ABSTRACT
<p><b>OBJECTIVE</b>To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro.</p><p><b>METHODS</b>Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential)and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1, which was markedly downregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation. Real-time PCR and Western blot were used to confirm the gene expression of PAG1 at mRNA and protein levels. Full-length coding sequence of human PAG1 was subcloned into plasmid pcDNA3.0 and the recombinant plasmids were stably transfected into PC-3M-1E8. The cell proliferation ability, anchorage-independent growth, cell cycle distribution, apoptosis rates and invasive ability were detected by MTT, and in addition, soft agar colony formation, flow cytometry analysis and matrigel invasion assay using Boyden chamber were also carried out respectively. All experiments contained pcDNA3.0-PAG1-transfected clones, vector transfected clones and non-transfected parental cells.</p><p><b>RESULTS</b>A total of 327 differentially expressed genes were obtained between the high and low metastatic sublines of PC-3M cells, including 123 upregulated and 204 downregulated genes in PC-3M-1E8. A total of 281 genes, including 167 upregulated and 114 downregulated genes were obtained in PG-BE1 cells. Nine genes were simultaneously downregulated and 8 genes were upregulated in both high metastatic cell lines of PC-3M and PG. The expression of PAG1 at mRNA and protein level were decreased in the high metastatic subline PC-3M-1E8. Western blot revealed that PAG1 protein was downregulated in PC-3M-1E8 cell line which was in agreement with the gene expression at mRNA level. The proliferation ability and clonogenicity of PAG1 overexpression cells by stable transfection were markedly decreased in comparing with that of the control cells (P < 0.05). Colonies formed in stably PAG1-transfected cells, the vector-transfected clones and parental cells were 26.7 ± 5.2, 47.2 ± 3.2 and 52.3 ± 3.4 respectively (P < 0.05). Flow cytometry analysis showed that the stable PAG1-transfected cells at G₀-G₁ phase were significantly more than that of the control cells (P < 0.05). However, no difference of the apoptosis rate was found between PAG1-transfected cells and control cells (P > 0.05). The number of cells passing through the matrigel and multipore membrane was also decreased in the stable PAG1-transfected cells (35.1 ± 4.9) compared with those of the vector-transfected clones (127.6 ± 6.6) and parental cells (135.0 ± 5.0, P < 0.05).</p><p><b>CONCLUSIONS</b>Using cDNA microarray technique and differential gene expression analysis of sublines of the parental cancer cell lines enable of revealing the metastasis-related genes, among which PAG1 represents one of those under-expressed genes in the high metastatic subline PC-3M-1E8. Transfection expression of PAG1 suppresses cell proliferation, anchorage-independent growth and invasive ability of PC-3M-1E8 cells in vitro. Conclusively, PAG1 may play an important role in inhibiting the proliferation, invasion and metastasis of the cancer cells.</p>
Subject(s)
Humans , Male , Adaptor Proteins, Signal Transducing , Genetics , Metabolism , Physiology , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Gene Expression Profiling , Membrane Proteins , Genetics , Metabolism , Physiology , Neoplasm Invasiveness , Neoplasm Metastasis , Oligonucleotide Array Sequence Analysis , Plasmids , Prostatic Neoplasms , Genetics , Metabolism , Pathology , RNA, Messenger , Metabolism , Recombinant Proteins , Genetics , Metabolism , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the mechanism of influence of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behavior of human prostatic cancer cell line PC-3M-1E8.</p><p><b>METHODS</b>The expression of GST-Raf1-RBD recombinant protein, a specific binding domain of active Ras (GTP-Ras), was induced by IPTG in JM109 bacterium. SDS-PAGE and coomassie brilliant blue staining were performed using cleaved product of the bacterium to determine the expression of the fusion protein. GST-pull down essay was designed to detect the level of active Ras in PGA1 and vector transfected, respectively and in the native PC-3M-1E8 cells. Western blotting was used to detect the expression levels of downstream proteins of Ras signal pathway which may be related to the function of PAG1. Phalloidine labeled by tetramethylrhodamine-5-(and-6) isothiocyanate (TRITC) was used for the staining of intracellular F-actin, Laser passing confocal microscopy was adopted for observing change of the cell morphology and the arrangement of F-actin.