Ror2-Src signaling in metastasis of mouse melanoma cells is inhibited by NRAGE.

The receptor tyrosine kinase (RTK) Ror2 plays important roles in developmental morphogenesis and mediates the filopodia formation in Wnt5a-induced cell migration. However, the function of Ror2 in noncanonical Wnt signaling resulting in cancer metastasis is largely unknown. Here, we show that Ror2 expression is higher in the highly metastatic murine B16-BL6 melanoma cells than in the low metastatic variant B16 cells. Overexpression of Ror2 increases the metastasis ability of B16 cells, and knockdown of Ror2 reduces the migration ability of B16-BL6 cells. Furthermore, the inhibition of Src kinase activity is critical for the Ror2-mediated cell migration upon Wnt5a treatment. The C-terminus of Ror2, which is deleted in brachydactyly type B (BDB), is essential for the mutual interaction with the SH1 domain of Src. Intriguingly, the Neurotrophin receptor-interacting MAGE homologue (NRAGE), which, as we previously reported, can remodel the cellular skeleton and inhibit cell-cell adhesion and metastasis of melanoma and pancreatic cancer, sharply blocks the interaction between Src and Ror2 and inhibits Ror2-mediated B16 cell migration by decreasing the activity of Src and focal adhesion kinase (FAK). Our data show that Ror2 is a potential factor in the tumorigenesis and metastasis in a Src-dependent manner that is negatively regulated by NRAGE.

NRAGE suppresses metastasis of melanoma and pancreatic cancer in vitro and in vivo.

We previously reported that human NRAGE could significantly alter the cellular skeleton and inhibit cell-cell adhesion, suggesting that human NRGAE play a potential role in cellular motility. Here, we report overexpression of human NRAGE in PANC-1 and B16-Bl6 cells could significantly suppress the metastasis of these cells in vitro and in vivo. Consistently, PANC-1 with stable silencing of NRAGE by RNA interference, exhibits a more metastatic phenotype than the native cell. Expression of epithelial proteins, including E-cadherin and beta-catenin is down regulated in siRNA-NRAGE PANC-1 cells. Further studies find that overexpression of human NRAGE suppresses the mRNA expression and activity of MMP2 significantly. Summary, our studies indicate for the first time that NRAGE could suppress metastasis of melanoma and pancreatic cancer probably through downregulation of MMP-2.

[Construction of recombinant adenovirus vector of hNRAGE gene and its effect on cell cycle of 293 cells].

AIM: To construct the recombinant adenovirus vector of hNRAGE gene and study its effect on the cell cycle of 293 cells. METHODS: hNRAGE gene was amplified by PCR and subcloned into the shuttle vector pAdTrack-CMV to construct a shuttle plasmid pAdTrack-CMV/hNRAGE. After sequencing, it was linearized with Pme I and cotransformed into E.coli BJ5183 cells with adenovirus genomic plasmid pAdEasy-1 by electroporation to achieve homologous recombination. After being digested with Pac I, the DNA of identified recombinant plasmid was transfected into QBI-293A cells by calcium phosphate transfection to package adenovirus. With the use of GFP gene expression in pAdTrack-CMV, the appearance of Ad-hNRAGE was observed and its concentration was measured. The expression of the target gene was detected by Western blot and its effect on cell cycle of 293 cells was examined by MTT colorimetry and FCM. RESULTS: The Ad-hNRAGE was successfully constructed and the expression of hNRAGE gene in 293 cells was proved by Western blot. After harvesting the virus particles, the concentration of Ad-hNRAGE was about 6.5x10(9) Ad/microL. The transfection of Ad-hNRAGE resulted in significantly lower proliferative rate of 293 cells compared with untransfected ones, with cell number of G0-G1 and G2-M phase increasing and that of S phase decreasing. CONCLUSION: hNRAGE gene can inhibit the growth of 293 cells.

Knockdown of c-Met by adenovirus-delivered small interfering RNA inhibits hepatocellular carcinoma growth in vitro and in vivo.

c-Met is highly expressed and constitutively activated in various human tumors. We employed adenovirus-mediated RNA interference technique to knock down c-Met expression in hepatocellular carcinoma cells and observed its effects on hepatocellular carcinoma cell growth in vitro and in vivo. Among the five hepatocellular carcinoma and one normal human liver cell lines we analyzed, c-Met was highly expressed and constitutively tyrosine phosphorylated in only MHCC97-L and HCCLM3 hepatocellular carcinoma cells. Knockdown of c-Met could inhibit MHCC97-L cells proliferation by arresting cells at G0-G1 phase. Soft agar colony formation assay indicated that the colony forming ability of MHCC97-L cells decreased by approximately 70% after adenovirus AdH1-small interfering RNA (siRNA)/met infection. In vivo experiments showed that adenovirus AdH1-siRNA/met inhibited the tumorigenicity of MHCC97-L cells and significantly suppressed tumor growth when injected directly into tumors. These results suggest that knockdown of c-Met by adenovirus-delivered siRNA may be a potential therapeutic strategy for treatment of hepatocellular carcinoma in which c-Met is overexpressed.

