Disturbance in function and expression of condensin affects chromosome compaction in HeLa cells.

Condensin, a major non-histone protein complex on chromosomes, is responsible for the formation of rod-shaped chromosome in mitosis. A heterodimer composed of SMC2 (structural maintenance of chromosomes) and SMC4 subunits constitutes the core part of condensin. Although extensive studies have been done in yeast, fruit fly and Xenopus to uncover the mechanisms and molecular nature of SMC proteins, little is known about the complex in mammalian cells. We have conducted a series of experiments to unveil the nature of condensin complex in human chromosome formation. The results show that overexpression of the C-terminal domain of SMC subunits disturbs chromosome condensation, leading to formation of swollen chromosomes, while knockdown of SMC subunits severely disturbs mitotic chromosome formation, resulting in chromatin bridges between daughter cells and multiple nuclei in single cells. The salt extraction assay indicates that a fraction of the condensin complex is bound to chromatin in interphase, but most of the condensin bind to chromatin at the onset of mitosis. Thus, disturbance in condensin function or expression affects chromosome condensation and influences mitotic progression.

Platelets and fibrinogen facilitate each other in protecting tumor cells from natural killer cytotoxicity.

The functions of platelets and fibrinogen in protecting tumor cells from natural killer cytotoxicity have been discussed for more than 20 years. However, their exact roles and relationships in the process are still not clear. In this study, we show that tumor cells prefer to adhere to fibrinogen than to platelets, and fibrinogen can enhance the adhesion of tumor cells to platelets. Beta3 integrin plays an important role in the adhesion of B16F10 to platelets enhanced by fibrinogen. In the presence of thrombin, fibrinogen forms dense fibrin(ogen) layers around tumor cells. Tumor cells can induce platelets to aggregate and form thrombin. Platelets, as well as thrombin, can help fibrinogen protect tumor cells from lethal contact with natural killer cells and natural killer cytotoxicity. Hirudin, a specific inhibitor of thrombin, can reverse the effect of platelets on fibrinogen in blocking natural killer cytotoxicity. Our results suggest that fibrinogen helps platelets to adhere to tumor cells, and platelets in turn promote more fibrinogen to aggregate around tumor cells by forming thrombin. They facilitate each other in protecting tumor cells from natural killer cytotoxicity.

c-Abl is required for the signaling transduction induced by L-selectin ligation.

Lymphocyte recruitment onto inflamed tissues requires cells tethering to and rolling on vascular surfaces under flow. L-selectin is constitutively expressed on leukocytes to mediate the leukocytes' initial capture and subsequent rolling along the vessel. Apart from its adhesive function, engagement of L-selectin also results in cell activation, which is related to the completed signaling transduction. Here we show that ligation of L-selectin with its mAb increases c-Abl kinase activity, and that the activated c-Abl kinase can be recruited to and phosphorylate the cytoplasmic domain of L-selectin. In addition, the activated c-Abl kinase can regulate Zap70 kinase by increasing the phosphorylation of the Y319 site of Zap70 kinase and connect with Zap70 kinase through its SH2 domain. These results indicate that c-Abl kinase plays an important role in accepting and transferring the upstream activation events induced by L-selectin ligation.

c-Abl tyrosine kinase activates p21 transcription via interaction with p53.

c-Abl non-receptor tyrosine kinase has been implicated in many cellular processes including cell differentiation, stress response and regulating gene transcription. The mechanism by which c-Abl is involved in the regulation of gene transcription remains to be elucidated. In this study, we investigated the functions of c-Abl in the activation of p21 promoter. Our results showed that overexpression of c-Abl tyrosine kinase activated p21 promoter and endogenous p21 transcription in U2OS cells. We found that p53 is involved in the activation of p21 promoter by c-Abl, and integrative structure of p53 is required for regulating p21 transcription. In addition, the chromatin immunoprecipitation study demonstrated that c-Abl and p53 can be recruited to the region containing p53 binding site of p21 promoter, and c-Abl increases the DNA binding activity of p53 to the p21 promoter. Furthermore, not only the activation of p21 promoter but also the recruitment to p21 promoter by c-Abl is dependent on the interaction between c-Abl and p53 protein.

Nuclear actin is involved in the regulation of CSF1 gene transcription in a chromatin required, BRG1 independent manner.

Actin is an important protein in nucleus and has been implicated in transcription, however, the mechanism of its function in transcription is still not clear. In this article, we studied the role of actin in the regulation of human CSF1 gene transcription. Our results showed that nuclear actin stimulates the activity of CSF1 promoter, and the role in augmenting CSF1 gene transcription requires the formation of chromatin and Z-DNA structure. The ATP binding motifs of nuclear actin are essential for its function in regulating CSF1 gene transcription, and upon actin overexpression, there is an increase in the ATPase activity of nuclear proteins. Further investigation revealed that nuclear actin regulates CSF1 gene transcription in a BRG1 independent manner. Together, these original results have provided evidence for further understanding the mechanism of nuclear actin in regulating gene transcription.

c-Abl is involved in the F-actin assembly triggered by L-selectin crosslinking.

