| OBJECTIVES In the entire proceeding, stomach carcinoma is affected with factors related to cell growth and differentiation, including hormones. To discover the behavior of hCG and its receptor, LH/hCG receptor, in the pathogenesis and evolvement of stomach carcinoma, the clinical implications and relationships of hCG, LH/hCG receptor, ER and PR expressed on stomach carcinoma and mutation of LH/hCG receptor were investigated, and effect of hCG on cell proliferation, cell phases and oncogenes related with stomach carcinoma as well. METHODS With built ligand-labeling affinity histocytochemistry for LH/hCG receptor and immunohistocytochemistry for hCG, ER and PR, their expression was studied in paraffin-embeded specimens of stomach adenocarcinoma. LH/hCG receptor, ER and PR mRNA was examined with real-time quantitive RT-PCR in serum-free cultured SGC-7901 cell, a stomach adenocarcinoma cell line, incubated with hCG. MTT and flow cytometric analysis was served to observe cell proliferation and cell cycle phases of SGC-7901 stimulated by hCG, respectively, at the same time, c-fos, c-erbB2, c-met and uPA mRNA was determined using real-time quantitive RT-PCR.mRNA of those genes was also assessed in the presence of inhibitors specified for PKA, PKC and CaM(PKAI, PKCI and CaMI) with or without hCG. Additionally, conjugated bilirubin was used to observe the influence of antioxidant on cell growth of SGC-7901 induced by hCG. Finally, LH/hCG receptor mRNA was amplified by RT-PCR with a pair of primers outside for full length(1-2101) and another pair inside for proofing the fidelity of PCR(478-2086). After cloned and sequenced, the cDNA was analyzed by OMIGAand blasted in Genbank.RESULTS 1. LH/hCG receptor stained with ligand-labeling affinityhistocytochemistry was identical to stained with immnohistocytochemistry. Thecases positive with ER and PR were significant higher than with hCG and LH/hCGreceptor(51.1 %, 40.0%; 20.0%, 22.2%). LH/hCG receptor had no relation with hCG,and only coexpressed with hCG in a small portion of cases (44.4%). ER or PR lied in77.8% or 66.7% of hCG positive cases, but only in 20.0% or 30.0% of LH/hCGreceptor positive cases. However, hCG had no relation with ER, or PR or thecoexpression of PR and LH/hCG receptor, except for a markedly relation with thecoexpression of ER and LH/hCG receptor. The rate of ER, PR, hCG and LH/hCGreceptor expressed in high-differentiated adenocarcinoma was higher than inpoor-differentiatial or mucinous adenocarcinoma. Cases with lymph nodemetastasis presented each of the four indices was higher than the ones withoutmetastasis. mRNA of ER and PR but not of LH/hCG receptor in SGC-7901 cell wasinduced by hCG in biphasic style. 2. At lower concentration of hCG (<8.93IU/ml), cellgrowth, cells of S, G2/M, and mRNA of c-erbB2, c-met and uPA increased inSGC-7901 cell, but decreased at higher concentration (>8.93IU/ml), and thedecrease was more intense than the increase, but cell growth were independent ofthe incubating time and bilirubin. PI (proliferation index) was linear to mRNA ofc-erbB2, c-met and uPA, whereas c-fos mRNA increased in an exponential typedependency on hCG concentration lower than 35.72 ID/ml. Analyzed by statistics, itshowed that the genes above interact mutually. 3. Under the stimulation of hCG,expression of uPA, c-met and c-fos mRNA was inhibited by PKCI, PKAI and CaMI,but promoted by CaMI, PKCI and PKCI, respectively. Both PKAI and PKCIenhenced induction of c-erbB2 mRNA by hCG. Any two of the three inhibitorscombined acted similarly to the results applying singly abovementioned. 4. Ahomozygous G to C transversion was identified at 1732 in exon 11 of LH/hCGreceptor in SGC-7901 cell, resulting in a Asp578His substitution located in thetransmembrane domain 6(TM6), and a homozygous polymorphism C to T transitionat 1065 was also found.CONCLUSIONS 1. Ligand-labeling affinity histocytochemistry was reliable, practical and e... |
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