</p><p><b>RESULTS</b>After IPTG induction, the GST-Raf1-RBD recombinant protein, with a molecular weight of 33 000, was noticed to be highly expressed in JM109 bacterium. GST-pull down assay revealed that the expression level of active Ras markedly decreased after PAG1-transfection while the total Ras remained unchanged. The expression of p-ERK and cyclin D1 in the PAG1-transfected cells decreased in accordance with the level of active Ras. However, the expression of p21(WAF1/CIP1) and p-Akt didn't show any variation. Additionally, the structure of F-actin was turbulent and the pseudopodia of cells diminished conspicuously after PAG1-transfection. There was a high expression of PAG1 in normal human prostate tissue, however, the positive rate of PAG1 immuno-staining decreased in cases of prostatic adenocarcinoma, correspondent with increasing of the grading index of cell differentiation established in the Gleason grading system for the diagnosis of prostate adenocarcinoma.</p><p><b>CONCLUSIONS</b>An over-expression of PAG1 in PC-3M-1E8 cells effectively suppresses the activation of Ras and ERK, as well as the cyclin D1 expression, leading to an inhibition of the proliferation ability of tumor cells. The turbulence of F-actin and reduction of pseudopodia of cells result in an impairment of cell mobility, invasiveness and metastatic capability. In human prostate and prostatic adenocarcinoma tissues, the expression of PAG1 is related to the cellular differentiation and malignancy.</p>
Subject(s)
Humans , Male , Actins , Metabolism , Adaptor Proteins, Signal Transducing , Metabolism , Adenocarcinoma , Metabolism , Pathology , Cell Cycle , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cyclin D1 , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Extracellular Signal-Regulated MAP Kinases , Metabolism , Membrane Proteins , Metabolism , Neoplasm Invasiveness , Prostate , Metabolism , Prostatic Neoplasms , Metabolism , Pathology , Proto-Oncogene Proteins c-akt , Metabolism , Transfection , ras Proteins , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of tumor metastasis-related gene TMSG-1 overexpression on the proliferation and invasion of a highly metastatic prostate cancer cell line in vitro.</p><p><b>METHODS</b>The eukaryotic expression plasmids containing full-length TMSG-1 cDNAs were stably transfected into the highly metastatic prostate cancer cell line PC-3M-1E8. Clones highly expressing TMSG-1 were identified by RT-PCR and Western Blot analysis after G418 screening. The cell proliferation was detected by cell growth curve, MTT assay and soft agar colony formation assay. The invasive potential of tumor cells in vitro was tested by Matrigel invasion assay.</p><p><b>RESULTS</b>Three TMSG-1 overexpression clones were selected. Cell growth curve and MTT assay showed that TMSG-1 overexpression clones exhibited a strong inhibition of proliferation compared with that of the parental cells or those transfected with vector alone from the third day of culture (P <0.05). In vitro analysis also showed that the TMSG-1 transfected clones exhibited a decreased clonogenicity in soft agar compared with that of the parental cells or those transfected with vector only (P < 0.05). TMSG-1 expression significantly suppressed cell invasion in vitro of TMSG-1-transfected PC-3M-IE8 cells (P < 0.05).</p><p><b>CONCLUSION</b>The TMSG-1 protein may serve as a tumor metastasis suppressor due to inhibiting cell proliferation and invasion of the highly metastatic prostate cancer cell line PC-3M-1E8.</p>
Subject(s)
