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Overexpression of p27kip1 induces growth arrest and apoptosis in lung cancer cell lines
Biology
198
without an MHC restriction. We conclude that MUCl-specific CTLs can be induced from any patient with different HLA haplotypes using one synthetic pepUde. It is expected to become an useful strategy for therapeutic treatment of lung cancer.
Wednesday, 13 September 2000 POSTER SESSION
Biology I-~-~ Quantification of MDR1, MRP1, and GSTpi gene expression levels in smokers with and without bronchial dysplasia or cancer J.Y. Hung, J.S. Cheng, S. Ralph, J. English, V. Ling, S. Lain. British
Columbia Cancer Agency, Vancouver, British Columbia; University of British Columbia, Vancouver, British Columbia, Canada The purpose of this study was to compare the levels of expression of MDR1, MRP1, and GSTpi in smokers with and without bronchial dysplasia or cancer to determine if these genes play a role in protecting airway epithelial cells from the effects of inhaled carcinogens. Bronchial brushings were collected from 30 current smokers and 30 ex-smokers without evidence of dysplasia or lung cancer. For comparison, bronchial brushings were obtained from 30 heavy smokers and 30 ex-smokers matched for age, sex, and smoking history with dysplasia or cancer on fluorescence bronchoscopy. In addition, paired normal bronchial fragments and tumor tissue were collected from 30 patients who underwent surgery for lung cancer. A multiplex competitive reverse transcriptase polymerase chain reaction assay was used to quantify the mRNA copy numbers of MDR1, MRP and GSTpi concurrently. RNA internal standards were constructed, and known copy numbers of the internal standards were introduced into the multiplex reverse transcriptase polymerase chain reactions. The RNA competitors were designed with a four to eight base deletion compared to the target mRNAs and had identical primer sites. Therefore, these standards competed with the targets for enzymes, nucleotides and primer molecules, but are distinguishable by separation in capillary electrophoresis. The amounts of target mRNAs in a sample were determined by extrapolating against the standard curve of the internal standards. The housekeeping gene, b2-microglobulin was used as an endogenous RNA control for the differences in the quality of total RNA and cDNA yields in each reaction. The relative expression levels of these genes and the relevance of these results relating to the hypothesis that these genes may play a role in the protection of airway epithelial cells against inhaled carcinogens will be discussed. (This work was supported by a grant awarded by the British Columbia Lung Association. J.Y.H is a recipient of the Medical Research Council Canada-British Columbia Lung Association Scholarship). I-6~
Overexpression of p27kip1 induces growth arrest and apoptosis in lung cancer cell lines
G. KobayashiI , I. Naruse 2, H. Hoshino2, N. Yana~]itani2, K. Minato2, A. Takise2, S.-i. Ishihara 2, N. Fueki2, T. Nomoto~, R. Saito 1,
1Department of Internal Medicine, National Nishi-Gunma Hospital, Shibukawa, Gunma; 2First Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan p27kip1 is a cyclin-dependent kinase inhibitor which controls the G1 phase of the cell cycle in conjunction with pRb. Some recent reports have described that p27 is associated with cell cycle arrest and apoptosis. In this study, we transferred the full-length human p27 cDNA using a replication-deficient recombinant adenovirus vector (Axp27) into lung cancer cell lines, and evaluated the potential of this strategy for anti-cancer gene therapy. After infection with Ax-p27, the
growth of H322, A549 and SQ-5 ceils, which express pRb, was almost completely suppressed, although no such effect was found in H69 and Lu-135 cells which do not express pRb. In addition, cell death from day 4 after infection with Ax-p27 was only observed in H322, A549 and SQ-5 cells, but not in H69 and Lu-135 cells. The cell cycle of H322 cells treated with Ax-p27 became arrested at the G1 phase from day 1 to day 3 despite the continued overexpression of p27. When we examined the changes in the expression levels of pRb and E2F-1, which play important roles in cell cycle progression from the G1 to S phase, downregulation of pRb expression was detected in H322 cells 3 days after infection with Ax-p27. These data suggest that; 1) the growth-inhibitory effect and induction of apoptosis by the overexpression of p27 require expression of the pRb protein, and 2) adenovirus-mediatad p27 gene transfer may have promise as a novel strategy in cancer gene therapy.
I - ~ - ] Clinical measurement and evaluation of CYERA21-1 as a new tumor marker for non small cell lung cancer Z. Jinchuan, C. Qi, C. Banzao, Y. Hande. Department of Respiratory
Medicine in South Building, General Hospital of PLA, Beijing 100853, RR. China This stud tried to investigate the effect of serum CYERA21-1 as a new lung cancer marker in clinical diagnosis and monitoring the prognosis of non small cell lung cancer (NSCLC), especially in squamous cell cancer (SQC). The values of serum CYERA21-1 and CEA (Carcinoembryonic antigen) were determined in 92 patients with lung cancer (64 cases) and benign lung dieses (28 cases) admitted in our hospital from August 1998 to April 1999, and compared the deference values of serum CYERA21-1 between different histological types, clinical stages be fore and after therapy of lung cancer. The positive rates of serum CYERA21-1 and CEA in lung cancer were 42.19% and 34.53% respectively significantly higher than in benign lung dieses; the values or positive rates of serum CYERA21-1 in SQC were higher significantly than adenocarcinoma (P < 0.01), higher in stage IV than stage Ill of SQC (P < 0.05), decreased after treatment in NSCLC. The serum CYERA21-1 is a useful diagnostic and monitoring tumor marker in lung cancer particularly for squamous cell lung cancer.
