Cancer The The Gene Alc1 Hcap1 Biological Function Of A Preliminary Study

This thesis studied the biological functions of two tumor associated-gene encoding proteins ALC1, HCAP1 and their interacting proteins.The gene ALC1 is often amplified in the chromosome of human hepatocellular carcinoma (HCC), so it is a candidate oncogene for liver cancer. The protein ALC1 has the characteristic structures of SNF Family. It was conformed interacting with NR4A1 (one protein of GR family) by yeast two-hybrid. Recombined and expressed along with protein GFP, GFP-ALC1 was localized in cell nucleus. Therefore, the protein ALC1 was conferred to have some functions related with remodeling of the chromosome or controlling of transcription.The N-terminal of protein ALC1 could interact with protein NTKL, which has a domain similar to protein kinase in the N-terminus. In addition, a new protein was found to interact with the protein NTKL, and was tentatively named NTKL-BP1. These interactions were confirmed by GST pull-down assay and coimmunoprecipitation experiment. Furthermore, the cDNA sequences of the human and mouse homologues were cloned and named as mNTKL-BP1 and hNTKL-BP1 independently. With RT-PCR and Northern blot methods, the expression patterns of mNTKL-BPl were observed in the normal tissues of mouse. Fused with EGFP and by immunofluorescent staining assay, NTKL and NTKL-BP1 were observed to distribute in plasma surrounding the nucleus. The result of colony forming assay and tumor formation in nude mice Balb/c demonstrated that both mNTKL-BPl and hNTKL-BPl could inhibit the proliferation of cancer cell and tumor formation. By means of fluroscence active cyto-sorting, we found that the number of cell at phase G2-M increased with the overexpression of mNTKL-BPl or hNTKL-BPl. All these results demonstrated that these proteins might control the cell proliferation.The HCAP1 gene is considered as a candidate tumor-suppressor gene and located in a LOH region on chromosome 17pl3.3. It contains one helix-loop-helix motif and one leucine zipper domain. With yeast two-hybrid screening, over ten proteins were identified as HCAP1-interacting proteins. The structures of theseproteins were analyzed with bioinformtic software and deletion analysis. It was demonstrated that the zinc-finger structure and Cys-rich region were responsible for the interaction with the C-terminal of HCAP1, which contains a leucine zipper domain. The interactions were further confirmed by GST pull-down, coimmunoprecipitation and immunofluorecsent staining assays.The NDP52 is an interacting protein of HCAP1 identified by yeast two-hybrid screening. The C-terminal of NDP52 has two zinc-finger regions, which were identified as the important regions responsible for the interaction with HCAP1. Moreover, immunofluorescent staining assay indicated that NDP52 was co-localized with HCAP1 in cytoplasm surrounding the nucleus. Demonstrated by Northern blot method, the NDP52 was widely expressed in the normal tissues. It was expressed in heart and muscle at high level and in lung and brain at low level. The result of colony forming assay supposed that NDP52 could inhibite the proliferation of cells. Further more, we found that the N-terminal of NDP52 could interact with the C-termianl of RB1CC1. The later were identified to control the expression of RBI. It was reported that the RB1CC1 was very important to control the development of muscles in mammals. The mutation and deletion of the gene were also observed in some tumor tissues.These results of this thesis supported some valuable informations for the determination of the function of these genes in cancer and tissue development.