[Association between A49T polymorphism of SRD5A2 gene and risk of prostate cancer].

OBJECTIVE: To investigate the association between A49T polymorphism of SRD5A2 gene and risk of prostate cancer. METHODS: PCR was used to examine the A49T polymorphisms of SRD5A2 gene in the tissues of prostate cancer resected from 112 patients (CaP group) and the specimens of benign prostate hyperplasia (BPH group) resected from 89 patients. The association of A49T polymorphism with age of onset, FPSA, TPSA, F/T, T stage, and Gleason score were analyzed. RESULTS: There was no significant difference in A49T polymorphism between the CaP and BPH groups (P > 0.05). The average age of CaP patients was significantly higher than that of the BPH patients (P < 0.05). In the CaP patients, the Gleason score was significantly higher, and the age of onset was significantly lower in the AT + TT genotype than in the AA genotype (both P < 0.05) 2. The age of onset of the AA + AT group was significantly lower than that of the AA group (P < 0.05). CONCLUSION: AA + AT genotype may be of worse prognosis, however, without significant difference. Rank scoring may reflect the relation between Gleason score and A49T genotype and estimate the prognosis better than two-level discrete evaluation.

[Prevention of cell death in LNCaP cells by overexpression of clusterin].

OBJECTIVE: To evaluate the effect of clusterin with and without leader sequence on overexpression preventing apoptosis in human prostate LNCaP cells. METHODS: The plasmid pIRES2-EGFP was used to generate the clusterin expression constructs with full-length or without the leader sequence (designated as pIRES2-EGFP/cluac, pIRES2-EGFP/clubc, respectively). Western blot analysis was employed to compare clusterin expression levels in the lysis and supernatant fluid of clusterin transfected LNCaP cells in vitro. The distribution of different functional domains of clusterin in cells was detected with Immunocytochemical staining. The clusterin's protective role of Na2SeO3-induced apoptosis in LNCaP cells was examined by flow cytometry (FCM) and fluorescence microscope. RESULTS: Clusterin expression was detected in the lysis and supernatant fluid of pIRES2-EGFP/cluac transfected LNCaP cells, while clusterin was found only in lysis liquid of pIRES2-EGFP/clubc transfected LNCaP cells, but not found in their supernatant fluid. The distribution of cluserin in the plasm of pIRES2-EGFP/cluac transfected cells was aggregative, and on the other hand, clusterin distributed dispersedly in pIRES2-EGFP/clubc transfected cells. Its anti-apoptotic property in LNCaP cells was proved by FCM and fluorescence microscope. CONCLUSION: It is apparent that clusterin plays an important role in preventing apoptosis in prostate cancer, and the presence of the leader sequence is necessary for clusterin's anti-apoptotic function.

[Association of polymorphisms in testosterone 5-alpha-reductase II genotype and prognosis factors of prostate cancer].

OBJECTIVE: The correlation were studied between testosterone 5-alpha-reductase II (SRD5A2) gene polymorphisms and prognosis factors. METHODS: V89L and A49T variants was identified with Mwo1 and Rsa1. The differences of V89L and A49T between cancer of prostate (CaP) and benign prostatic hyperplasia (BPH) were studied. In addition, we also researched the association of polymorphisms with age of onset, free prostate specific antigen (FPSA), total PSA (TPSA), FPSA/TPSA (F/T), Gleason score, and T stage in cancer group. RESULTS: We found no differences of V89L and A49T polymorphisms between CaP and BPH. In CaP group the A49T variant was associated with lower age of onset (P = 0.03) and higher Gleason score (P = 0.015). There were no differences between VV and VL+LL polymorphisms with any of the characteristics studied. When the characteristics above were regarded as two-level discrete variable, there were no differences by A49T and V89Lvariants. CONCLUSION: In CaP group, the AT+TT genotype was perhaps associated with poor prognosis. VL+LL genotype has no relation with prognosis.

[Expression of human-mouse chimeric antibody ch-BD1 and its affinity to human bladder cancer in vitro and in vivo].

