Research Progress in the Structure and Functions of Prohibitin

Prohibitin is named due to the negative regulatory role of its gene products in cell proliferation. Prohibitin gene is located at q21 of chromosome 17 in human beings and the protein is found at mitochondria, nucleus and cytoplasm. Due to its size and ring-shaped structure, prohibitin protein defines functional subcompartments in mitochondria. Its subunits, PHB1 and PHB2, suppress cell proliferation as in the protein itself Nevertheless, recent investigation suggests that prohibitin protein enhances cell proliferation as well. It has also been found to suppress cell apoptosis by reducing cytochrome C release via the avoidance of mitochondrial crista remodeling which is facilitated through type 1 optic atrophy protein （OPAl）. Acting as a binding site for ubiquitin, prohibitin protein regulates protein degradation by proteasome. Examples are the degradations of sperm mitochondria in a fertilized ovum or those of an abnormal sperm.     

Proteome analysis of hepatocellular carcinoma by laser capture microdissection

Hepatocellular carcinoma （HCC） is one of the most frequent visceral neoplasia worldwide and is a multifactorial and multistage pathogenesis that finally leads to the deregulation of cell homeostasis. Laser capture microdissection （LCM） may allow a more ready identification of differences in protein expression in selected cell types or areas of tissue, and microscopic regions as small as 3-5 microm in diameter can be sampled. Here we applied the LCM to the proteomic study of hepatitis B-related HCC and surrounding non-tumor tissues. Proteome alterations were observed using 2-DE and ESI-MS/MS, and alterations in the proteome were examined. Twenty protein spots were selected, of which 11 proteins were significantly altered in the HCC compared with the surrounding non-tumor tissues. Of the proteins that were selected, peroxiredoxin 2, apolipoprotein A-Ⅰ precursor, 3-hydroxyacyl-CoA dehydrogenase type Ⅱ , and 14.5-kDa translational inhibitor protein appear to be novel candidates as useful hepatitis B-related HCC markers.     

Novel mutation of the cyclin-dependent kinase 4 gene in a Chinese patient with intimal sarcoma of the pulmonary artery

The long arm of human chromosome 12 contains a region that has been found to be amplified in a number of different tumors, including osteosarcomas and soft tissue sarcomas. There are more than 5 genes located in this area such as CDK2, CDK4, WNT1, MDM2 and WNTIOb. CDK4 gene consists of eight exons, of which the start codon is located in the beginning of exon 2 and the stop codon in the a member of the Ser-Thr catalytic domain extends beginning of exon 8. CDK4 is protein kinase family and its from amino acid 6 to 295.     

P53 Gene Mutation and Expression of MDM2, P53, P16 Protein and their Relationship in Human Glioma

To investigate the effect of P53 protein accumulation and p53 gene mutation in the pathogenesis of glioma and to study the role of MDM2, P53 and P16 protein in glioma formation and progression and their relationship with each other, LSAB immunohistochemical staining method and non-isotopic PCR-SSCP techniques were used to detect the expression of MDM2, P53 and P16 protein and p53 gene mutation in 48 cases of gliomas. The results showed that the positive expression rate of MDM2, P53 and the negative rate of P16 was 22.9 %, 41.7 % and 60.4 %, respectively. The latter two in high grade （grade Ⅲ , Ⅳ） gliomas had a significantly higher rate than in the low grade （grade Ⅱ ） gliomas. Moreover, the co-expression of MDM2 and P53 protein was confirmed in only 1 of 48 cases. No significant difference was found in the rate of the expression of MDM2 between high grade and low grade gliomas （P〉0.1） . PCR-SSCP results showed that mutation of 5 --8 exons of p53 gene was detected in 17 out of 48 cases （35.42 %） . Mutation was detected in 16 of 20 cases of positive p53 expression, and another one was detected in 28 cases of negative expression cases. The correlation between p53 mutation and p53 immunopositivity was observed in 89.6 % of the cases. P53 gene mutation and the level of MDM2, P53 and PI6 protein were not related to age, gender of the patients, tumor location and size. It is concluded that the mutation of p53 and deletion of p16 might play important roles in the tumorigenesis of gliomas and it was significantly associated with the grade of tumor differentiation. P53 protein accumulation can indirectly reflect p53 mutation. MDM2 amplification and overexpression might be an early event in the growth of human gliomas.     

