ABSTRACT
In order to screen the genes differentially expressed in two human prostate cancer cells with different metastasis potentials, suppression subtractive hybridization (SSH) was done twice on human prostate cancer cell line with high potential of metastasis PC3M-1E8 and its synogenetic cell line PC3M-2B4 with low metastasis potential. In the first subtraction PC3M-2B4 was used as tester and PC3M-1E8 as driver and the forward subtractive library was constructed. In the second on the tester and driver were interchanged and the reverse subtractive library was constructed. The screened clones of both libraries were sequenced and Gene Bank homology search was performed. Some clones were confirmed by quantitative real-time PCR. The results showed that two subtractive libraries containing 238 positive clones were constructed. Analysis of 16 sequenced clones randomly picked from two libraries showed that 4 differentially expressed gene fragments were identified as new EST with unknown functions. It was concluded that two subtractive libraries of human prostate cancer cell lines with different metastasis potentials were constructed successfully.
Subject(s)
Cell Line, Tumor , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Library , Neoplasm Metastasis/genetics , Nucleic Acid Hybridization/methods , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathologyABSTRACT
In order to screen the genes differentially expressed in two human prostate cancer cells with different metastasis potentials, suppression subtractive hybridization (SSH) was done twice on human prostate cancer cell line with high potential of metastasis PC3M-1E8 and its synogenetic cell line PC3M-2B4 with low metastasis potential. In the first subtraction PC3M-2B4 was used as tester and PC3M-1E8 as driver and the forward subtractive library was constructed. In the second one the tester and driver were interchanged and the reverse subtractive library was constructed. The screened clones of both libraries were sequenced and Gene Bank homology search was performed. Some clones were confirmed by quantitative real-time PCR. The results showed that two subtrac-tive libraries containing 238 positive clones were constructed. Analysis of 16 sequenced clones ran-domly picked from two libraries showed that 4 differentially expressed gene fragments were identi-fied as new EST with unknown functions. It was concluded that two subtractive libraries of human prostate cancer cell lines with different metastasis potentials were constructed successfully.
ABSTRACT
To investigate the reliability and feasibility of human papillomavirus (HPV) DNA test in cervical scraping smears with polymerase chain reaction (PCR), 131 cases of cervical scraping specimens were collected, and the positive rates and accuracy of HPV infection were determined in normal subjects and cervical cancer patients. GP5+/GP6+ and E7 primer pairs designed for detecting HPV L1 and HPV type 16 E7 were tested in this study. Our results showed that positive rates of HPV DNA in normal population and cervical cancer patients were 32.99 % and 73.53 % respectively and there was significant difference between them (P＜0. 001). In normal subjects, detection rates of HPV DNA with GP5+/GP6+ and E7 primer pairs were 27.84 % and 16.49 % respectively, with statistically significant difference between them (P＞0.05). However the detection rates in cervical cancer patients were 38.24 % and 67.65 % for the two markers, with a significant difference found between them (P＜0.05). It is concluded that HPV DNA test with PCR for cervical scraping smears was feasible. GP5+/GP6+ primer pairs may be a useful probe to screen HPV infection in normal population, but they are not sensitive enough in cervical cancer patients. It is suggested that high risk type HPV DNA test was very useful in population with high risk of cervical cancer.
ABSTRACT
The relationship of connexin43 (Cx43) and bystander effect in ovarian tumor cells in herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in vitro was explored and the effect of all-trans retinoic acid (RA) on the expression of Cx43 and bystander effect investigated. The Cx43 expression was detected by flowcytometry, Western blot, and immunofluorescence in two ovarian tumor cell lines OVCAR3, CaOV3 before and after RA treatment. Bystander effect was determined by the cells growth inhibitory rate with methyl thiazolyl tetrazolium. Following exposure to ganciclovir, there was much greater bystander killing in OVCAR3 than that in CaOV3 (P<0.05). The expression of Cx43 was detected in OVCAR3 by flowcytometry and Western blot, but it could not be detected in CaOV3. The expression of Cx43 in both cell lines could be induced by RA. Immunofluoresence staining showed that Cx43 protein of OVCAR3 was located on membrane surface, whereas CaOV3 in cytoplasm. RA could not change the location of Cx43 protein in both cell lines. There is relationship between Cx43 expression and HSV-TK/GCV bystander effect. HSV-TK/GCV bystander effect can be enhanced by RA in ovarian cancer.
