Transgenic Arabidopsis thaliana plants expressing a ß-1,3-glucanase from sweet sorghum (Sorghum bicolor†L.) show reduced callose deposition and increased tolerance to aluminium toxicity.

Seventy-one cultivars of sweet sorghum (Sorghum bicolor L.) were screened for aluminium (Al) tolerance by measuring relative root growth (RRG). Two contrasting cultivars, ROMA (Al tolerant) and POTCHETSTRM (Al sensitive), were selected to study shorter term responses to Al stress. POTCHETSTRM had higher callose synthase activity, lower ß-1,3-glucanase activity and more callose deposition in the root apices during Al treatment compared with ROMA. We monitored the expression of 12 genes involved in callose synthesis and degradation and found that one of these, SbGlu1 (Sb03g045630.1), which encodes a ß-1,3-glucanase enzyme, best explained the contrasting deposition of callose in ROMA and POTCHETSTRM during Al treatment. Full-length cDNAs of SbGlu1 was prepared from ROMA and POTCHETSTRM and expressed in Arabidopsis thaliana using the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Independent transgenic lines displayed significantly greater Al tolerance than wild-type plants and vector-only controls. This phenotype was associated with greater total ß-1,3-glucanase activity, less Al accumulation and reduced callose deposition in the roots. These results suggest that callose production is not just an early indicator of Al stress in plants but likely to be part of the toxicity pathway that leads to the inhibition of root growth.

[Anaplastic lymphoma kinase fusion gene expression, clinical pathological characteristics and prognosis in 95 Chinese patients with non-small cell lung cancer].

OBJECTIVE: To examine the prevalence of anaplastic lymphoma kinase (ALK) fusion gene in Chinese patients with non-small cell lung cancer (NSCLC). METHODS: In this study, 95 patients with NSCLC and corresponding clinical information and formalin-fixed paraffin-embedded (FFPE) tissue blocks were included. Hematoxylin & eosin (HE) staining, conventional ALK immunochemistry (IHC) staining and intercalated antibody-enhanced polymer (iAEP) IHC staining, and dual-color split fluorescence in situ hybridization (FISH) for ALK fusion gene were performed. RESULTS: Eight ALK-positive cases were detected using anti-ALK immunohistochemistry with the iAEP method, and FISH analyses revealed 4 patients of them who harbored the ALK fusion gene (4.2%, 4/95), including 2 cases of female patients with solid signet-ring cell adenocarcinoma and 2 cases of male patients with adenosquamous carcinoma. The positive cases were all non-smokers without EGFR/KRAS mutations. Furthermore, the positive cases all survived, and the overall postsurgery survival time of 2 cases was more than 5 years. CONCLUSION: ALK IHC with the iAEP method is better than conventional ALK IHC, and the percentage of the positive cells is more important than that of the intensity. ALK translocations were infrequent in the entire NSCLC patient population (<5%) with better prognosis.

[Clinicopathologic features and prognostic factors of malignant phyllodes tumors].

OBJECTIVE: To study the clinicopathologic features of malignant phyllodes tumors (PT) by histopathologic analyses, immunohistochemical profiling and DNA content assay, and evaluation of the clinical outcome. METHODS: Ten patients with malignant PT from 1999 to 2013 who were treated by surgery were enrolled in this study. The morphologic characteristics were studied under light microscope, standard two-step EnVision method of immunohistochemical staining was used to assess the expression of CK5/6, CKpan, 34ß E12, desmin, p63, ER-α, PR, Ki-67, CD34, SMA, p53, p16, bcl-2 and CD117 in the tumors. The corresponding paraffin blocks were also used for flow cytometric DNA content assay. These data were correlated with the follow-up results. RESULTS: The median age of onset was 46.5 years old. The mean tumor size was 7.4 cm (2.0-25.0 cm). At the end of the follow-up period (22 to 125 months), there were tumor recurrences in 3/8 patients and the median time of recurrence was 24 months. Metastasis occurred in 3/8 patients who all died of the tumors. PT had heterogeneous histology, with stromal overgrowth with leaf-like projections, periductal stromal overgrowth, and most commonly, diffuse stromal overgrowth with sarcomatous differentiation. The mean positive index of Ki-67 was 11.4%. The stromal tumor cells were positive for CD34, SMA, p53, p16, and bcl-2 in 3/10, 9/10, 6/10, 8/10, and 4/10 cases, respectively. CD117,ER-α and PR were negative. Interpretable DNA histograms were obtained in nine cases with triploidy in two cases. CONCLUSIONS: The diagnosis of malignant PT should be considered based on the diversity of growth patterns and heterogeneous histology.Ki-67 and CD34 are valuable diagnostic and prognostic factors in patients with malignant PT. Tumors with diffuse stromal overgrowth, heterologous elements, Ki-67 ≥ 20% or aneuploidy are more likely to metastasize.

