Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells

Bisphosphonates are used for prevention of osteoporosis and metastatic bone diseases. Anti-invasive effects on various cancer cells have also been reported, but the mechanisms involved are not well-understood. We investigated the effects of the nitrogen-containing bisphosphonate alendronate (ALN) on the regulation of actin cytoskeleton in PC-3 cells. We analyzed the ALN effect on the organization and the dynamics of actin, and on the cytoskeleton-related regulatory proteins cofilin, p21-associated kinase 2 (PAK2), paxillin and focal adhesion kinase. Immunostainings of cofilin in ALN-treated PC-3 cells and xenografts were performed, and the role of cofilin in ALN-regulated F-actin organization and migration/invasion in PC-3 cells was analyzed using cofilin knockdown and transfection. We demonstrate that disrupted F-actin organization and decreased cell motility in ALN-treated PC-3 cells were associated with decreased levels of total and phosphorylated cofilin. PAK2 levels were also lowered but adhesion-related proteins were not altered. The knockdown of cofilin similarly impaired F-actin organization and decreased invasion of PC-3 cells, whereas in the cells transfected with a cofilin expressing vector, ALN treatment did not decrease cellular cofilin levels and migration as in mock transfected cells. ALN also reduced immunohistochemical staining of cofilin in PC-3 xenografts. Our results suggest that reduction of cofilin has an important role in ALN-induced disruption of the actin cytoskeleton and inhibition of the PC-3 cell motility and invasion. These data also support the idea that the nitrogen-containing bisphosphonates could be efficacious in inhibition of prostate cancer invasion and metastasis, if delivered in a pharmacological formulation accessible to the tumors.</p

Unsatisfactory gene transfer into bone-resorbing osteoclasts with liposomal transfection systems

BACKGROUND: Bone-resorbing osteoclasts are multinucleated cells that are formed via fusion of their hematopoietic stem cells. Many of the details of osteoclast formation, activation and motility remain unsolved. Therefore, there is an interest among bone biologists to transfect the terminally differentiated osteoclasts and follow their responses to the transgenes in vitro. Severe difficulties in transfecting the large, adherent osteoclasts have been encountered, however, making the use of modern cell biology tools in osteoclast research challenging. Transfection of mature osteoclasts by non-viral gene transfer systems has not been reported. RESULTS: We have systematically screened the usefulness of several commercial DNA transfection systems in human osteoclasts and their mononuclear precursor cell cultures, and compared transfection efficacy to adenoviral DNA transfection. None of the liposome-based or endosome disruption-inducing systems could induce EGFP-actin expression in terminally differentiated osteoclasts. Instead, a massive cell death by apoptosis was found with all concentrations and liposome/DNA-ratios tested. Best transfection efficiencies were obtained by adenoviral gene delivery. Marginal DNA transfection was obtained by just adding the DNA to the cell culture medium. When bone marrow-derived CD34-positive precursor cells were transfected, some GFP-expression was found at the latest 24 h after transfection. Large numbers of apoptotic cells were found and those cells that remained alive, failed to form osteoclasts when cultured in the presence of RANKL and M-CSF, key regulators of osteoclast formation. In comparison, adenoviral gene delivery resulted in the transfection of CD34-positive cells that remained GFP-positive for up to 5 days and allowed osteoclast formation. CONCLUSION: Osteoclasts and their precursors are sensitive to liposomal transfection systems, which induce osteoclast apoptosis. Gene transfer to mononuclear osteoclast precursors or differentiated osteoclasts was not possible with any of the commercial transfection systems tested. Osteoclasts are non-dividing, adherent cells that are difficult to grow as confluent cultures, which may explain problems with transfection reagents. Large numbers of α(v)β(3 )integrin on the osteoclast surface allows adenovirus endocytosis and infection proceeds in dividing and non-dividing cells efficiently. Viral gene delivery is therefore currently the method of choice for osteoclast transfection

Connexin-mimetic peptide Gap 27 decreases osteoclastic activity

BACKGROUND: Bone remodelling is dependent on the balance between bone resorbing osteoclasts and bone forming osteoblasts. We have shown previously that osteoclasts contain gap-junctional protein connexin-43 and that a commonly used gap-junctional inhibitor, heptanol, can inhibit osteoclastic bone resorption. Since heptanol may also have some unspecific effect unrelated to gap-junctional inhibition we wanted to test the importance of gap-junctional communication to osteoclasts using a more specific inhibitor. METHODS: A synthetic connexin-mimetic peptide, Gap 27, was used to evaluate the contribution of gap-junctional communication to osteoclastic bone resorption. We utilised the well-characterised pit-formation assay to study the effects of the specific gap-junctional inhibitor to the survival and activity of osteoclasts. RESULTS: Gap 27 caused a remarked decrease in the number of both TRAP-positive mononuclear and multinucleated rat osteoclasts cultured on bovine bone slices. The decrease in the cell survival seemed to be restricted to TRAP-positive cells, whereas the other cells of the culture model seemed unaffected. The activity of the remaining osteoclasts was found to be diminished by measuring the percentage of osteoclasts with actin rings of all TRAP-positive cells. In addition, the resorbed area in the treated cultures was greatly diminished. CONCLUSIONS: On the basis of these results we conclude that gap-junctional communication is essential for the action of bone resorbing osteoclasts and for proper remodelling for bone

The Cyprinodon variegatus genome reveals gene expression changes underlying differences in skull morphology among closely related species

Genes in durophage intersection set at 15 dpf. This is a comma separated table of the genes in the 15 dpf durophage intersection set. Given are edgeR results for each pairwise comparison. Columns indicating whether a gene is included in the intersection set at a threshold of 1.5 or 2 fold are provided. (CSV 13 kb