Subject(s)
Female , Humans , Pregnancy , Antiviral Agents , Pharmacology , Bystander Effect , Cell Line, Tumor , Connexin 43 , Genetics , Ganciclovir , Pharmacology , Genes, Transgenic, Suicide , Genetic Therapy , Ovarian Neoplasms , Metabolism , Therapeutics , Simplexvirus , Genetics , Thymidine Kinase , Genetics , Tretinoin , PharmacologyABSTRACT
To evaluate the specific inhibition of antisense u-PAR on the u-PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u-PAR recombinant was transfected into highly invasive cell subclones. The u-PAR expression in neo-resistant cells was examined by RT-PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u-PAR in transfected cells decreased sharply, and the rate of inhibition was 53% and 73%, respectively, indicating that an antisense u-PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u-PAR on invasion in highly invasive cell subclones of human prostate carcinoma.
Subject(s)
Humans , Male , Cell Line, Tumor , Cloning, Molecular , Neoplasm Invasiveness , Plasmids , Prostatic Neoplasms , Metabolism , Pathology , RNA, Antisense , Genetics , Receptors, Cell Surface , Genetics , Receptors, Urokinase Plasminogen Activator , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Urokinase-Type Plasminogen Activator , Genetics , MetabolismABSTRACT
To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P < 0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.
Subject(s)
Animals , Humans , Male , Mice , Antisense Elements (Genetics) , Genetics , Pharmacology , Cell Division , Cloning, Molecular , Matrix Metalloproteinase 9 , Genetics , Metabolism , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Prostatic Neoplasms , Metabolism , Pathology , RNA, Antisense , Receptors, Cell Surface , Genetics , Metabolism , Receptors, Urokinase Plasminogen Activator , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tumor Cells, CulturedABSTRACT
Objective: To observe the inhibiting effects of an antisense u-PAR vector on invasion by highly invasive PC-3M cell subclones. Methods: The effects of an antisense vector on invasion by highly invasive PC-3M cell subclones were observed and compared in vitro by monolayer invasion assay and soft agar clone. Then, both a quantitative RT-PCR and zymography were used to exam the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones. Furthermore, the tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. Results: It is found that the speed of growth in vitro was slowing down by highly invasive PC-3M cell subclones transfected with the antisense u-PAR, and the ability of anchorage-independent growth of those cell subclones was also decreasing sharply,and the inhibiting rate was 79% and 60%, respectively. Although the antisense u-PAR didn′t change MMP-9 gene transcription, but they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups reached statistical significance ( P
ABSTRACT
AIM: To investigate the cell cycle arrest ind uced by hypoxia, hypoxia inducible factor-1 and their possible mechanism in huma n ovarian cancer cell line SW626. METHODS: CoCl 2, a chemical inducer of hypoxia and hypoxic cell culture chamber were used to induce chemical and physical hypoxia in human ovar ian cancer cell line SW626. The method of ‘decoy’ was used to block the functi on of HIF-1? because it acts as the core sequence of the target gene as a compe titor combined to the HIF-1?. The cells were divided into group A1 (normal oxyg en), A2 (normal oxygen plus HIF-1? decoy), B1 (CoCl 2), B2 (CoCl 2 plus HIF-1 ? decoy), C1 (hypoxia) and C2 (hypoxia plus HIF-1?). The expression of the HIF -1? protein, mRNA and cell cycle analysis were detected by Western blotting, RT -PCR and flow cytometry (FCM). RESULTS: The expression level of HIF-1? protein in group B1 (3 .75?1.31) and group C1 (3.48?1.01) was significantly higher than that in g roup A1 (0.97?0.31) (P0.05). FCM showed that the G 0/ G 1 phase was markedly increased in group B1 (81.78?24.33) and group C1 (77 .62?22.76) and was significantly higher than that in group A1 (49.49?18.54 ) (P0.05). CONCLUSION: Both CoCl 2 and physical hypoxia could distinctly i nduce cell cycle arrest in G 0/G 1 phase and the expression of HIF-1? in huma n ovarian cancer cell line SW626. HIF-1? plays an important role in cell cycle arrest induced by hypoxia in human ovarian cancer cell line SW626.