[Silencing of tumor metastasis suppressor gene 1 promotes invasion of prostate cancer cell in vitro and its molecular mechanisms].

OBJECTIVE: To explore the effect of small interference RNA (siRNA) targeting homosapiens longevity assurance homologue 2(LASS2, or TMSG1) on the invasion of PC-3M-2B4 (a variant subline of human prostate carcinoma cell line PC-3M with low metastatic potential) and its molecular mechanisms. METHODS: PC-3M-2B4 cells were transfected with siRNA by using lipofectamine 2000. The expression of LASS2 mRNA and protein was detected after transfection by real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blot to screen the effective siRNA fragment. The V-ATPase activity of PC-3M-2B4 cells was detected by V-ATPase activity assay kit. The concentration of extracellular hydrogen ion was measured by pH-sensitive fluorescence probe bis-carboxyethyl-carboxyfluorescein (BCECF). The matrix metalloproteinase-2 (MMP-2) protein in the supernatant and cells was analyzed by Western blot. The activity of MMP-2 was examined by Gelatin zymography. Furthermore, the migration and invasion of cells were evaluated by in vitro wound migration assay and invasion assay. RESULTS: RFQ-PCR and Western blot revealed dramatic reduction (84.5% and 60% ) in the levels of LASS2 mRNA and protein after transfection of siRNA-2 in PC-3M-2B4 cells. The V-ATPases activity and extracellular hydrogen ion concentration were significantly increased in PC-3M-2B4 cells transfected with the siRNA-2 compared with other control groups (P<0.05); There were no differences in the expression and secretion of MMP-2 protein between LASS2-siRNA treated cells and other control groups. However, the activity of MMP-2 was up-regulated in LASS2-siRNA treated cells compared with other control groups( P<0.05); and the capacity for migration and invasion in LASS2-siRNA treated cells was significantly higher than in other control groups (P<0.05). CONCLUSION: Silencing of LASS2 can promote invasion of prostate cancer cells in vitro through the increase of the V-ATPases activity, extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2, indicating that LASS2 is a novel tumor metastasis suppressor gene.

[Transcriptional activation of TMSG-1 by complex of KLF6 and Sp1].

OBJECTIVE: To investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1. METHODS: Luciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1. Co-immunoprecipitation (CoIP) was performed to analyze the interaction between KLF6 and Sp1. TMSG-1 and wt-KLF6 mRNA expressions in cells with different metastatic capacities were quantitated by real-time PCR. Cell invasive capability was determined by Matrigel invasion assay. RESULTS: A 63 bp inducible regulatory region (+59 bp - +123 bp) in exon 1 was identified by luciferase assay using reporter plasmids with a series of TMSG-1 regulatory region deletions. Mutations in KLF6/Sp1 binding sites of this region resulted in a decrease of luciferase activity, while cotransfection with KLF6 or Sp1 expressing plasmids led to a remarkable increase of luciferase activity. EMSA and ChIP demonstrated that KLF6 as well as Sp1 interacted with this region. CoIP also indicated a possible interaction between KLF6 and Sp1 proteins. In the highly metastatic cell sublines, a low level of wild type KLF6 was associated synchronously with a low TMSG-1 level. Prostate carcinoma cells overexpressing KLF6 exhibited a higher TMSG-1 level and a lower invasive capability. CONCLUSIONS: Transcription factor complex of KLF6 and Sp1 may participate in the inducible transcriptional regulation of TMSG-1, and a decreased wild type KLF6 expression is likely associated with a low TMSG-1 level in the highly metastatic cell sublines.