[A DNA vector-based RNAi technology to inhibit the activity of the telomerase of cell line HCCLM3].

OBJECTIVE: To develop a DNA vector-based RNA interference (RNAi) technology that inhibits the activity of the telomerase of the hepatocellular carcinoma cell line HCCLM3 in order to suppress the proliferation of the cells. METHODS: Hepatocellular carcinoma cells of the line HCCLM3 were cultured. mRNA interfering double-stranded DNA vector PSG-AS targeting the mRNA of human telomerase reverse transcriptase (hTERT) and the control vector PSG-CTR were constructed respectively, and then were transfected into the HCCLM3 cells. The expression of hTERT of the transfected cells was determined by Western blotting and the activity of telomerase was determined by telomeric repeat amplification-ELISA (TRAP-ELISA). Flow cytometry was used to detect the apoptosis of transfected cells and MTS method was used to measure the growth curve of the cells so as to observe the effect of the PSG-AS on the proliferation of HCCLM3 cell. RESULTS: TRAP-ELISA showed that the inhibition rate of PSG-AS on the telomerase activity was 76%. The apoptotic rate of the PSG-AS group was significantly higher than that of the PSG-CTR group (t = 11.48, P < 0.001). Western blotting showed a remarkable inhibition of hTERT protein in the PSG-AS group. CONCLUSION: Capable of suppressing the hTERT expression and the activity of telomerase, RNA interfering technology can be applied to treatment of tumors.

The association of calmodulin with central spindle regulates the initiation of cytokinesis in HeLa cells.

Calmodulin is a major cytoplasmic calcium receptor that performs multiple functions in the cell including cytokinesis. Central spindle appears between separating chromatin masses after metaphase-anaphase transition. The interaction of microtubules from central spindle with cell cortex regulates the cleavage furrow formation. In this paper, we use green fluorescence protein (GFP)-tagged calmodulin as a living cell probe to examine the detailed dynamic redistribution and co-localization of calmodulin with central spindle during cytokinesis and the function of this distribution pattern in a tripolar HeLa cell model. We found that calmodulin is associated with spindle microtubules during mitosis and begins to aggregate with central spindle after anaphase initiation. The absence of either central spindle or central spindle-distributed calmodulin is correlated with the defect in the formation of cleavage furrow, where contractile ring-distributed CaM is also extinct. Further analysis found that both the assembly of central spindle and the formation of cleavage furrow are affected by the W7 treatment. The microtubule density of central spindle was decreased after the treatment. Only less than 10% of the synchronized cells enter cytokinesis when treated with 25 microM W7, and the completion time of furrow regression is also delayed from 10 min to at least 40 min. It is suggested that calmodulin plays a significant role in cytokinesis including furrow formation and regression, The understanding of the interaction between calmodulin and microtubules may give us insight into the mechanism through which calmodulin regulates cytokinesis.

hNRAGE, a human neurotrophin receptor interacting MAGE homologue, regulates p53 transcriptional activity and inhibits cell proliferation.

hNRAGE, a neurotrophin receptor p75 interacting MAGE homologue, is cloned from a human placenta cDNA library. hNRAGE can inhibit the colony formation of and arrest cell proliferation at the G1/S and G2/M stages in hNRAGE overexpressing cells. Interestingly, hNRAGE also increases the p53 protein level as well as its phosphorylation (Ser392). Further studies demonstrated that hNRAGE does not affect the proliferation of mouse p53-/- embryonic fibroblasts, suggesting that p53 function is required for hNRAGE induced cell cycle arrest. Moreover, the cell cycle inhibiting protein p21(WAF) is induced by hNRAGE in a p53 dependent manner. The data provide original evidence that hNRAGE arrests cell growth through a p53 dependent pathway.

[The distribution of calmodulin with midbody and its involvement in cytokinesis regulation].

Calmodulin (CaM) is a major cytoplasmic Ca2+ receptor and performs a multiplicity of functions in the cell. By using GFP-CaM fusion protein, we have studied the detailed dynamic redistribution of CaM during cytokinesis in HeLa cells. CaM associates with midbody in late cytokinesis phase. When the cells were treated with Ca2+/CaM inhibitor W7, the dissolving of the midbody was delayed. Moreover, we have found that gamma-tubulin colocalized with CaM at the midbody during cytokinesis. W7 treatment could affect the dissociation of gamma-tubulin from midbody. These results suggest that CaM may involve in the regulation of midbody microtubules disassembly and may thus affect the completion of cytokinesis.