Expression Of TSG101 And Its Correlation With MDM2 In Squamous Cell Carcinoma And Adenocarcinoma Of Lung

IntroductionTumor 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, TSG101 protein contains N termination like ubiquitin conjugase,a proline-rich domain,C-termination coiled-coil domain(231-302 amino acids site) and a steady box SBwhich regulates its steady state. TSG101 protein contains N- termination like ubiquitin conjugase but it is short of cysteine which has an activity site. The finding has shown that TSG101 is a transcription inhibition factor of ubiquitin conjugase, TSG101 protein that contains Ctermination coiled-coil domain may act as transcription inhibition factor regulating transcription activation. Acting as transcription inhibition factor, TSG101'S target gene is MDM2. TSG101 affects MDM2 catalytic domain of ubiquitin conjugase by UBC domain, it forms a independent ubiquitin feedback control loop to affect MDM2's stability. TSG101 participates in protein degradation Mediated by MDM2, which indicates TSG101participates in the lung cancer carcinogenesis. So we investigate the expression of TSG101 and MDM2 by IHC and Westen Blot method 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. Samples76 lung cancer tissue 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 1980-2001. The other 26 fresh lung cancer tissue samples and 26 fresh non-cancerous tissue were obtained from the First Affiliated Hospital of China Medical University between May to July, 2006.2. ReagentThe anti-human TSG101 mouse monoclonal antibody (sc-7964) and the anti-human MDM2 (sc-965) were 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 Ultro-sensitive S-P kit (SP-900/9001/9002) were provided by Fujian mai xin biotechnology co.(1) Immunohistochemistry: Detect the TSG101and MDM2 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, MDM2 is located in nucleus and/or cytoplasm. 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.MDM2 staining result determination:≧10％That the Buffy fine particle appear in ??cytoplasm is regarded as positive.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.①Electrophoresis;②transfer proteins from gel to PVDF membrane;③blocking;④incubation with primary antibody;⑤enzyme conjugateincubation;⑥substrate incubation;⑦DAB staining.3. Statistical analysisAll the data are analyzed with SPSS for Windows 13.0 software.We useχ~2 test to analyze the correlation between clinicopathologic parameters of lung cancer and expression of TSG101 and MDM2, we use the permutation test for the Spearman correlation coefficient to analyze the correlation between expression of TSG101 and MDM2.The result of Western Blot is analyzed by t test. A P value of less than 0.05 is accepted as statistically significant.Result1. 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, it is expressed strongly in normal tissues. TSG101 is not expressed in poorly differentiated lung cancer or is expressed weakly in cancer cell's cytoplasm.MDM2 is not expressed in normal lung tissue, it is expressed in well-differentiated squamous cell carcinoma of lung and adenocarcinoma of lung.MDM2 is expressed strongly in poorly differentiated lung cancer. MDM2 is located in nucleus and/or cytoplasm, the Buffy fine particle appear in intra-cellular.2. Clinical significance of TSG101There was no relationship between TSG101 expression and age, sex, tumor size, histological type and P-TNM stages(P＞0.05); otherwise, there was 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 was significantly higher than that in poorly differentiated cells (P＜0.05, P＜0.01) The positive rate of MDM2 protein was correlated with the cellular differentiation, lymph node metastases and P-TNM stages(P＜0.05), we find that MDM2 is expressed in 32 of 47 with expressed TSG101, the expression level of TSG101 is negatively correlated with MDM2 (P＜0.05).3. Western Blot resultTSG101 is expressed differently in 26 lung cancer tissues and normal lung tissues, it is expressed obviously lower than that in the normal tissues(t=8.578,＜0.05).DiscussionTumor susceptibility gene 101(TSG101) was identified in a random mutagenesis screen for potential tumor suppressors in NIH 3T3 cells. Restoration its function, the cell grow normally. We find much splicing transcripts of TSG101 in a lot of tumor tissues.so TSG101 becomes a critical gene in tumorigenesis.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 it 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.MDM2 (murine double minute-2, MDM2) is an oncogene, regulating cell growth. It strengthens cells longevity, prolonging cells life span, promting Cellular proliferation and tumor growth. It exists in much tisues both in human and murine.MDM2 is correlated with P53, it is expressed highly in gastric cancer, carcinoma of bladder and fleshy tumor, but it is expressed lower in neighbour non-cancerous tissues. It shows: MDM2 may play a role in the process of tumorigenesis。In the present study, we find that MDM2 is expressed in in squamous cell carcinoma of lung, adenocarcinoma of lung, the nucleus expressing rate is 77.21％, there is closely relationship between MDM2 expression and cellular differentiation, lymph node metastases and P-TNM stages, the expression of MDM2 protein in tumor tissues is significantly higher than that in normal lung tissues, the expression rate of MDM2 in poorly differentiated cells is significantly higher than that in well and moderately differentiated cells, the expression rate of MDM2 in patients without lymphatic metastasis is significantly lower than that in those with lymphatic metastasis, the result is agreement with the documents. The finding has shown that TSG101 is a transcription inhibition factor of ubiquitin conjugase, TSG101 protein that contains C- termination coiled-coil domain may act as transcription inhibition factor regulating transcription activation. Acting as transcription inhibition factor, TSG101'S target gene is MDM2. TSG101 affects MDM2 catalytic domain of ubiquitin conjugase by UBC domain, it forms a independent ubiquitin feedback control loop to affect MDM2's stability. TSG101 participates in protein degradation Mediated by MDM2, which indicates TSG101participates in the lung cancer carcinogenesis, the expression level of TSG101 is negatively correlated with MDM2.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 negatively correlated with MDM2 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 cue malignant tumor in time.Conclusion1. TSG101 is expressed strongly in normal lung cancer tissue, it alters coincidentally accompanied with tumor grade.2. MDM2 is not expressed in normal lung tissue, it is expressed opposite with tumor cell differeation.3. In lung cancer, expression of TSG101 and MDM2 shows a significantly negative correlation.