[Detection of human epidermal growth factor receptor 2 gene in invasive breast cancer by fluorescence in situ hybridization and research of its association with tumor characteristics].

OBJECTIVE: To investigate the concordance of detecting human epidermal growth factor receptor 2 (HER-2) status of patients with invasive breast cancer by fluorescence in situ hybridization (FISH) for gene amplification and immunohistochemistry (IHC) for protein overexpression and to evaluate the association between tumour characteristics and HER-2 gene amplification. METHODS: The HER-2 status was evaluated in 41 invasive ductal breast carcinomas by both FISH and IHC. The associations between HER-2 gene amplification and other clinicopathological factors, tumour grade, estrogen receptor (ER), tumor emboli, lymph node status, were evaluated using the chi-square test. RESULTS: HER-2 amplifications by FISH were seen in 15 patients, including 8 IHC+++ patients (88.89%, 8/9 of all+++ cases), 6 IHC++ patients (42.68%, 6/14 of all++ cases), 1 IHC+ patient (8.33%, 1/12 of all+ cases) and none IHC negative patient (0%, 0/6 of all negative cases). ER negative or weak positive breast cancers were more often HER-2 FISH positive than ER strong positive cancers (P=0.018). HER-2 amplifications were more often negative in tumor emboli negative or lymph node negative patients than in tumor emboli positive or lymph node positive patients (P=0.000, P=0.025). There was no statistical difference in HER-2 gene amplification among different grades of tumors(P=0.095). CONCLUSION: HER-2 amplification in breast cancer can be determined by FISH accurately. ER is negatively associated with HER-2 amplification, whereas tumor emboli and lymph node status are positively associated with HER-2.

[Identification of nucleolar localization signal sequence of tumor metastasis suppressor gene-1].

OBJECTIVE: To identify the putative specific localization signal sequence of tumor metastasis suppressor gene-1 (TMSG-1) and to explore the mechanism of subcellular localization of TMSG-1 protein. METHODS: Vectors expressing green fluorescence protein (GFP) tagged different TMSG-1 fragments were generated and transfected into human embryo kidney 293 (HEK293) cells. The expression of those fusion proteins was detected by Western blotting and their subcellular localizations were observed by laser confocal microscope. RESULTS: GFP was fused with the native TMSG-1(aa1-380) or different fragments including T1 (aa1-70), T2 (aa1-128), T3 (aa129-380), T4 (aa71-128), T5 (aa71-179) and T6 (aa71-380). Anti-GFP Western blotting showed that these fusion proteins were successfully expressed. Under laser confocal microscope, GFP fused with fragment T4 (aa71-128) localized mainly in the nucleolus; GFP fused with fragment T6 (aa71-380) localized diffusely in the nucleus; while other fusion proteins with TMSG-1 (aa1-380) or fragment T1 (aa1-70), T2 (aa1-128), T3 (aa129-380) and T5 (aa71-179) localized in the cytoplasm. Fragment T4(Δ119-128) was generated from T4 with deletion of 10 amino acid of the C terminal. GFP fused with fragment T4(Δ119-128) remained in the nucleus, but no longer in the nucleolus. CONCLUSIONS: There is a nucleolar localization signal (aa119-128 RRRRNQDRPS) within TMSG-1. This finding may have laid the foundation for further investigations into subcellular localization and function of TMSG-1.

[Influence of phosphoprotein associated with glycosphingolipid microdomains 1 on biologic behavior of human prostatic cancer cell line in-vitro].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

TMSG-1 and its roles in tumor biology.