Correlation of the nuclear accumulation of CTNNB1 and colonic tumorigenesis

CTNNB1 （or beta-catenin） is regarded as a central effecter in molecules of the wingless/Wnt signalling pathway, It is cadherin mediated cell to forms a complex with a key component of the cell adhesion system and the protein product of adenomatous polyposis coil （APC）, glycogen synthase kinase 3β （GSK3β） and conductin. One mutation of APC is also responsible for activation of wingless/Wnt signalling pathway and accumulation of free beta-catenin in the cell. Beta-catenin upregulates oncogenes, such as cyclin D1 and c-myc. Beta-catenin expression in cytoplasm and nuclei was reported to increase in many cases of intestinal tumorigenesis. In addition, the hyperexpression of integrin linked kinase （ILK） in colonic polyposis has been demonstrated.     

A Second Protein Marker of Caveolae：Caveolin-2

Caveolin-2,a protein about 20 kD,is a major component of the inner surface of caveolae,small invaginations of the plasma membrane.Similar with caveolin-1 and caveolin-3,it serves as a protein marker of caveolae.Caveolin-1 and-2 are located next to each other at 7q31.1 on human chromosome,the proteins encoded are co-localized and form a stable hetero-oligomeric complex,distributing similarly in tissue and cultured cells.Caveolin-3 is located on different chromosomes but confirmed to interact with caveolin-2.Caveolin-2 is similar to caveolin-1 in many respects but differs from the latter in functional domains,especially in G-protein binding domain and caveolin scaffolding domain.The mRNAs of both caveolin-1 and caveolin-2 are most abundantly expressed in white adipose tissue and are induced during differentiation of 3T3-L1 cells to adipocytes.Caveolin-2-deficinet mice demonstrate clear pulmonary defects,with little or no change in caveolin-1 expression and caveolae formation,suggesting that caveolin-2 plays a selective role in lung functions.Caveolin-2 is also involved in lipid metabolism and human cancers.     

A possible receptor for β2 glycoproteinⅠon the membrane of hepatoma cell line smmc7721

Objectives To study the interaction of beta-2-glycoprotein Ⅰ (β2GP Ⅰ) with the membrane of hepatocytes and determine whether β2GP Ⅰ participates in HBV infection.Methods Ligand blotting, fluorescence microscopy, and fluorescence activated cell sorter (FACS) analysis were used to detect the specific interaction of β2GP Ⅰ with the hepatoma cell line smmc7721, the gastric carcinoma cell line SGC7901, and the lymphoma cell line HL-60.Results A specific 40 kDa β2GP Ⅰ band was observed by ligand blotting in the case of smmc7721 cells. No such band was observed in SGC7901 or HL-60 cells. Fluorescence microscopy also revealed specific binding of FITC-β2GP Ⅰ to smmc7721 cells, but neither to SGC7901 nor HL-60 cells. FACS analysis demonstrated that the binding rate of FITC-β2GP Ⅰ to smmc7721 cells was significantly higher than these in SGC7901 and HL-60 cells （P<0.01）. The binding rate to smmc7721 cells did not increase with increasing amounts of FITC-β2GP Ⅰ.Conclusions There is a specific β2GP Ⅰ-binding protein on the membrane of hepatoma cells in cell line smmc7721 which as the β2GP Ⅰ receptor may participate in HBV infection of hepatocytes.     

Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

Summary： To investigate the expression of cyclooxygenase-2 （COX-2） in ovarian cancer cell lines, RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a Basis for the chemoprevention of ovarian cancer.     

Expression of MDM2 Protein and mRNA in Condyloma Acuminata

In order to investigate the role of murine double minute 2 (MDM2) in cell proliferation and carcinogenesis in condyloma acuminatum (CA), immunohistochemistry and in situ hybridization were used to detect the expression of MDM2 protein and mRNA in normal skin and skin lesions of CA of vulva. PCR was also used to detect HPV types. The results showed that in 32 observed CA specimens, the expression of MDM2 protein and mRNA was detected in 18(56.25 %)and 22 (68.75 %)respectively, while the co-expression of MDM2 protein and mRNA was found in 14. PCR results revealed that HPV6/11 and HPV16/18 subtypes were shown in 28(87.5 %)and 4 (12.5 % )respectively out of 32 CA specimens. Out of the 18 positive specimens expressing MDM2 protein, HPV6/11 subtypes were shown in 15 and HPV16/18 subtypes in 3. In 22 positive specimens expressing MDM2 mRNA, HPV6/ll subtypes were shown in 18 and HPV16/18 subtypes in 4. No expression of MDM2 protein and MDM2 mRNA was observed in normal skin. Our study indicated that the overexpression of MDM2 might be involved in malignant proliferation and carcinogenesis of CA.     