[6-•
Expression of type 1 and type 2 cytokines in lung cancer cell lines T. lizasa, T. Fujisawa, M. Suzuki, S. Yokoi, T. lida, S. Motohashi, M. Otsuji, Y. Sekine, K. Shibuya, Y. Saitoh, M. Baba. Department of
Surgery, Institute of Pulmonary Cancer Research, Chiba University School of Medicine, Chiba, Japan We investigated production of type 1 and type 2 cytokines in lung cancer cell lines which are yielded by CD4+ T lymphocytes (Thl, and Th2). A series of 15 cell lines derived from lung cancers (4 adenocarcinomas, 5 squamous cell carcinomas, 2 large cell carcinomas, and 4 small cell carcinomas) were used in this study. Expression of mRNA of type 1 and type 2 cytokines (IL-2, IFN-y, IL-4, IL-5, IL-6, IL-10, IL13, TNF-6, and TGF-~) was examined using RT-PCR. Culture supernatant concentrations of most of the cytokines (IL-2, IL-4, IL-5, IL-6, and IL-10) were measureded with a ELiSA. No cytokines except for IL-6 was observed in mRNA and culture supernatant of either cell lines. Twelve of 15 lung cancer cell lines (3/4 adenocarcinomas, 4/5 squamous cell carcinomas, 2/2 large cell carcinomas, and 3/4 small cell carcinomas) have expressed mRNA of IL-6 in RT-PCR. Eight of 14 cell lines (2/4 adenocarcinomas, 4/5 squamous cell carcinomas, 1/2 large cell carcinomas, and 1/3 small cell carcinomas) have yielded IL-6 in culture supernatants. Correlation between the expression of mRNAs and the concentration of culture supernatants in IL-6 was noted. We concluded that lung cancer cell lines frequently produced IL-6 in the type 1 and type 2 cytokines, the production of IL-6 might have influenced on host immune systems of the tumor burden.
198
without an MHC restriction. We conclude that MUCl-specific CTLs can be induced from any patient with different HLA haplotypes using one synthetic pepUde. It is expected to become an useful strategy for therapeutic treatment of lung cancer.
Wednesday, 13 September 2000 POSTER SESSION
Biology I-~-~ Quantification of MDR1, MRP1, and GSTpi gene expression levels in smokers with and without bronchial dysplasia or cancer J.Y. Hung, J.S. Cheng, S. Ralph, J. English, V. Ling, S. Lain. British
Columbia Cancer Agency, Vancouver, British Columbia; University of British Columbia, Vancouver, British Columbia, Canada The purpose of this study was to compare the levels of expression of MDR1, MRP1, and GSTpi in smokers with and without bronchial dysplasia or cancer to determine if these genes play a role in protecting airway epithelial cells from the effects of inhaled carcinogens. Bronchial brushings were collected from 30 current smokers and 30 ex-smokers without evidence of dysplasia or lung cancer. For comparison, bronchial brushings were obtained from 30 heavy smokers and 30 ex-smokers matched for age, sex, and smoking history with dysplasia or cancer on fluorescence bronchoscopy. In addition, paired normal bronchial fragments and tumor tissue were collected from 30 patients who underwent surgery for lung cancer. A multiplex competitive reverse transcriptase polymerase chain reaction assay was used to quantify the mRNA copy numbers of MDR1, MRP and GSTpi concurrently. RNA internal standards were constructed, and known copy numbers of the internal standards were introduced into the multiplex reverse transcriptase polymerase chain reactions. The RNA competitors were designed with a four to eight base deletion compared to the target mRNAs and had identical primer sites. Therefore, these standards competed with the targets for enzymes, nucleotides and primer molecules, but are distinguishable by separation in capillary electrophoresis. The amounts of target mRNAs in a sample were determined by extrapolating against the standard curve of the internal standards. The housekeeping gene, b2-microglobulin was used as an endogenous RNA control for the differences in the quality of total RNA and cDNA yields in each reaction. The relative expression levels of these genes and the relevance of these results relating to the hypothesis that these genes may play a role in the protection of airway epithelial cells against inhaled carcinogens will be discussed. (This work was supported by a grant awarded by the British Columbia Lung Association. J.Y.H is a recipient of the Medical Research Council Canada-British Columbia Lung Association Scholarship). I-6~
Overexpression of p27kip1 induces growth arrest and apoptosis in lung cancer cell lines
G. KobayashiI , I. Naruse 2, H. Hoshino2, N. Yana~]itani2, K. Minato2, A. Takise2, S.-i. Ishihara 2, N. Fueki2, T. Nomoto~, R. Saito 1,