TMSG-1 is a newly discovered tumor metastasis suppressor gene, which plays important roles in promoting apoptosis and inhibiting invasion and metastasis of tumor cells. The inhibitory function of TMSG-1 in tumor cells may be related to vacuolar H+-ATPase and ceramide, but the underlying mechanism remains unknown. Studies on TMSG-1 are limited worldwide, and only a research group in Shanghai and our group have recently studied on it. As a new research field, the function of TMSG-1 remains to be explored. This review discusses the discovery of TMSG-1, structure of its encoded protein, its roles and possible mechanism in inhibiting tumor invasion and metastasis.

[Screening of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) by cDNA microarray and influence of overexpression of PAG1 on biologic behavior of human metastatic prostatic cancer cell line in vitro].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Extracellular ATP enhances in vitro invasion of prostate cancer cells by activating Rho GTPase and upregulating MMPs expression.

We previously found that in addition to anti-proliferation function, extracellular ATP had a pro-invasion effect on prostate cancer cells, and probably serves as an important regulator of invasion in local microenvironment. However, the underlying mechanism remains unclear. In this study, we demonstrated that ATP increased the motility of prostate cancer cells, and promoted formation of lamellipodia and filopodia. We also found that ATP induced activation of Rac1 and Cdc42, and promoted expression of MMP-3 and MMP-13. These data suggest that extracellular ATP enhances the invasion of prostate cancer cells by activating Rho GTPases Rac1 and Cdc42 and upregulating MMPs expression.

[Roles of p57KIP2 immunohistochemistry and flow cytometry in diagnosis of molar pregnancy].

OBJECTIVE: To study the value of combined use of paternally imprinted gene product p57(KIP2) immunohistochemistry and flow cytometry in the differential diagnosis of placental hydropic diseases. METHODS: A total of 32 cases of hydropic placenta with DNA polymorphism information were collected, and the genetic results were used as basis for the diagnosis of complete hydatidiform moles (CHM), partial hydatidiform moles (PHM) or hydropic abortions. All cases were examined by histology, p57(KIP2) immunohistochemical staining (EnVision method) and flow cytometry DNA ploidy analysis. The p57(KIP2) immunohistochemical staining and DNA ploidy results were compared with the genetic results. RESULTS: In CHM, p57(KIP2) negative rates were 95.2% (20/21), whereas all the 11 cases of non-CHM (7 cases PHM and 4 cases hydropic abortions) were positive (11/11). In 11 p57(KIP2) -positive cases, 7 cases with triploidy and 4 cases with diploidy by flow cytometry were proven to be PHM and hydropic abortions by genetic analysis, respectively. Overall, 96.9% (31/32) cases of hydropic placentas were correctly diagnosed by combined use of p57(KIP2) immunohistochemistry and flow cytometry. CONCLUSIONS: p57(KIP2) immunohistochemical negativity is a reliable index for the diagnosis of CHM. Combined flow cytometry DNA ploidy and p57(KIP2) immunohistochemistry are useful in the pathological differentiation of CHM, PHM and hydropic abortions.

[Malignant giant cell tumor of the tendon sheaths in the hand].

OBJECTIVES: To retrospectively study on malignant giant cell tumor of tendon sheath (MGCTTS) in the hand, and to evaluate its clinical, histologic, immunohistochemical features and biologic evolution. METHODS: Between January 1991 and December 2001, 10 patients with histologically proven MGCTTS were treated. The clinical material, radiographs and hematoxylin and eosin-stained sections were reviewed. Immunohistochemical studies and nuclear suspensions for flow cytometry were done on paraffin embedded tissue. All patients were followed up. RESULTS: Three of 10 patients in which the diagnosis of MGCTTS was originally considered were excluded after the slides reviewed and immunohistochemical examination performed. In the other 7 patients, one showed malignant and aggressive nature: the lesion recurred several times and the patient eventually died with pulmonary metastases. The immunohistochemical profile of the patient was similar to that reported in benign GCTTS, and the flow cytometry DNA analysis detected aneuploidy. Six cases presented histologic features of malignancy, 4 of them undertook the immunohistochemical examination and their profiles were similar to that reported in benign GCTTS. An aneuploidy DNA pattern was detected in one case on flow cytometry evaluation, diploidy DNA pattern was detected in 3 cases, and their S-phase fraction was 4.5%, 11.6% and 2.6% respectively. All of them had a benign clinical features, they were alive and without evidence of disease from 1.5 to 7.5 years (averagely, 4.5 years) after complete surgical excision or resections with wide surgical margins. None of them had received chemotherapy or radiation therapy. CONCLUSIONS: Malignant giant cell tumor of tendon sheath is an extremely rare malignant tumor, some cases have a poor outcome, the others, despite the histologically malignant features, have a good prognosis if wide surgical excision ablates the tumor completely.