OBJECTIVE: To investigate the expression of human-mouse chimeric antibody ch-BD1 against human bladder cancer and its affinity to human bladder cancer in vitro and in vivo. METHODS: Three kinds of mutated fragments of dihydrofolate reductase (DHFR) gene were created by techniques of molecular biology to decrease the transcriptional activities and then cloned into pDHL-BD1 so as to construct the vectors pWSD1-BD1, pWSD2-BD1, and pWSD3-BD1 expressing the human-mouse chimeric antibody ch-BD1 against human bladder cancer with decreased expression of DHFR gene. These vectors and pDHL-BD1 were transfected into Chinese hamster ovary cell (CHO)/DHFR- cell respectively. 72 hours later Northern blotting was used to examine their DHFR gene expression. Methotrexate (MTX) of increasing concentrations was added into the culture of transfected CHO/DHFR-cells. The ch-BD1 levels in the supernatants were measured. Purified ch-BD1 was labeled by (99m)TcO(4)(-) and added into the serially diluted solutions of human bladder cancer EJ cells to examine their radioactivities and calculate their affinity constants. EJ cells were injected into the roots of hind limb of 3 Balb/C mice. Four weeks later, (99m)TcO(4)(-)-labeled antibody ch-BD1 were injected into the rats' caudal veins. Radioimmunoimaging was conducted to examine the distribution of the antibody. RESULTS: The sequence of DHFR gene expression levels from strong to weak in the constructed vectors was as follows: pDHL-BD1 > pWS1-BD1 > pWS3-BD1 > pWS2-BD1. When the concentration of MTX was 10(-6) mol/L the expression level of ch-BD1 was significantly correlated to the expression level of DHFR gene, the lower the baseline expression level of DHFR gene the higher the expression level of ch-BD1. After the serially diluted EJ cells were co-incubated with the (99m)TcO(4)(-)-labeled antibody ch-BD1 the immunoactivity ratio of ch-BD1 was 76%, and that of murine monoclonal antibody was 81%; the affinity constant of ch-BD1 was 3.56 x 10(9) M(-1), and that of murine monoclonal antibody BD1 was 1.22 x 10(9) M(-1). 6 hours after injection of the (99m)TcO(4)(-)-labeled antibody ch-BD1 into the mice body it was mainly distributed in the tumor, 22 hours later, it was specifically distributed in tumor, and 24 hours later it was still concentrated here. CONCLUSION: Decrease of the baseline expression level of DHFR gene effectively increases the amplification of MTX to exogenous gene. The human-mouse chimeric antibody ch-BD1 shows an ideal affinity activity to human bladder cancer in vivo and in vitro and has a certain clinical prospect.

[Construction and screening of suppression subtractive hybridization library of renal cell carcinoma].

OBJECTIVE: To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[In vitro and in vivo functional evaluation of anti-human bladder tumor human-mouse chimeric antibody ch-BDI].

OBJECTIVE: To evaluate the in vitro and in vivo function of anti-human bladder tumor human-mouse chimeric antibody ch-BDI and its future clinical application. METHODS: With ch-BDI in high-expression cell-line medium, affinity chromatography was used for the purification. Labeled with (99m)Tc through reduction method, its immunoreactive fraction and association constant were measured. The constant was injected into nude mice with xenografted human bladder tumor. The biodistribution of the labeled ch-BDI was studied with radioimmunoimaging. RESULTS: ch-BDI showed desirable immunoreactive fraction (76%) and association constant (3.56 x 10(9) M(-1)) in vitro and a terrific specific targeting effect in vivo. CONCLUSION: ch-BDI has fairly good function against human bladder tumor both in vitro and in vivo, and is promising in clinical use.

[Screening of novel genes differentially expressed in human renal cell carcinoma by suppression subtractive hybridization].

BACKGROUND AND OBJECTIVE: Identifying the differentially expressed genes in renal cell carcinoma (RCC) contributes to the elucidation of its genetic basis. However, the above knowledge has not yet been fully understood. The aims of this experiment were to screen novel genes differentially expressed in RCC tissues by suppression subtractive hybridization (SSH) and clone RCC-specific related genes. METHODS: To construct SSH library of RCC by using the mRNA from RCC tissues and matched normal kidney tissues as tester and driver, respectively. Partial positive clones in the library were selected randomly and sequenced, then analyze the sequences with the BLAST software. To confirm the location of the fragments of interest in human chromosome through comparing their sequences with the human genome draft. mRNA levels of the novel genes in RCC and matched normal kidney tissues were determined by Northern blot and semi-quantitative RT-PCR analysis. RESULTS: The SSH library contained 414 positive clones. Random analysis of 280 clones with enzyme restriction showed that 265 clones contained cDNA fragments distributed mainly between 300-900bp. Among 80 arbitrary clones with were derived from above 265 clones and sequenced, No. 28, 158, 170, and 249 clones are previously unknown genes and located in human chromosome 21q22, 4p15.3, 9q34, and 22q11.2 by electronic mapping, respectively. The consequence of semi-quantitative RT-PCR demonstrated that mRNA levels of the two novel genes were overexpressed in RCC compared to matched normal tissues by more than 2-6 folds. Northern blot analysis confirmed the above results. CONCLUSIONS: SSH is a reliable strategy for screening novel genes differentially expressed in RCC. The novel gene fragments can be used to clone their full length and further to study their functions.

[Correlation and significance of E-cadherin and N-cadherin expression in prostate cancer].

BACKGROUND & OBJECTIVE: Decreased expression of E-Cadherin correlated with many kinds of cancer, but inappropriate expression of N-cadherin has been reported in breast cancer recently, which has a more distinct and direct role in promoting cancer cell motility than E-cadherin. The aim of this study was to investigate the correlation between E-Cadherin and N-Cadherin expression with the grade and stage of prostate cancer and their relationship with prostate specific antigen (PSA). METHODS: E-Cadherin and N-Cadherin expression in 56 prostate cancer samples were determined by immunohistochemical staining. RESULTS: Twenty-four patients (43%) were positive and 32 patients (57%) were negative in E-Cadherin expression. Eighteen patients (32%) were negative and 38 patients (68%) were positive in N-Cadherin expression. E-Cadherin and N-Cadherin were significantly related to the grade and stage of cancer and the change of F/T ratio (free prostate specific antigen/total prostate specific antigen ratio). There was no significant relationship between Cadherin expression and total prostate specific antigen (tPSA) or free prostate specific antigen (fPSA). CONCLUSION: E-Cadherin and N-Cadherin abnormal expressions may serve as the indicators to malignant degree and prognosis of the prostate cancer.