.1. Man2c1 The Suppression Of Gene Expression By Mitotic Arrest And Apoptosis Induced Inhibition Of Esophageal Cancer Cell Growth The 2.8b7 Ghost Cell Protein Localization

The first part of this thesis is "Inhibition of a-mannosidase Man2c1 gene expression suppresses growth of esophageal carcinoma cells through mitotic arrest and apoptosis".Man2c1 cDNA encoding a humanα-mannosidase was cloned in our laboratory in 2000 with a GenBank accession number AF044414.The cDNA is 3123 bp long,which encodes a 1040-aaα-mannosidase with a molecular weight of 116 kDa under de-glycosylation.Ju Jiyu,one of the Ph.D.students in this lab developed two cell lines, B7 and D2,with profound Man2c1 suppression from EC9706 human esophageal carcinoma cell line using RNAi technique mediated by adenovirus.We observed that growth in vitro,colony formation and tumorigenesis in nude mice of B7 and D2 cells was significantly inhibited comparing to that of controls.Flow cytometric analysis showed accumulation of cells in S and G2-M phases,as well as a sub-G0/G1 peak, showing cell apoptosis.In exploring mechanisms,I examined cell mitosis first.It was found that mitosis of B7 and D2 cells was arrested at the M checkpoint.Since a number of crucial biological processes such as cell division were associated with cytoskeleton in eukaryocytes, eytoskeleton was examined by immuno fluorescence staining with mAb anti-α-tubulin as a probe.Although control cells showed a nest-like microtubule network,the microtubule network in B7 and D2 cells was vague and condensed at the perinuclear region.Some cells with Man2c1 suppression had large protrusions of cytoplasm.Western blotting showed that a-tubulin polymerization was inhibited.The data imply that induction of mitotic arrest and consequent apoptosis resulted from microtubule disorganization appears to be one of the major cellular mechanisms by which Man2c1 suppression gene causes growth inhibition of EC9706 esophageal carcinoma cells.In addition,enhanced expression of E-eadherin,α-catenin andβ-catenin caused by Man2c1 suppression implies that the E-cadherin-catenin complex might also play a role in the growth inhibition of EC9706 cells.The second part of this thesis is "Subcellular localizations of 8B7 spectrin".8B7 cDNA was cloned from a human tonsil cellλgt11 cDNA library in our laboratory in 1998 with an accession number AF043290 in GenBank.8B7 cDNA is 1835 bp long,encoding a 363-aa protein.Since 8B7 protein contains spectrin repeats,it is named 8B7 spectrin by us.8B7 spectrin is a new member of Nesprin family.Blast shows that the nucleotide sequence of 8B7 cDNA is 100%identical to the 3' 1835 bp nucleotides of Enaptin,Nesprin-1,Myne-1 and Syne-1 cDNA and the 363 aa 8B7 spectrin is completely homologous to the C-terminal 363 amino acids of Enaptin,Nesprin-1,Myne-1 and Syne-1 protein.More than 20 members including Syne-1,Syne-2,Nesprin-1,Nesprin-2,Myne-1,Myne-2,Enaptin and Nuauce were found in Nesprin protein family.All the proteins are generated by alternative splicing of nesprin-1 giant gene and nesprin-2 giant gene.8B7 cDNA is encoded by exon 141-147.Northern blot analysis showed existence of 8B7 mRNA in human tissue.In order to examine existence of 8B7 spectrin,I prepared a mAb anti-8B7 spectrin.Western blotting demonstrated the existence in cells.Nesprin family protein contains both KASH domain and actin binding domain in the molecule, which determines localization of the protein at nuclear envelope.Although 8B7 spectrin contains only KASH domain at the C-terminal and lacks actin binding domain at the N-terminal,it locates at nuclear envelope,indicating it is the KASH domain which determines sub-cellular localization of 8B7 spectrin.Nesprins located at nuclear envelop were found to function in taking part in: breakdown and reconstruction of nuclear envelop;nuclear division;construction and regulation of cytoskeleton;cross reaction with inner nuclear envelop,conformation of a complex at the nuclear envelop,and connection of nucleus to cytoskeleton through cross linking.More than 30 proteins which are possibly interacted with 8B7 spectrin were found in a yeast two hybridization experiment performed by Miss Liu Rong,a master student in this lab.Interaction of 8B7 spectrin with Dynactin(P62),Stathmin,Emerin, Lamin A and found Lamin A was examined by means of co-immunoprecipitation test by me.Only Lamin A was observed to be able to be co-immunoprecipitated with 8B7 spectrin.Lamin A is a component in lamin and functions as a common anchoring site for many proteins.It plays important roles in maintaining normal morphology of nuclear envelope,presenting anchoring site for chromatin and reconstructing of nucleus.8B7 spectrin may function through interaction with Lamin A.