Characterization of the cancer/testis antigen CABYR

• Main Authors: Yen-Tzu Tseng, 曾彥慈
• Other Authors: 楊秋英
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/54004897572536045659

博士 === 國立中興大學 === 分子生物學研究所 === 99 === Calcium binding tyrosine phosphorylation-regulated protein (CABYR) family composed of five isoforms resulted from alternative splicing was first identified as a testis specific gene. A phagotope, which may be derived from CABYR, was affinity-selected by antiserum against hepatocellular carinoma HepG2 cells from phage display peptide libraries. Expressions of CABYR-c/d in HepG2 as well as several other carcinoma cell lines were confirmed by RT-PCR. In this study, the expressions of different CABYR isoform in cancer cell lines and tissues from cancer patients were examined by RT-PCR and Western blot analysis. CABYR-a/b and CABYR-c/d transcripts were obtained in all tested cell lines. CABYR-a/b transcripts were detected more frequently in tumor tissues than in adjacent noncancerous tissues; CABYR-c/d was more frequently detected in tumor tissues of lung and esophageal cancers but was also detectable in most liver tissues from liver cancer patients. In contrast, CABYR-e was only detected in lung and esophageal cancers but not in liver cancer. Immunoreactive bands corresponding to CABYR-a/b, CABYR-c and CABYR-d were detected in human cell lines and the liver tumor tissues. However, the levels of transcripts and protein were not correlated in tested samples. Surprisingly, CABYR transcripts were detected in fresh isolated peripheral leukocytes but not in umbilical cord blood leukocyte. CABYR-a and CABYR-c proteins were also expressed in leukocyte, like in cancer cells, protein and transcripts levels were not correlated to each other. To reveal the CABYR expression pattern in leukocyte, flow cytometry was employed to determine the percentage of CABYR+ cells in different cell types. CABYR was detected in 34.3%~65.8% of CD14+, 5.7%~8.5% of CD19+, 6.5%~29% of CD4+, 20.6%~72.6% of CD8+, and 21.8%~61.9% of CD26+ cells. Together with undetectable CABYR in UCB leukocytes suggesting that the expression of CABYR may be induced by antigen stimulation. To realize whether the mRNA stability may cause the inconsistency between mRNA and protein levels, the mRNA stability was compared in cancer cells and leukocytes which were cultured in presence or absence of actinomycin D. After 6 hr-treatment, 35%, 17% and 7% mRNA was remained in THP-1, PNC076 and PBMC, respectively. Furthermore, 5’ RACE was performed to determine CABYR gene transcriptional initiation site. Five transcripts (A-E) different in initiation sites were identified and the free energies of predicted 5’UTR secondary structures were calculated. The transcript C with the lowest free energy implicating higher stability was only presented in cancer cells. However, this transcript contains three untranslated AUG which may affect translation efficiency. This may be one of the explanations for the discrepancy on the expression of mRNA and protein. As an initial attempt to elucidate the biological function of CABYR, immunoprecipitation was performed to isolate the interacting partner(s) of CABYR-a and c. Alpha-enolase was co-precipitated with CABYR-a but not CABYR-c and which interacted with CABYR-a aa181-315 fragment. CABYR-a and part of alpha-enolase were colocalized in cytoplasm implicating that CABYR-a may plays a role in the regulation of cellular energy. In summary, CABYR protein isoforms were detected in tissues from cancer patients but also detected in adult leukocytes. The protein expression pattern of various CABYR isoforms is important with regard to the consideration of using CABYR as a target antigen for the development of vaccines for cancer therapy.