第壹部份：人類B型血球抗原BAT3片段之氮端53個殘基引發細胞"hummingbird"型態變化之功能性研究；第貳部份：人類肝癌致病基因Granulin之功能性研究

Abstract

PartI：Abstract Ricin, consists of A chain (RTA) and B chain (RTB), is a toxic protein which was isolated from the seeds of the castor beans Ricinus communis. It is a member of type Ⅱ ribosome-inactivating protein (RIP). The RTA possesses a RNA N-glycosidase activity which can inactivate ribosomes by removing the 4324th adenine residue from the 28S rRNA and inhibit protein synthesis to cause cell death. Apoptosis, also named program cell death, was found in ricin-treated HeLa cells. By using RTA as bait in a yeast two-hybrid system, a RTA-interacting protein was isolated and designed as human HLA-B-associated transcript 3 (BAT3) in the previous study. It had been demonstrated that BAT3 was cleaved by ricin-induced caspase-3 activity and released a c-terminal 131-residue fragment (CTF-131). Overexpression of the CTF-131 fragment in HeLa cell causes several characteristic apoptotic morphologies such as phosphatidylserine (PS) exposure, cell rounding and shrinkage, nuclear condensation and actin disruption. It was identified that CTF-131 contains a Bcl-2-associated athanogene (BAG) domain and this BAG domain was responsible for inducing the apoptotic morphologies. Interestingly, we identified a fragment, the N-terminal 53 amino acids (N53) of CTF-131 (1002-1054 residues), which had dramatic effects on the cell morphology and led to distinct morphologic changes of cells which resembled the “hummingbird” phenotype. By fractionation of F-/G-actin using sucrose density gradient centrifugation, F-actin accumulation was discovered in the N53 transfectants but not in those control groups. The results suggest that the elongated cell type resulted from N53 transfection could be due to the accumulation of F-actin. To study the roles of the N53 in the morphologic changes and accumulation of F-actin, identification the proteins interacting with N53 was carried out by using yeast two-hybrid system, and to study whether these interacting proteins involved in morphologic change. An actin binding proteins, coronin, was found to be the N53 interacting protein. It suggests that N53 interacted with coronin and caused the decrease of dissociation of F-actin. PartII：Abstract Hepatocellular carcinoma (HCC) is one of the most common human malignant tumors in the world. The disease is highly prevalent in Asia but relatively rare in developed countries. In Taiwan, it ranks first in terms of cancer mortality. Different aetiological factors such as hepatitis viral infection , dietary aflatoxins, or chemical carcinogens are associated with the development of liver cancer. Nevertheless, the molecular mechanism remains to be clarified. A full-length cDNA library of HCC was constructed from a 65-year-old male patient infected by hepatits C virus. The sequencing data were collected and analyzed for their sequence similarity against the GeneBank nucleotide library maintained by the NCBI. By the BLAST program, we had got 24 deletion clones, including Granulin (GRN). GRN was identified as a putative growth factor which is characterized by a unique and highly conserved cysteine-rich motif of 12 cysteines. Cloning of the cDNA for the GRN showed that it encoded a 63 kDa protein that contained a signal peptide (designed as progranulin, proGRN). Previous investigation indicates that expression of GRN in cancer cells may play a critical role in tumor formation. In this study, the mutated GRN is deleted in-frame from 331-465 base pair in its coding region (shortened 135 bps). This mutated GRN was cloned, and wild type GRN was cloned into pcDNA3 vector for mammalian cells transfection. In colony formation assay, wild type proGRN conferred SW 13 cells better ability of anchorage-independent growth. Further study will be needed to elucidate the relationship between the proGRN and HCC.