1Department of Internal Medicine, National Nishi-Gunma Hospital, Shibukawa, Gunma; 2First Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan p27kip1 is a cyclin-dependent kinase inhibitor which controls the G1 phase of the cell cycle in conjunction with pRb. Some recent reports have described that p27 is associated with cell cycle arrest and apoptosis. In this study, we transferred the full-length human p27 cDNA using a replication-deficient recombinant adenovirus vector (Axp27) into lung cancer cell lines, and evaluated the potential of this strategy for anti-cancer gene therapy. After infection with Ax-p27, the
growth of H322, A549 and SQ-5 ceils, which express pRb, was almost completely suppressed, although no such effect was found in H69 and Lu-135 cells which do not express pRb. In addition, cell death from day 4 after infection with Ax-p27 was only observed in H322, A549 and SQ-5 cells, but not in H69 and Lu-135 cells. The cell cycle of H322 cells treated with Ax-p27 became arrested at the G1 phase from day 1 to day 3 despite the continued overexpression of p27. When we examined the changes in the expression levels of pRb and E2F-1, which play important roles in cell cycle progression from the G1 to S phase, downregulation of pRb expression was detected in H322 cells 3 days after infection with Ax-p27. These data suggest that; 1) the growth-inhibitory effect and induction of apoptosis by the overexpression of p27 require expression of the pRb protein, and 2) adenovirus-mediatad p27 gene transfer may have promise as a novel strategy in cancer gene therapy.
I - ~ - ] Clinical measurement and evaluation of CYERA21-1 as a new tumor marker for non small cell lung cancer Z. Jinchuan, C. Qi, C. Banzao, Y. Hande. Department of Respiratory
Medicine in South Building, General Hospital of PLA, Beijing 100853, RR. China This stud tried to investigate the effect of serum CYERA21-1 as a new lung cancer marker in clinical diagnosis and monitoring the prognosis of non small cell lung cancer (NSCLC), especially in squamous cell cancer (SQC). The values of serum CYERA21-1 and CEA (Carcinoembryonic antigen) were determined in 92 patients with lung cancer (64 cases) and benign lung dieses (28 cases) admitted in our hospital from August 1998 to April 1999, and compared the deference values of serum CYERA21-1 between different histological types, clinical stages be fore and after therapy of lung cancer. The positive rates of serum CYERA21-1 and CEA in lung cancer were 42.19% and 34.53% respectively significantly higher than in benign lung dieses; the values or positive rates of serum CYERA21-1 in SQC were higher significantly than adenocarcinoma (P < 0.01), higher in stage IV than stage Ill of SQC (P < 0.05), decreased after treatment in NSCLC. The serum CYERA21-1 is a useful diagnostic and monitoring tumor marker in lung cancer particularly for squamous cell lung cancer.
[6-•
Expression of type 1 and type 2 cytokines in lung cancer cell lines T. lizasa, T. Fujisawa, M. Suzuki, S. Yokoi, T. lida, S. Motohashi, M. Otsuji, Y. Sekine, K. Shibuya, Y. Saitoh, M. Baba. Department of
Surgery, Institute of Pulmonary Cancer Research, Chiba University School of Medicine, Chiba, Japan We investigated production of type 1 and type 2 cytokines in lung cancer cell lines which are yielded by CD4+ T lymphocytes (Thl, and Th2). A series of 15 cell lines derived from lung cancers (4 adenocarcinomas, 5 squamous cell carcinomas, 2 large cell carcinomas, and 4 small cell carcinomas) were used in this study. Expression of mRNA of type 1 and type 2 cytokines (IL-2, IFN-y, IL-4, IL-5, IL-6, IL-10, IL13, TNF-6, and TGF-~) was examined using RT-PCR. Culture supernatant concentrations of most of the cytokines (IL-2, IL-4, IL-5, IL-6, and IL-10) were measureded with a ELiSA. No cytokines except for IL-6 was observed in mRNA and culture supernatant of either cell lines. Twelve of 15 lung cancer cell lines (3/4 adenocarcinomas, 4/5 squamous cell carcinomas, 2/2 large cell carcinomas, and 3/4 small cell carcinomas) have expressed mRNA of IL-6 in RT-PCR. Eight of 14 cell lines (2/4 adenocarcinomas, 4/5 squamous cell carcinomas, 1/2 large cell carcinomas, and 1/3 small cell carcinomas) have yielded IL-6 in culture supernatants. Correlation between the expression of mRNAs and the concentration of culture supernatants in IL-6 was noted. We concluded that lung cancer cell lines frequently produced IL-6 in the type 1 and type 2 cytokines, the production of IL-6 might have influenced on host immune systems of the tumor burden.