• Tuberculosis and Respiratory Diseases
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• 제44권4호
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• pp.796-805
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• 1997

연구배경 : 세포주기의 활성화, 그 중에서도 특히 $G_1$/S 이행에 관여하는 세포주기관련 단백질들은 암발생에 있어서 매우 중요한 역할을 하는 것으로 알려져 있다. $G_1$ 세포주기 관련 단백질 중의 하나인 cdk4 (cyclin dependent kinase 4)의 억제제로 알려져 있는 p16 유전자는 최근에 밝혀진 종양억제유전자중의 하나로서 MTS1 (multiple tumor suppressor 1)이라고도 불린다. p16 유전자는 지금까지 알려진 어느 종양관련 유전자보다도 유전자변이의 빈도가 높은 암억제유전자인데, 특히 비소세포폐암인 경우는 70% 이상의 세포주에서 p16 단백질의 발현이 없는 것으로 밝혀져 있어 p16 유전자는 비소세포폐암 발생에 매우 중요한 역할을 할 것이라고 알려져 있다. 본 연구에서는 비소세포폐암에서 p16을 이용한 유전자치료의 타당성을 입증하기 위하여 다음과 같은 연구를 시행하였다. 방 법 : p16이 결여된 비소세포폐암 세포주 (NCI-H441)에, 정상섬유아세포에서 총 RNA를 추출하여 역전사효소 및 DNA 중합효소반응으로 증폭된 p16 cDNA를 유핵세포 발현 vector인 pRC-CMV plasmid에 subcloning하여 구축된 pRC-CMV-p16 plasmid vector를 lipofectin을 이용하여 유전자 이입한 후, 단백질을 추출하여 Western blot 분석과 면역침전법으로 $G_1$ 세포주기관련 단백질의 변동을 관찰하고, colony 형성능을 비교함으로써 암억제효과를 확인하였다. 결 과 : p16이 유전자주입된 NCI-H441 세포주에서 p16과 cdk4가 복합체를 형성하고 있고 인산화 Rb가 대조 세포주에 비해 감소되어 있음을 확인할 수 있어, p16이 cdk4와 결합함으로써 cdk4에 의한 Rb의 인산화를 방해하고 이에 따른 $G_1$ 세포주기 정체에 의해 종양억제효과가 나타난다는 설명을 뒷받침할 수 있었다. Clonogenic assay 결과는 p16 유전자주입된 NCI-H441 세포주의 colony 형성능이 대조 세포주에 비하여 현격히 감소함을 관찰하였다. 결 론 : 이상의 결과로 p16(MTS1) 유전자를 p16 단백질을 발현하지 못하는 비소세포폐암 세포주에 주입할 경우, 주입한 유전자에서 생성되는 p16 단백질이 cdk와 결합하여 Rb 단백질의 인산화를 저하시켜 궁극적으로 암억제 효과를 일으킬 수 있음이 확인되었고, 이는 향후 비소세포폐암의 유전자치료에 있어서 p16 유전자의 이용 가능성을 확인한 기초자료가 된다고 생각된다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권5호
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• pp.1839-1843
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• 2015

Background: MicroRNAs are a class of noncoding RNAs which regulate multiple cellular processes during tumor development. The purpose of this report is to investigate the clinicopathological and prognostic significance of miR-218 in human gliomas. Materials and Methods: Quantitative RT-PCR (qRT-PCR) was conducted to detect the expression of miR-218 in primary normal human astrocytes, three glioma cell lines and 98 paired glioma and adjacent normal brain tissues.Associations of miR-218 with clinicopathological variables of glioma patients were statistically analyzed. Finally, a survival analysis was performed using the Kaplan-Meier method and Cox's proportional hazards model. Results: The expression level of miR-218 in primary normal human astrocytes was significantly higher than that in glioma cell lines (p<0.01). Also, the expression level of miR-218 in glioma tissues was significantly downregulated in comparison with that in the adjacent normal brain tissues (p<0.001). Statistical analyses demonstrated that low miR-218 expression was closely associated with advanced WHO grade (p=0.002) and low Karnofsky performance score (p=0.010) of glioma patients. Kaplan-Meier analysis with the log-rank test showed that patients with low-miR-218 expression had poorer disease-free survival and overall survival (p=0.0045 and 0.0124, respectively). Multivariate analysis revealed that miR-218 expression was independently associated with the disease-free survival (p=0.009) and overall survival (p=0.004) of glioma patients. Conclusions: Our results indicate that miR-218 is downregulated in gliomas and that its status might be a potential valuable biomarker for glioma patients.