[Overexpression of human tumor metastasis-related gene TMSG-1 suppresses cell proliferation and invasion of a highly metastatic prostate cancer cell line PC-3M-1E8 in vitro].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[Correlation of expression of RhoC with invasiveness of prostate cancer cell line PC-3M in vitro].

OBJECTIVE: To investigate the expression of RhoC in prostate cancer cells with different metastatic potential and the correlation with invasiveness. METHODS: Expression of RhoC mRNA and protein in human prostate cancer cell subclones PC-3M-2B4 with non-metastatic potential and PC-3M-1E8 with highly metastatic potential was detected by RT-PCR and Western blot. Eukaryotic expression plasmids of RhoC were constructed and transfected into PC-3M-2B4. The biological effects were observed, including in vitro invasion by Boyden chamber assay, motility by would healing assay, alteration of microfilament network by the staining of TRTIC-phalloidin, activities of matrix metalloproteinases (MMP) by gelatin zymography analysis and expression of p-Akt by Western blot assay. RESULTS: The expression levels of RhoC mRNA and protein varied in the two different metastatic subclones of human prostate cancer cell. RhoC was significantly upregulated in the highly metastatic subclones in comparison to the nonmetastatic counterpart (P < 0.01). As shown in Boyden chamber assay, the invasive capacity of transfected cells overexpressing RhoC was significantly promoted as reflected by more penetrating cells (125.21 +/- 10.43) than the antisense transcripts (46.22 +/- 8.12), the negative (53.77 +/- 8.56) and blank controls (57.68 +/- 7.25). Further study by would healing assay indicated that cells overexpressing RhoC were more motile in actin-based active movement (would healing ratio after 16 h of the sense transcripts, antisense transcripts, negative controls and blank controls was 62.38 +/- 2.36, 29.47 +/- 1.86, 32.23 +/- 2.43, 31.88 +/- 2.67 respectively). The TRITC-phalloidin staining revealed less actin filament bundles and a fuzzy network of shorter filaments in the sense transcripts. In addition, MMP-2 activity and p-Akt expression level were upregulated in the sense transcripts. CONCLUSION: RhoC overexpression could promote the invasive capacity of human prostate cancer cell in vitro and it's expression level correlated positively with the metastatic capacity of human prostate cancer cell, so RhoC may be a potential target in the development of a novel strategy for treating metastasis of prostate cancer.

[Correlation of expression of RhoC with invasiveness of breast cancer cells in vitro].

OBJECTIVE: To investigate the expression of RhoC in breast cancer cells with different metastatic potential and its correlation with invasiveness. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Overexpression of tumor metastasis suppressor gene 1 suppresses proliferation and invasion, but enhances apoptosis of human breast cancer cells MDA-MB-231 cells].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[P2Y purinergic receptor activated PI-3K/Akt signaling pathway in regulation of growth and invasion of prostatic cancer].

