tumor undergo such pathology process including multiple genes, many procedures, multistage, which requires lots of gene.TSG101 expresses lower in lung cancer, the expression level of TSG101 is positively correlated with P300/CBP and P21^WAF1/CIPI. Human pay close attention to the function of TSG101 and its aberrant splicing in tumor tissues and normal tissues. As it is a new candidate tumor suppressor gene, we try to find their changing law and provide important clues to detect and cure malignant tumor in time.Conclusions1. The expression of TSG101 in lung cancer is much lower than that in normal lung tissues, it changes consistently with the level of cell differentiation.2. In lung cancer, expression of TSG101, P21(^WAF1/CIPI) and P300/CBP show a significantly positive correlation.3. TSG101, P21^WAF1/CIPI and P300/CBP is positively correlated to cell differentiation and lymphnode metastasis, P21^WAF1/CIP and P300/CBP is positively correlated to p-TNM stage.