Investigation of Function of Novel Sperm Binding Protein HBRP in Human

Objective To investigate the biology function of novel protein related to bovineseminal plasma protein in human testis.Methods Recombination pcDNA3/HBRP was constructed and transfected to HEK293cell and permanently expression cell line was established. The activity of proteinkinase C (PKC) of the cell line was detected by autoradiography method.Results The stable expression cell line of HBRP was obtained. The HBRP inhibitedthe activity of PKC significantly.Conclusion One of the new functions of novel sperm binding protein in human is theinhibitor action on activity of PKC. It may be involved in the sperm capacitation andacrosome reaction.     

Expression of cyclooxygenase-2 mRNA in drug-sensitive cell and drugresistant strains of ovarian cancer cell lines

Objective： To investigate the expression of cyclooxygenase-2 （COX-2） mRNA in drug-sensitive cell and drugresistant clones of ovarian cancer cell lines. Methods： RT-PCR and immunocytochemistry were used to investigate the expression of cyclooxygenase-2 in 3 clones drug-sensitive and 5 clones drug-resistant ovarian cancer cell. Results： Strong COX-2 mRNA expressions were detected in 3 clones of drug-sensitive cell and weak expressions were detected in 5 clones of drug-resistant cell. The protein expression of COX-2 in drug-sensitive cell was strongly positive reaction in immunocytochemistry stain and there was a weak positive reaction in 5 clones of drug-resistant cell. Conclusion： The expression of COX-2 mRNA in drug-sensitive cell strains is much higher than that in drugresistant strains of ovarian cancer cell lines, providing a basis of the chemoprevention for ovarian cancer.     

Effect of proline rich domain of an RNA-binding protein Sam68 in cell growth process, death and B cell signal transduction

Background Sam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line. Methods By using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometric analyses were performed to investigate the Sam68 functions. Results A slower growth rate （2.1 hours growth elongation） and longer S phase （1.7 hours elongation） was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells. Conclusions The proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.     

Patrinia scabiosaefolia Inhibits Growth of 5-FU-Resistant Colorectal Carcinoma Cells via Induction of Apoptosis and Suppression of AKT Pathway

Objective: To investigate the effects of ethanol extract of Patrinia scabiosaefolia(EEPS) on chemo-resistance of colorectal cancer cells(CRC) and explore the possible molecular mechanisms. Methods: 5-fluorouracil(5-FU)-resistant human colorectal carcinoma cell line(HCT-8/5-FU) and its parental cells HCT-8 were treated with EEPS(0, 0.25, 0.50, 1 or 2 mg/mL), or 5-FU(0, 100, 200, 400, 800 or 1600 μmol/L). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to evaluate the cell viability. Cell density was observed by phase-contrast microscope, cell counting and colony formation assay were used to determine the cell proliferation of HCT-8/5-FU cells treated with 0, 0.5, 1 or 2 mg/mL EEPS. Cell apoptosis was determined by Hoechst staining. Western-blot was performed to detect the phosphorylation of AKT as well as the protein expression level of B-cell CLL/lymphoma 2(Bcl-2) and Bcl-2-associated X protein(Bax). Results: Compared with HCT-8 cells, MTT assay results indicated that HCT-8/5-FU cells were resistant to 5-FU treatment(P0.05), and sensitive to EEPS treatment(P0.05). Moreover, compared with untreated HCT-8/5-FU cells, 1 and 2 mg/mL of EEPS treatment significantly reduced cell density, cell number, inhibited cell survival(P0.05), and induced apoptosis in HCT-8/5-FU cells. Furthermore, 1 and 2 mg/mL of EEPS significantly decreased the phosphorylation level of p-AKT and Bcl-2 protein expression, and increased the expression of Bax protein(P0.05). Conclusion: EEPS is a promising therapeutic agent that may overcome chemo-resistance in cancer cells, likely through suppression of the AKT pathway and promotion of cancer cell apoptosis.     

Antitumor activity of F90, an epidermal growth factor receptor tyrosine kinase inhibitor, on glioblastoma cell line SHG-44

Background Over-expression of epidermal growth factor receptor （EGFR） is thought to be related to cell proliferation, invasion, metastasis, resistance to chemoradiotherapy and poor prognosis of various human cancers. Forty percent to fifty percent of glioblastoma multiforme （GBM） possess deregulated EGFR, which may contribute to the aggressive and refractory course of GBM. Therefore, blockade of EGFR signal transduction may be a promising treatment strategy for GBM. Methods MTT assay, cell growth curve assay and tumor xenograft model were used to evaluate the antitumor activity of F90 against SHG-44 in vitro and in vivo. Western blot assay was applied to evaluate the expression of p-EGFR, p-ERK1, p-JNK, p-P38, Bcl2 and P53 proteins. Results F90 inhibited the cell proliferation in a dose-dependent manner in vitro. The growth of SHG-44 tumor xenografts was suppressed by F90 at a high dose level （100 mg.kg^-1·d^-1）. Phosphorylation of EGFR and activated downstream signaling proteins, such as ERK1, JNK and P38, were found to be depressed after incubation with F90 for 48 hours in vitro. Down-regulated Bcl2 protein and up-regulated P53 protein were also observed. Conclusions The results demonstrate that F90 is effective in inhibiting the proliferation of SHG-44 cells in vitro and tumor growth in vivo, suggesting that F90 may be a new therapeutic option for treatment of GBM.     

