No abstract

We examined the influence of connexin (Cx) expression on the development of apoptosis in HeLa parental cells (coupling deficient cell line) and HeLa cells expressing wild-type Cx43 and Cxs fused with enhanced green fluorescent protein (EGFP). EGFP was attached to the C-terminus of Cx32 and Cx43, Cx32-EGFP and Cx43-EGFP, respectively, and to the N-terminus of Cx32, EGFP-Cx32. All fusion proteins assembled into junctional plaques (JPs) at areas of cell-cell contact, but only the C-terminal fusion proteins formed functional gap junction (GJ) channels as well as hemichannels. In each cell line, apoptosis was induced by treatment with various agents including anisomycin, camptothecin, cis-platinum, colchicine, cycloheximide, etoposide, staurosporin and taxol. Using fluorescence microscopy, time-lapse imaging and dual whole-cell voltage clamp techniques, we correlated the changes in functional properties of GJ channels and Cx distribution with the progression of apoptosis based on cells' labeling with acridine orange and ethidium bromide (EB). The early phase of apoptosis (a viable apoptotic (VA) state) was characterized by shrinkage of the cells and by increased internalization of JPs accompanied by decreased cell-cell coupling. The apoptotic reagents had no direct effect on electrical cell-cell coupling. Transformation from a VA to a nonviable apoptotic (NVA) state was faster in HeLa cells expressing Cx43 or Cx43-EGFP than in HeLa parental cells. The potent GJ uncoupler, octanol, slowed the transition of HelaCx43-EGFP cells into a NVA state. In the absence of apoptotic reagents, the rate of EB uptake was higher in HeLaCx43-EGFP than in HeLa parental cells consistent with the presence of open Cx43-EGFP hemichannels. However, in both cell lines the rate of EB uptake decreased proportionally during the development of apoptosis suggesting that membrane permeability ascribed to Cx hemichannels is reduced. Cells expressing Cx32-EGFP and EGFP-Cx32 demonstrate the same apoptotic patterns as HeLaCx43-EGFP and HeLa parental cells, respectively. Intracellular levels of ATP in HeLaCx43-EGFP cells were substantially lower than in HeLa parental cells, and ATP added to the medium abolished the accelerated transition from a VA to a NVA state in HeLaCx43-EGFP cells. In summary, Cx32 or Cx43 accelerates transformation of cells into a NVA state or secondary necrosis and this depends on the ability of Cxs to form functional GJ channels and hemichannels.

No abstract

Improvement in the yield of adult organism stem cells, and the ability to manage their differentiation and survival potential are the major goals in their application in regenerative medicine and in the adult stem cell research. We have demonstrated that adult rabbit muscle-derived cell lines with an unlimited proliferative potential in vitro can differentiate into myogenic, osteogenic, adipogenic and neurogenic lineages. Studies of cell survival in vitro showed that differentiated cells, except neurogenic ones, are more resistant to apoptosis inducers compared to proliferating cells. Resistance to death signals correlated with the level of protein kinase AKT phosphorylation. Skeletal muscle-derived cell lines can be multipurpose tools in therapy. Enhanced resistance of differentiated cells to certain types of damage shows their potential for long-term survival and maintenance in an organism. This article was published online on 29 January 2013. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 6 March 2013.

Daunorubicin (as well as other anthracyclines) is known to be toxic to heart cells and other cells in organism thus limiting its applicability in human cancer therapy. To investigate possible mechanisms of daunorubicin cytotoxicity, we used stem cell lines derived from adult rabbit skeletal muscle. Recently, we have shown that daunorubicin induces apoptotic cell death in our cell model system and distinctly influences the activity of MAP kinases. Here, we demonstrate that two widely accepted antagonistic signalling pathways namely proapoptotic JNK and prosurvival PI3K/AKT participate in apoptosis. Using the Western blot method, we observed the activation of JNK and phosphorylation of its direct target c-Jun along with inactivation of AKT and its direct target GSK in the course of programmed cell death. By means of small-molecule kinase inhibitors and transfection of cells with the genes of the components of these pathways, c-Jun and AKT, we confirm that JNK signalling pathway is proapoptotic, whereas AKT is antiapoptotic in daunorubicin-induced muscle cells. These findings could contribute to new approaches which will result in less toxicity and fewer side effects that are currently associated with the use of daunorubicin in cancer therapies.