• Asian Pacific Journal of Cancer Prevention
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• 제16권8호
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• pp.3447-3450
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• 2015

Objective: To explore the sensitivity of gastric cancer cells to chemotherapy drugs in elderly patients and its correlation with cyclooxygenase-2 (COX-2) expression in cancer tissue. Materials and Methods: Forty-three elderly patients with gastric cancer (observation group) and 31 young patients with gastrointestinal tumors (control group) who were all diagnosed by pathology and underwent surgery in the 89th Hospital of Chinese People's Liberation Army were selected. Drug sensitivity testing of tumor cells in primary culture was carried out in both groups using a methyl thiazolyl tetrazolium (MTT) method, and the expression of COX-2 and the factors related to multi-drug resistance (MDR) in cancer tissue were assessed by immunohistochemistry. Results: The inhibition rates (IR) of vincristine (VCR), 5-fluorouracil (5-FU), oxaliplatin (L-OHP), mitomycin (MMC) and epirubicin (eADM) on tumor cells in the observation group were dramatically lower than in the control group, with statistical significance (P<0.05 or P<0.01). The positive rates of COX-2, glutathione s-transferase-${\pi}$ (GST-${\pi}$) and P glycoprotein (P-gp) expression in cancer tissue in the observation group were all higher than in control group (P<0.05), while that of DNA topoisomerase $II{\alpha}$ ($TopoII{\alpha}$) expression lower than in the control group (P<0.01). In the observation group, COX-2 expression in cancer tissue had a significantly-positive correlation with GST-${\pi}$ and P-gp (r=0.855, P=0.000; r=0.240, P=0.026), but a negative correlation with $TopoII{\alpha}$ (r=-0.328, P=0.002). In the control group, COX-2 expression in cancer tissue was only correlated with P-gp positively (r=0.320, P=0.011). Bivariate correlation analysis displayed that COX-2 expression in cancer tissue in the observation group had a significantly-negative correlation with the IRs of 5-FU, L-OHP, paclitaxel (PTX) and eADM in tumor cells (r=-0.723, P=0.000; r=-0.570, P=0.000; r=-0.919, P=0.000; r=-0.781, P=0.000), but with hydroxycamptothecine (HCPT), VCR and 5-FU in the control group (r=-0.915, P=0.000; r=-0.890, P=0.000; r=-0.949, P=0.000). Conclusions: Gastric cancer cells in elderly patients feature stronger MDR, which may be related to high COX-2 expression.

• Asian Pacific Journal of Cancer Prevention
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• 제16권16호
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• pp.6851-6855
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• 2015

Background: This study aimed to examine the clinical significance of fatty acid synthase (FASN) expression in gastric cancer (GC), and investigate any prognostic role. Materials and Methods: FASN expression was assessed in gastric cancers by immunohistochemistry using 60 paraffin-embedded tissue specimens, and clinical data were collected by retrospective chart review. Moreover, FASN mRNA expression in 15 fresh resected specimens was evaluated by the reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical staining of PTEN was performed to assess the correlation of PTEN with FASN in gastric cancer. Results: Increased expression of FASN was noted in gastric cancers. The frequency of FASN gene amplification was also significantly higher in gastric cancer than in adjacent normal tissue. FASN expression in human gastric cancer tissues was significantly correlated with patient TNM stage and peritoneal dissemination (p<0.05). Moreover, higher FASN expression significantly correlated with shorter overall survival (p<0.05). Here, upregulation of FASN negatively correlated with PTEN expression in gastric cancer. Conclusions: These findings indicate that FASN expression is upregulated in gastric cancer, and increased FASN may be critical to th peritoneal metastasis and survival. Our results suggest that FASN upregulation and PTEN downregualtion may be involved in peritoneal dissemination for gastric cancer progression.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1362-1368
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• 2006

A new constitutive episomal expression vector, pGAPZ-E, was constructed and used for initial screening of eukaryotic target gene expression in Pichia pastoris. Two reporter genes such as beta-galactosidase gene and GFPuv gene were overexpressed in P. pastoris. The expression level of the episomal pGAPZ-E strain was higher than that of the integrated form when the beta-galactosidase gene was used as the reporter gene in P. pastoris X33. The avoiding of both the integration procedure and an induction step simplified the overall screening process for eukaryotic target gene expression in P. pastoris. Nine human protein targets from the Core 50, family of Northeast Structural Genomics Consortium (http://www.nesg.org), which were intractable when expressed in E. coli, were subjected to rapid screening for soluble expression in P. pastoris. HR547, HR919, and HR1697 human proteins, which had previously been found to express poorly or to be insoluble in E. coli, expressed in soluble form in P. pastoris. Therefore, the new episomal GAP promoter vector provides a convenient and alternative system for high-throughput screening of eukaryotic protein expression in P. pastoris.