Animals , Humans , Male , Mice , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Membrane Proteins , Genetics , Metabolism , NIH 3T3 Cells , Neoplasm Invasiveness , Prostatic Neoplasms , Metabolism , Pathology , RNA, Messenger , Metabolism , Sphingosine N-Acyltransferase , Transfection , Tumor Suppressor Proteins , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of RhoC in breast cancer cells with different metastatic potential and its correlation with invasiveness.</p><p><b>METHODS</b>Expression of RhoC mRNA and protein in human breast cancer cells MCF-7 with low metastatic potential and MDA-MB-231 with high metastatic potential was detected by RT-PCR, Western blot, immunohistochemistry and immunofluorescence staining. Eukaryotic expression plasmids of RhoC were constructed and transfected into MCF-7 cells. The biological effects were observed, including in vitro invasion by Boyden charmber assay, motility by would healing assay, alteration of microfilament network by TRTIC-phalloidin staining and expression of p-Akt by Western blot assay.</p><p><b>RESULTS</b>The expression levels of RhoC mRNA and protein varied in the two different metastatic breast cancer cell lines. RhoC was significantly up-regulated in the highly metastatic cells in comparison to the weakly metastatic counterpart (P < 0.01). As shown by Boyden charmber assay, the invasive capacity of transfected cells overexpressing RhoC was significantly promoted as reflected by more penetrating cells (56.88 +/- 4.18) than that of the antisense transcripts (23.12 +/- 3.22), the negative (23.77 +/- 3.64) and blank controls (28.44 +/- 2.48). Further study by would healing assay indicated that cells overexpressing RhoC were more motile in actin-based active movement. The wound healing ratio after 24 h of the sense transcripts, antisense transcripts, negative controls and blank controls was 58.28% +/- 2.14%, 22.36% +/- 2.73%, 28.23% +/- 2.62%, 30.18% +/- 2.86%, respectively. The TRITC-phalloidin staining revealed less actin filament bundles and a reorganized cytoskeleton within the sense transcripts. In addition, p-Akt expression level was upregulated in the sense transcripts.</p><p><b>CONCLUSION</b>RhoC overexpression may promote the invasive capacity of human breast cancer cells in vitro and its expression level is positively correlated with the metastatic capacity of those cells. So RhoC may be a potential target in the development of a novel strategy for treating metastasis of breast cancer.</p>
Subject(s)
Female , Humans , Breast Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Movement , Gene Expression Regulation, Neoplastic , Neoplasm Invasiveness , Phosphorylation , Plasmids , Proto-Oncogene Proteins c-akt , Metabolism , RNA, Messenger , Metabolism , Transfection , Up-Regulation , rho GTP-Binding Proteins , Genetics , Metabolism , rhoC GTP-Binding ProteinABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of tumor metastasis suppressor gene 1 (TMSG-1) overexpression on the proliferation, invasion and apoptosis of breast cancer cells and to determine possible correlations of TMSG-1 and metastasis of breast cancer.</p><p><b>METHODS</b>Full-length human TMSG-1 coding sequences were cloned into plasmid pcDNA3.0-FLAG. The recombinant plasmids constructs were transfeced into MDA-MB-231, a highly malignant breast cancer cell line. Parental, vector-only stable transfectant and TMSG-1 stable transfectant clones were tested by MTT, soft agar colony formation and Boyden chamber assays. At twenty-four hours and forty-eight hours post transient transfection, double staining with Annexin-V-FITC and PI were employed to distinguish apoptotic cells from living cells by flow cytometry analysis.</p><p><b>RESULTS</b>Three TMSG-1 overexpression clones were selected. Compared with the control cells, TMSG-1 overexpression MDA-MB-231 cells showed strong inhibition of proliferation and decreased clonogenicity in soft agar (P<0.05). Transfection of TMSG-1 into MDA-MB-231 cells significantly suppressed the cell invasion ability in vitro (decreased numbers of cells trespassing the matrigel in three experiments: 72.3+/-8.1, 85.0+/-4.2, and 73.5+/-7.8) in comparison with nave cells without transfection (187.5+/-2.1) and cells transfected with the control vector (162.3+/-6.8) (P<0.01). Transient transfection of TMSG-1 into MDA-MB-231 cells could promote cell apoptosis at 24 and 48 hours after transfection (P<0.05).</p><p><b>CONCLUSIONS</b>TMSG-1 protein may have multiple functions in the regulation of proliferation, invasion and apoptosis of metastatic breast cancer cells, likely as a metastasis suppressor gene.</p>
