ABSTRACT
Jumonji Domain Containing 2A (JMJD2A) may be a cancer-associated gene involved in human breast cancer. With a view to investigating expression of JMJD2A in human breast cancer and benign lesion tissues as well as relationship between JMJD2A and tumor related proteins, histological and immunohistochemical analysis, Western blot and quantitative real-time PCR in infiltrating duct carcinoma and fibroadenoma for JMJD2A and immunohistochemical analysis and quantitative real-time PCR in infiltrating duct carcinoma for tumor related proteins (ARHI, p53, ER, PR and CerbB-2) were performed. Histological examination validated the clinical diagnosis. The JMJD2A positive rate of infiltrating duct carcinoma was significantly higher than fibroadenoma by immunohistochemical analysis. The mean optical density of JMJD2A in infiltrating duct carcinoma was higher than fibroadenoma by western blot. JMJD2A mRNA level in infiltrating duct carcinoma was higher than fibroadenoma by quantitative real-time PCR. Spearman correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from immunohistochemical results respectively. Pearson correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from quantitative real-time PCR results respectively. Expression of JMJD2A in infiltrating duct carcinoma was higher, and associated with ARHI, p53 and ER. The results may take JMJD2A as a potential diagnostic and therapeutic target in human breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Cell Line, Tumor , Female , Fibroadenoma/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Jumonji Domain-Containing Histone Demethylases/biosynthesis , Receptor, ErbB-2/biosynthesis , Receptors, Estrogen/biosynthesis , Receptors, Progesterone/biosynthesis , Tumor Suppressor Protein p53/biosynthesis , rho GTP-Binding Proteins/biosynthesisABSTRACT
<p><b>OBJECTIVE</b>To study the in vitro expression of 6 novel missense mutations (R270G, P275A, F121L, A156P, E183G, I324N) and a previously described R408Q mutation of phenylalanine hydroxylase (PAH) gene and explore the genotype-phenotype correlation through comparison of protein levels and residual enzyme activities.</p><p><b>METHODS</b>Seven expression vectors containing PAH cDNA were constructed with a site-directed mutagenesis kit. The plasmids were extracted and sequenced to confirm the target mutations. pcDNA3.0 containing PAH cDNA was transfected into COS-7 cells and total proteins were extracted 48 h after transfection. The quantities of proteins and residual enzyme activities of the 7 mutants were assessed with the wild-type PAH gene as reference.</p><p><b>RESULTS</b>Relative quantities of PAH proteins for R270G, P275A, F121L, A156P, E183G, I324N and R408Q were 10.5%, 56.6%, 54.3%, 8.7%, 8.5%, 67.3% and 85.4%, respectively. The residual enzyme activities were 7.7%, 27.6%, 19.0%, 10.4%, 9.1%, 50.6% and 40.2%, respectively.</p><p><b>CONCLUSION</b>PAH residual enzyme activities of 7 PAH mutants were all significantly reduced.</p>
Subject(s)
Animals , Humans , Amino Acid Sequence , COS Cells , Chlorocebus aethiops , Genetic Association Studies , Methods , Molecular Sequence Data , Mutation, Missense , Phenylalanine Hydroxylase , Genetics , Sequence AlignmentABSTRACT
<p><b>OBJECTIVE</b>To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC).</p><p><b>METHODS</b>Poly A(+) RNA was isolated from RCC lines 786-O (tester) and renal cell (RC) lines HK-2 (driver), respectively. SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit (Clontech), and PCR products were cloned into pT-Adv vector and transformed E. coli TOP10F'. All positive clones picked out were digested and some of which were sequenced.</p><p><b>RESULTS</b>The SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb. Among 50 clones sequenced randomly, 2 represented unknown genes and the other 48 derived from 36 known genes.</p><p><b>CONCLUSION</b>The quality of the SSH library of human RCC is reliable and its construction is the basis for further screening differentially expressed genes of RCC.</p>
Subject(s)
Humans , Adenocarcinoma, Clear Cell , Genetics , Cell Line, Tumor , Gene Library , Kidney Neoplasms , Genetics , Nucleic Acid Hybridization , MethodsABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression and significance of Clusterin in normal prostate, benign prostate hyperplasia (BPH) and prostate cancer.</p><p><b>METHODS</b>Clusterin expression in samples of 12 normal prostate, 15 BPH, and 56 prostate cancer were studied by immunohistochemical stain.</p><p><b>RESULTS</b>Of 83 cases, 67 are positive or weak positive (81%). The rate of positive or weak positive for normal prostate, BPH and prostate cancer was 17% (2/12), 73% (11/15), and 96% (54/56) respectively. The expression level of Clusterin in prostate cancer was much higher than in normal prostate (t = 8.82, P < 0.01). BPH (t = 7.63, P < 0.01) was related positively with pathological grade (r = 0.649, P < 0.01) and stage (r = 0.609, P < 0.01) of prostate cancer.</p><p><b>CONCLUSION</b>Clusterin may play an important role in the biological characteristics of prostate cancer by the anti-apoptosis pathway.</p>