• Asian Pacific Journal of Cancer Prevention
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• 제16권2호
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• pp.513-517
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• 2015

Background: Growing evidence suggests that the members of the ubiquitin-proteasome system (UPS) are important for tumorigenesis. HERC4, one component, is a recently identified ubiqutin ligase. However, the expression level and function role of HERC4 in lung cancer remain unknown. Our objective was to investigate any correlation between HERC4 and development of lung cancer and its clinical significance. Materials and Methods: To determine HERC4 expression in lung cancer, an immunohistochemistry analysis of a tissue microarray containing samples of 10 lung normal tissues, 15 pulmonary neuroendocrine carcinomas, 45 squamous epithelial cancers and 50 adenocarcinomas was conducted. Receiver operating characteristic (ROC) curve analysis was applied to obtain a cut-off point of 52.5%, above which the expression of HERC4 was regarded as "positive". Results: On the basis of ROC curve analysis, positive expression of HERC4 was detected in 0/10 (0.0%) of lung normal tissues, in 4/15 (26.7%) of pulmonary neuroendocrine carcinomas, in 13/45 (28.9%) of squamous epithelial cancers and in 19/50 (38.0%) of adenocarcinomas. It showed that lung tumors expressed more HERC4 protein than adjacent normal tissues (${\chi}^2$=4.675, p=0.031). Furthermore, HERC4 positive expression had positive correlation with pT status (${\chi}^2$=44.894, p=0.000), pN status (${\chi}^2$=43.628, p=0.000), histological grade (${\chi}^2$=7.083, p=0.029) and clinical stage (${\chi}^2$=72.484, p=0.000), but not age (${\chi}^2$=0.910, p=0.340). Conclusions: Our analysis suggested that HERC4 is likely to be a diagnostic biomarker for lung cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8633-8636
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• 2016

Background: To investigate the expression of insulin-like growth factor-I receptor (IGF-IR) and sushi domain containing protein 3 (SUSD3) in breast cancer tissue, and analyze their relationship with clinical parameters and the correlation between the two proteins. Materials and Methods: The expression of IGF-IR and SUSD3 in 100 cases of breast cancer tissues and adjacent normal breast tissues after surgery was detected by immunohistochemical technique MaxVisionTM, and the relationship with clinical pathological features was further analyzed. Results: The positive rate of IGF-IR protein was 86.0% in breast cancer, higher than 3.0% in adjacent normal breast tissue (P<0.05). The positive expression rate of SUSD3 protein was 78.0% in breast cancer, higher than 2.0% in adjacent normal breast tissue (P<0.05). The expression of IGF-IR and SUSD3 was related to estrogen receptor and pathological types (P<0.05),but not with age, stage, the expression of HER-2 and Ki-67 (P>0. 05). The expression of IGF-IR and SUSD3 in breast cancer tissue was positively related (r=0.553, P<0.01). Conclusions: The expression of IGF-IR and SUSD3 may be correlated to the occurrence and development of breast cancer. The combined detection of IGF-IR, SUSD3 and ER may play an important role in judging prognosis and guiding adjuvant therapy after surgery of breast cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제16권9호
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• pp.3973-3976
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• 2015

Objectives: To explore the expression of aquaporin 1 ($AQP_1$) in bladder uroepithelium cell carcinoma (BUCC) and its relevance to recurrence. Materials and Methods: Tissue samples from 45 BUCC patients who underwent total cystectomy or transurethral resection of bladder tumor (TURBT) and from 40 patients with non-bladder cancers who underwent special detection or treatments were collected. The level of expression of $AQP_1$ in BUCC tissues and normal bladder tissues was assessed by immunohistochemistry so as to analyze the relevance to pathological patterns and time of recurrence in BUCC patients. Results: The expression levels of $AQP_1$ normal bladder tissues and BUCC tissues were $2.175{\pm}0.693$ and $3.689{\pm}0.701$, respectively, and the difference was significant (t=9.99, P<0.0001). Marked increase was noted with BUCC histological grade and pathological stage (P<0.01). Moreover, the expression of $AQP_1$ was evidently higher in cancerous tissues with lymph node metastasis than in those without (P<0.01). With short-term recurrence, the positive cell expression rate of $AQP_1$ was higher in primary tissues, which increased obviously after recurrence. Additionally, the recurrent time of BUCC was negatively associated with the positive cell expression rate of $AQP_1$ and the difference between the expression of $AQP_1$ before and after recurrence (r=-0.843, F=39.302, P=0.000; r=-0.829, F=35.191, P=0.000). Conclusions: $AQP_1$, which reflects the grade, stage, lymph node metastasis and recurrence of BUCC, has potential guiding significance in the diagnosis and treatment of bladder cancarcinoma.