Subject(s)
Female , Humans , Apoptosis , Breast Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Membrane Proteins , Genetics , Metabolism , Physiology , Neoplasm Invasiveness , Plasmids , Recombinant Proteins , Metabolism , Sphingosine N-Acyltransferase , Genetics , Metabolism , Physiology , Transfection , Tumor Suppressor Proteins , Genetics , Metabolism , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To determine the expression level of survivin in androgen-independent prostate carcinoma, and to investigate the biological role of survivin in invasion and metastasis of androgen-independent prostate carcinoma.</p><p><b>METHODS</b>Highly metastatic prostatic cancer cell line PC-3M-1E8 was stably transfected with pSilencer plasmid targeting survivin expression by RNA interference. The biological effects were observed, including anchorage-independent growth, in vitro invasion by soft agar colony formation and Boyden chamber assay, and also in vivo tumorigenesis in nude mice. Cell cycle and apoptosis indices were evaluated by flow cytometry and Western blot analysis of bioactive fragments of caspase 3.</p><p><b>RESULTS</b>The expression of survivin in transfected PC-3M-1E8 cells was markedly depressed at both mRNA and protein levels (about 78% to 80%) as compared with control. The growth of tumor cells was retarded by anchorage-independent growth assay. The survivin transfectants formed smaller and fewer colonies (14.33 +/- 3.51) than the negative (52.33 +/- 6.81) and blank controls (54.00 +/- 6.00). Inhibition of survivin expression was correlated with enhanced apoptosis of tumor cells (percentages of apoptotic cells of the negative control, blank control and experimental groups were 5.88 +/- 0.99, 6.97 +/- 1.60, 16.40 +/- 1.95 respectively), along with an increased expression of activated caspase 3, and cell cycle inhibition at G(0)/G(1) phase (the relative number of cells at G(0)/G(1) phase were 43.65 +/- 3.44, 43.59 +/- 1.83 and 52.71 +/- 1.10, respectively). In addition, multinucleated giant cells were observed along with a marked inhibition of invasion as reflected by fewer penetrating cells by Boyden chamber assay (46.07 +/- 9.97, 47.87 +/- 9.58 and 38.67 +/- 6.59, respectively).</p><p><b>CONCLUSIONS</b>Survivin expression is high in androgen-independent prostate cancer cells and likely may be related to the apoptosis, growth and invasion of the tumor cells. Targeting the survivin pathway by RNA interference appears to be a promising approach for clinical treatment of androgen-independent prostate cancer.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Androgens , Metabolism , Apoptosis , Blotting, Western , Caspase 3 , Metabolism , Cell Cycle , Cell Line , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Green Fluorescent Proteins , Genetics , Metabolism , Inhibitor of Apoptosis Proteins , Mice, Inbred BALB C , Mice, Nude , Microtubule-Associated Proteins , Genetics , Metabolism , NIH 3T3 Cells , Neoplasm Metastasis , Neoplasm Proteins , Genetics , Metabolism , Neoplasm Transplantation , Prostatic Neoplasms , Genetics , Metabolism , Pathology , RNA Interference , Transfection , Transplantation, HeterologousABSTRACT
<p><b>OBJECTIVE</b>In order to clarify the exact molecular weight of tumor metastasis suppressor gene-1 (TMSG-1) protein and its cellular localization, a monoclonal antibody against TMSG-1 was prepared, characterized and applied to evaluate the metastatic potential of human tumors.</p><p><b>METHODS</b>A dominant epitope-TMSG-1(15)-derived from TMSG-1 was synthesized based on Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen preparation was used to immunize BAL B/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, which were further characterized by western blotting and immunohistochemical staining.