1.hTERT phosphorylation by PKC is essential for telomerase activation and the holoenzyme assembly in oral cancer cells 2. Identification of highly invasive genes in nasopharyngeal carcinoma using proteomics technology

• Main Authors: Ya-ching Lu, 呂雅情
• Other Authors: Ann-joy Cheng
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/76009510227318280372

碩士 === 長庚大學 === 醫學生物技術研究所 === 92 === Part 1: Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the chromosome ends. We have previously found that all the six telomerase subunit proteins (hTERT, hTR, TEP1, hsp90, p23 and dyskerin) are needed for the full enzyme activity. Telomerase activity has been reported up-regulated by PKC but the detail mechanism is not clear. In this study, we examine how PKC regulates telomerase activity in oral cancer cells. Exposure cells to a PKC inhibitor, bisindolylmaleimide I (BIS), resulted in the inhibition of telomerase activity in a dose-dependent manner, but the mRNA levels of telomerase core subunits were not influenced, suggesting a post-translational regulatory mechanism may exist. RNA interference studies reveal that inhibition of PKC isoforms α, β, δ, ε, ζ specifically reduced telomerase activity. In vitro phosphorylations by PKC α, βI, βII, δ, ε, ζ can restore BIS-inhibited telomerase activity, and the phosphorylation target is on hTERT. To investigate whether this hTERT phosphorylation may affect on telomerase holoenzyme integrity, cells were exposed to hsp90 C-terminal and N-terminal inhibitors, novobiocin and geldanamycin, to disturb the association between hsp90 and other proteins. Telomerase activities were reduced by both agents, but only novobiocin-inhibited activity could be restored by PKC activator. These results indicate hTERT phosphorylation is essential for telomerase subunit assembly through interaction with hsp90 at it’s C-terminal domain, and this holoenzyme integrity is crucial for telomerase activity. Immunoprecipitation and immunoblot analyses further demonstrated that the disruption of hTERT-hsp90 can be reassociated after hTERT phosphorylation by PKC. In conclusion, PKC α, βI, βII, δ, ε, ζ can phosphorylate hTERT in oral cancer cells. This phosphorylation is essential for telomerase holoenzyme assembly and the structure integrity is crucial for telomerase with full enzyme activity. Part 2: The highest risk areas in the world of developing nasopharyngeal carcinoma (NPC) are found in South East Asia. In Taiwan, the incidence of NPC has become the 13th leading cancer, the 10th leading cancer in male. The standard treatment for patients with NPC is surgery or radiation therapy or a combination of the two. Because of the high rate of distant metastasis, patient’s survival rate decrease dramatically with the progress of cancer. The better understanding of molecular mechanism of invasion is important in the advances of early detection or therapeutics to control the malignant disease. Highly invasive NPC subclones have been successfully established to further investigate invasive related genes. In this study, there were 15 proteins identified by MALDI-TOF Mass Spectrometry. These proteins differentially expressed differently in parental and highly invasive subclones, suggesting their involvement in invasive ability. Functions of genes should be further verified by using molecular and cell methods. These results have provided fundamental knowledge for the future application in clinical diagnosis, prognosis and therapeutics.