Study of the SH3-domain GRB2-like 2 gene expression in laryngeal carcinoma

Background Laryngeal carcinoma is a common malignant tumor of the upper respiratory tract, and in 95% of cases the tumor is laryngeal squamous cell carcinoma （LSCC）. The abnormity of SH3-domain GRB2-1ike 2 （SH3GL2） gene was found in LSCC. In order to clarify the relationship between SH3GL2 gene and LSCC, we evaluated the expression of the SH3GL2 gene in LSCC. Method Real-time PCR, immunohistochemistry and Western blotting were used to detect the mRNA and protein expression and find the various rules of SH3GL2 gene in LSCC. Results The result of real-time PCR showed that the expression level of SH3GL2 mRNA in LSCC tissue was apparently down-regulated; immunohistochemical analysis showed that SH3GL2 protein was mainly located in cytoplasm, the rate of positive cells and SH3GL2 protein expression level were fluctuated with the pathological classification of LSCC; the result of Western blotting showed that SH3GL2 protein was down-regulated significantly in LSCC samples, especially in metastatic lymph nodes. Conclusions These results suggest that SH3GL2 is a LSCC related gene and its expression level is fluctuated with the pathological classification which indicate that SH3GL2 participates in the development and progression of LSCC. And it may be considered as a novel tumor marker to find both a new anti-oncogene and relative factors of invasion and metastasis of laryngeal carcinoma.     

SPECIFIC BINDING OF HUMAN BONE MORPHOGENETIC PROTEIN （2A） WITH MOUSE OSTEOBLASTIC CELLS

Human bone morphogenetic protein 2A (hBMP2A) cDNA terminal 567 nucleotides were cloned and expressed in a phage display vector pCSM2I. Hulnata BMP2A C-terminal peptide displayed on the surface of the phage can bind specifically to the sttrface of mouse osteoblastie cell (MC3T3) membrane. ELISA assay showed a positive signal of the binding by using antibody against M13 phage gene 8 protein. After labeling with ^3HTdR,the counts of the binding groups were 3 to 10 times higher than the control groups. It suggests that the‘surface of MC3T3 cells exist the recepzor for hBMP2A.     

EXPRESSION AND SUBCELLULAR LOCALIZATION OF P9-ZFD PROTEIN IN PATIENTS WITH MYASTHENIA GRAVIS

Objective To express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-related gene P9 ( P9-ZFD ) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG. Methods The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET24a, and the P9-ZFD recombinant protein was induced via E.coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied. Results The molecular weight of purified P9-ZFD protein was about 30kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control. Conclusion P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.     

Identification and validation of novel C/EBPβ-regulated genes in preadipocyte proliferation

Background CCAAT/enhancer-binding protein β（C/EBPβ） is required for mitotic clonal expansion （MCE） during adipogenesis. It is still unclear how C/EBPp regulates MCE in the earlier differentiation programs of 3T3-L1 preadipocytes. The purpose of this paper was to understand why C/EBPβ is required for preadipocyte proliferation, and identify new target genes of C/EBPP with chromatin immunoprecipitation （ChlP）-on-chip. Methods Postconfluent growth-arrested 3T3-L1 preadipocytes were induced to differentiation using a standard differentiation protocol. Chip was performed at 20 hours after induction with specific anti-C/EBPβ antibodies. The precipitated DNA was amplified, labeled and hybridized with a mouse promoter microarray. Compared with the control in which the ChIP experiment was performed with non-specific antibody, only the genes with a signal increasing more than 2 fold were considered as candidate genes.Results A total of 110 candidate genes were identified. BTG3 associated nuclear protein （SMAR1, Banp） and tripartite motif-containing 35 （Hls5, trim35） were two target genes among the 110 candidate genes which are involved in cell cycle regulation; the binding of C/EBP3 to the promoter of banp and trim35 was verified by ChlP-PCR. Conclusion C/EBPβ may regulate preadipocyte proliferation through activation of banp and trim35.