</p><p><b>RESULTS</b>One hybridoma cell line secreting anti-TMSG-1 antibody, designated as C8, was eventually established after primary ELISA screening, followed by rapid limited dilution procedure. It was confirmed that C8 was of IgM isotype. Result of competitive inhibition assay showed that the antibody was TMSG-1 specific. Using this antibody, an expected protein band of about 45,000 (relative molecular mass) was detected in the non-metastatic variants PC(3)-2B4 and PG-LH7 cells by Western blotting, but not in the isogenetic metastatic variants of PC3-1E8 and PG-BE1 cells. Immunohistochemistry using C8 showed a positive staining of cell membrane and cytoplasm of 2B4 and LH7 cells, whereas 1E8 and BE1 cells were non-reactive. Immunostaining using C8 of paraffin sections of 52 breast carcinomas and 41 colon cancers demonstrated a strong positivity in non-metastatic tumors, but none to weakly reactive in metastatic tumors (P < 0.05).</p><p><b>CONCLUSION</b>C8 monoclonal antibody against the synthetic peptide is TMSG-1 specific and is effective for Western blot and immunohistochemistry assays to detect TMSG-1 expression in cancer cells. TMSG-1 protein is about 45 000 (relative molecular mass) at cell membrane and cytoplasm of tumor cells. Expression of TMSG-1 protein correlates well, inversely with the tumor metastatic potential.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Antibodies, Monoclonal , Allergy and Immunology , Breast Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Membrane , Metabolism , Colonic Neoplasms , Metabolism , Pathology , Cytoplasm , Metabolism , Gene Expression Regulation, Neoplastic , Hybridomas , Allergy and Immunology , Bodily Secretions , Membrane Proteins , Allergy and Immunology , Metabolism , Mice, Inbred BALB C , Neoplasm Metastasis , Sphingosine N-Acyltransferase , Tumor Suppressor Proteins , Allergy and Immunology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors.</p><p><b>METHODS</b>By using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fusion protein was used to immunize BALB/c mice. The monoclonal antibody against G3BP was produced through conventional hybridoma method and characterized by ELISA, Western blot and immunohistochemical staining.</p><p><b>RESULTS</b>A hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the antibody was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively with the presence of lymph node metastasis and c-erbB2 expression.</p><p><b>CONCLUSIONS</b>The G3BP-specific monoclonal antibody derived from recombination protein can be used in ELISA, Western blot and immunohistochemical assay. It may provide an important tool in analysis of G3BP in in vitro and in vivo experiments. Besides, G3BP may serve as another prognostic marker for breast cancer.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Antibodies, Monoclonal , Allergy and Immunology , Biomarkers, Tumor , Breast Neoplasms , Metabolism , Pathology , Carrier Proteins , Genetics , Allergy and Immunology , Metabolism , DNA Helicases , Genetic Vectors , Hybridomas , Bodily Secretions , Lymphatic Metastasis , Mice, Inbred BALB C , Poly-ADP-Ribose Binding Proteins , RNA Helicases , RNA Recognition Motif Proteins , Receptor, ErbB-2 , Metabolism , Recombinant Fusion Proteins , Genetics , Metabolism , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To study effects of alternative transcripts of ING1 transfection on human cancer cell lines.</p><p><b>METHODS</b>p47/ING1A and p33/ING1B expression vehicles were constructed and introduced into a human breast cancer cell line MCF-7 and a human lung cancer cell line PAa, both expressing wild-type p53 protein. Growth characteristics of the transfectants and potentially related genes were analyzed.</p><p><b>RESULTS</b>The levels of p47/ING1A and p33/ING1B protein elevated respectively in tumor cells of MCF-7 and PAa after transfected with p47/ING1A and p33/ING1B, and the latter was much higher than that of the former. Ectopic overexpression of p33/ING1B effectively blocked tumor cell growth and arrested cells in the G(0) approximately G(1) phase of the cell cycle (P < 0.01), while p47/ING1A gave no effect on cell growth or cell cycle. Tumor cells overexpressing p33/ING1B contained more p21(WAF1) protein than that of the control cells, with undisturbed p53 protein level.</p><p><b>CONCLUSIONS</b>Expression of two different transcripts of ING1 may have different effects on tumor cell growth. p33/ING1B may cooperate with p53 in stimulating expression of p21(WAF1) gene, thus to arrest cell cycle and to inhibit tumor cell growth. p33/ING1B may be considered to be a candidate as a partner of p53 in gene therapy.</p>