L-selectin is a cell adhesion molecule mediating the initial capture and subsequent rolling of leukocytes along the endothelial cells expressing L-selectin ligands. In addition to its action in adhesion, an intracellular signaling role for L-selectin has been recognized. Its cytoplasmic domain is involved in signal transduction following antibody crosslinking and in the regulation of receptor binding activity in response to intracellular signals. In this work, we demonstrated that L-selectin crosslinking led to F-actin polymerization and redistribution in human neutrophils. Using immuno-fluorescence microscopy, we observed that F-actin redistribution spatiotemporally related to the polarization of L-selectin. STI571, a specific inhibitor for cytoplasmic tyrosine kinase c-Abl, can inhibit F-actin polymerization and c-Abl redistribution in the activated neutrophils. Furthermore, we determined that c-Abl redistributed to the region where L-selectin polarized and associated with L-selectin in the activated neutrophils. The association between L-selectin and c-Abl was reduced by cytochalasin B. These results suggested that c-Abl was involved in the F-actin alteration triggered by L-selectin crosslinking in human neutrophils.

Chemically modified heparin inhibits the in vitro adhesion of nonsmall cell lung cancer cells to P-selectin.

PURPOSE: Several independent studies have indicated that tumor metastasis can be inhibited by chemically modified heparin with low anticoagulant activity in the different tumor models. The mechanism of inhibition by the heparin derivatives in part accounts for the interference of tumor cell-platelet interaction mediated by P-selectin. METHODS: In the present study, we demonstrated that both heparin and chemically modified heparins inhibited the adhesion of nonsmall cell lung cancer (NSCLC) cells to P-selectin under static or flow conditions in vitro. RESULTS: Flow cytometric analysis with the heparan sulfate-specific monoclonal antibody revealed that both NSCLC cells express heparan sulfate-like proteoglycans. Furthermore, heparinase treatment impaired P-selectin binding, indicating that heparan sulfate-like proteoglycans on the tumor cell surface are implicated in the adhesion of NSCLC cells to P-selectin. CONCLUSIONS: These findings suggest that some chemically modified heparins with low anticoagulant activity may deserve further testing in the experimental NSCLC treatment protocols.

Selectively desulfated heparin inhibits P-selectin-mediated adhesion of human melanoma cells.

Accumulating evidence has suggested that one of the mechanisms by which heparin inhibits metastasis is by blocking the P-selectin-based interaction of platelets with tumor cells. Here we demonstrate that the sulfate groups at C6/N and especially C6, but not C2 and C3, of heparin play a critical role in P-selectin recognition and that 2-O,3-O-desulfated heparin can block P-selectin-mediated A375 human melanoma cell adhesion. Our findings show that chemical modification of heparin, especially 2-O,3-O-desulfation, may result in a therapeutic agent that is anti-metastatic because it blocks unwanted P-selectin-dependent adhesion but that lacks dose-limiting anticoagulant effects.

c-Abl tyrosine kinase regulates c-fos gene expression via phosphorylating RNA polymerase II.

c-Abl tyrosine kinase, predominantly distributed in nucleus, has been implicated in many important cellular processes including the regulation of gene transcription. In this study, we showed that c-Abl promoted the transcription of c-fos gene, both exogenously and endogenously. The nuclear localization and tyrosine kinase activity of c-Abl were required for the activation of c-fos gene. c-Abl was associated with RNA polymerase II (RNAP II) in vivo and augmented the tyrosine phosphorylation of the largest subunit of RNAP II. In addition, c-Abl and RNAP II could be recruited to the region of c-fos promoter. The combined results suggest that c-Abl plays an important role in the transcriptional regulation of c-fos gene and the tyrosine phosphorylation of the largest subunit of RNAP II by c-Abl is involved in the regulating process.

Modified heparin inhibits P-selectin-mediated cell adhesion of human colon carcinoma cells to immobilized platelets under dynamic flow conditions.

Accumulating evidence indicates that the formation of tumor cell platelet emboli complexes in the blood stream is a very important step during metastases and that the anti-metastasis effects of heparin are partially due to a blockade of P-selectin on platelets. In this study, heparin and chemically modified heparins were tested as inhibitors of three human colon carcinoma cell lines (COLO320, LS174T, and CW-2) binding to P-selectin, adhering to CHO cells expressing a transfected human P-selectin cDNA, and adhering to surface-anchored platelets expressing P-selectin under static and flow conditions. The aim was to screen for heparin derivatives with high anti-adhesion activity but negligible anticoagulant activity. In this study, four modified heparins with high anti-adhesion activity were identified including RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin. NMR analysis proved the reliability of structure of the four modified heparins. Our findings suggested that the 6-O-sulfate group of glucosamine units in heparin is critical for the inhibition of P-selectin-mediated tumor cell adhesion. Heparan sulfate-like proteoglycans on these tumor cell surfaces are implicated in adhesion of the tumor cells to P-selectin. Some chemically modified heparins with low anticoagulant activities, such as 2/3ODS-heparin, may have potential value as therapeutic agents that block P-selectin-mediated cell adhesion and prevent tumor metastasis.