• Asian Pacific Journal of Cancer Prevention
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• 제15권20호
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• pp.8911-8916
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• 2014

Background: The aim of the present study was to determine the predictive/prognostic value of the secreted protein, acidic and rich in cysteine (SPARC) in cases of unresectable, locally advanced, non-small cell lung cancer. Materials and Methods: The study included 84 patients with Stage IIIA-B non-small cell lung cancer, undergoing simultaneous chemoradiotherapy including radiotherapy at a dose of 66 Gy and weekly docataxel ($20mg/m^2$) and cisplatin ($20mg/m^2$). SPARC expression was studied in biopsy material by immunohistochemical methods and correlations with treatment responses or survival were evaluated. Results: Median overall survival was $16{\pm}2.73$ (11.55-20.46) months for low expression vs $7{\pm}1.79$ months (7.92-16.08) months for high expression (p=0.039), while median local control was $13{\pm}2.31$ (8.48-17.5) months for low expression vs $6{\pm}0.85$ (4.34-7.66) months for high expression (p=0.045) and median progression-free survival was $10{\pm}2.31$ (5.48-14.5) months for low expression vs $6{\pm}1.10$ (3.85-8.15) months for high expression (p=0.022). In both univariate and multivariate analyses, high SPARC expression was associated with significantly shorter overall survival (p=0.003, p=0.007, respectively), local control (p=0.008, p=0.036) and progression-free survival (p=0.004, p=0.029) when compared to low SPARC expression. No significant difference was detected between high and low SPARC expression groups regarding age, sex, T stage, N stage, histopathology and stage-related patient characteristics. Conclusions: High SPARC expression was identified as a poor prognostic factor in cases with locally advanced NSCLC treated with concurrent chemoradiotherapy.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제22권2호
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• pp.164-173
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• 2000

The p16 protein is a cyclin dependent kinase inhibitor that inhibits cell cycle progression from $G_1$ phase to S phase in cell cycle. Many p16 gene mutations have been noted in many cancer-cell lines and in some primary cancers, and alterations of p16 gene function by DNA methylation have been noticed in various kinds of cancer tissues and cell-lines. There have been a large body of literature has accumulated indicating that abnormal patterns of DNA methylation (both hypomethylation and hypermethylation) occur in a wide variety of human neoplasma and that these aberrations of DNA methylation may play an important epigenetic role in the development and progression of neoplasia. DNA methylation is a part of the inheritable epigenetic system that influences expression or silencing of genes necessary for normal differentiation and proliferation. Gene activity may be silenced by methylation of up steream regulatory regions. Reactivation is associated with demethylation. Although evidence or a high incidence of p16 alterations in a variety of cell lines and primary tumors has been reported, that has been contested by other investigators. The precise mechanisms by which abnormal methylation might contribute to carcinogenesis are still not fully elucidated, but conceivably could involve the modulation of oncogene and other important regulatory gene expression, in addition to creating areas of genetic instability, thus predisposing to mutational events causing neoplasia. There have been many variable results of studies of head and neck squamous cell carcinoma(HNSCC). This investigation was studied on 13 primary HNSCC for p16 gene status by protein expression in immunohistochemistry, and DNA genetic/epigenetic analyzed to determine the incidence, the mechanisms, and the potential biological significance of its Inactivation. As methylation detection method of p16 gene, the methylation specific PCR(MSP) is sensitive and specific for methylation of any block of CpG sites in a CpG islands using bisulfite-modified DNA. The genomic DNA is modified by treatment with sodium bisulfate, which converts all unmethylated cytosines to uracil(thymidine). The primers designed for MSP were chosen for regions containing frequent cytosines (to distinguish unmodified from modified DNA), and CpG pairs near the 5' end of the primers (to provide maximal discrimination in the PCR between methylated and unmethylated DNA). The two strands of DNA are no longer complementary after bisulfite treatment, primers can be designed for either modified strand. In this study, 13 paraffin embedded block tissues were used, so the fragment of DNA to be amplified was intentionally small, to allow the assessment of methylation pattern in a limited region and to facilitate the application of this technique to samlples. In this 13 primary HNSCC tissues, there was no methylation of p16 promoter gene (detected by MSP and automatic sequencing). The p16 protein-specific immunohistochemical staining was performed on 13 paraffin embedded primary HNSCC tissue samples. Twelve cases among the 13 showed altered expression of p16 proteins (negative expression). In this study, The author suggested that low expression of p16 protein may play an important role in human HNSCC, and this study suggested that many kinds of genetic mechanisms including DNA methylation may play the role in carcinogenesis.