OBJECTIVE: To investigate P2Y purinergic receptor activated PI-3K/Akt signaling pathway in the regulation of growth and invasion of prostate cancer in vitro. METHODS: Western blot was used to detect phosphorylation of Akt (a downstream target molecule of PI-3K) by P2Y receptor agonist in 1E8 cells (a highly metastatic subclone derived from PC-3 prostatic cancer cell line). Cell counts, flow cytometry, Matrigel invasion assay, wound healing assay and gelatin zymography were used to detect changes of biological behaviors of 1E8 cells after P2Y receptor activation. RESULTS: AMP-PNP, one non-hydrolysis ATP analogue and P2Y receptor agonist, induced significant phosphorylation of Akt in a time- and dose-dependent manner in IE8 cells. LY294002, a specific inhibitor of PI-3K, effectively blocked Akt phosphorylation induced by AMP-PNP. Continuous exposure to AMP-PNP induced significant growth inhibition of 1E8 cells (inhibition rate at 50.2% at the 8th day), and this inhibition was mainly due to an arrest at S phase of the cell cycle (the S phase fraction of AMP-PNP treated cells was 22.3% higher than that of the control). Application of LY294002 did not reverse the growth inhibition effect of AMP-PNP. Matrigel invasion assay showed that AMP-PNP stimulation increased invasive ability of 1E8 cells, and this effect was effectively blocked by LY294002. No significant changes in the activation of MMP-2 and MMP-9 were detected by gelatin zymography, although wound healing assay showed 21.2% increase in cell migration after AMP-PNP treatment. CONCLUSIONS: PI-3K/Akt signaling pathway participates in P2Y receptor-stimulated prostate cancer invasion by enhancing cell motility, rather than up-regulating MMP-2 and MMP-9 activities. PI-3K signaling pathway plays an important role in prostate cancer proliferation, but is not involved in P2Y receptor mediated growth inhibition.

Magnesium enhances aluminum-induced citrate secretion in rice bean roots (Vigna umbellata) by restoring plasma membrane H+-ATPase activity.

We demonstrated that magnesium (Mg) can alleviate aluminum (Al) toxicity in rice bean [Vigna umbellata (Thunb.) Ohwi & Ohashi] more effectively than is expected from a non-specific cation response. Micromolar concentrations of Mg alleviated the inhibition of root growth by Al but not by lanthanum, and neither strontium nor barium at the micromolar level alleviates Al toxicity. Aluminum also induced citrate efflux from rice bean roots, and this response was stimulated by inclusion of 10 microM Mg in the treatment solution. The increase in the Al-induced citrate efflux by Mg paralleled the improvement in root growth, suggesting that the ameliorative effect of Mg might be related to greater citrate efflux. Vanadate (an effective H+-ATPase inhibitor) decreased the Al-induced citrate efflux, while addition of Mg partly restored the efflux. Mg addition also increased the activity of Al-reduced plasma membrane H+-ATPase, as well as helping to maintain the Mg and calcium contents in root apices. We propose that the addition of Mg to the toxic Al treatment helps maintain the tissue Mg content and the activity of the plasma membrane H+-ATPase. These changes enhanced the Al-dependent efflux of citrate which provided extra protection from Al stress.

[Manganese uptake and transportation as well as antioxidant response to excess manganese in plants].

Manganese (Mn) is an essential micronutrient throughout all stages of plant development. Mn plays an important role in many metabolic processes in plants. It is of particular importance to photosynthetic organisms in the chloroplast of which a cluster of Mn atoms at the catalytic centre function in the light-induced water oxidation by photosystem II, and also function as a cofactor for a variety of enzymes, such as Mn-SOD. But excessive Mn is toxic to plants which is one of the most toxic metals in acid soils. The knowledge of Mn(2+) uptake and transport mechanisms, especially the genes responsible for transition metal transport, could facilitate the understanding of both Mn tolerance and toxicity in plants. Recently, several plant genes were identified to encode transporters with Mn(2+) transport activity, such as zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, natural resistance-associated macrophage protein (Nramp) transporters, cation/H(+) antiporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPase. In addition, excessive Mn frequently induces oxidative stress, then several defense enzymes and antioxidants are stimulated to scavenge the superoxide and hydrogen peroxide formed under stress. Mn-induced oxidative stress and anti-oxidative reaction are very important mechanisms of Mn toxicity and Mn tolerance respectively in plants. This article reviewed the transporters identified as or proposed to be functioning in Mn(2+) transport, Mn toxicity-induced oxidative stress, and the response of antioxidants and antioxidant enzymes in plants to excessive Mn to facilitate further study. Meanwhile, basing on our research results, new problems and views are brought forward.