C. D. Anuradha

Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells

It has been suggested that oxidative stress plays a major role in various forms of cell death, including necrosis and apoptosis. We have previously reported that fluoride (NaF) induces apoptosis in HL-60 cells by caspase-3 activation. The main focus of this investigation was to arrive at a possible pathway of the apoptosis induced by NaF upstream of caspase-3, because the mechanism is still unknown. The present study showed that after exposure to NaF, there was an increase in MDA and 4-HNE and a loss of mitochondrial membrane potential (deltaPsi(m)) was also observed in NaF-treated cells. There was a significant increase in cytosolic cytochrome c, which is released from the mitochondria. We have reported a downregulation of Bcl-2 protein in NaF-treated cells. The antioxidants N-acetyl cysteine (NAC), glutathione (GSH) protected the cells from loss of deltaPsi(m), and there was no cytochrome c exit or Bcl-2 downregulation, and we suggest that these antioxidants prevent apoptosis induced by NaF. These results suggested that perhaps NaF induced apoptosis by oxidative stress-induced lipid peroxidation, causing loss of deltaPsi(m), and thereby releasing cytochrome c into the cytosol and further triggering the caspase cascade leading to apoptotic cell death in HL-60 cells.

RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation.

RGD motif-containing peptides have been used in various studies of cell adhesion and growth. We report that RGD triggered apoptosis at a concentration of 1 mmol/L, whereas RAD-containing peptides failed to induce apoptosis in HL-60 cells. RGD-treated cells revealed internucleosomal DNA fragmentation. Western blot reveals caspase-3 activation in RGD peptide-treated cells. A caspase-3 inhibitor z-VAD-FMK completely blocked the apoptosis, but a caspase-1 inhibitor (Ac-YVAD-CMK) and caspase-2 inhibitor (z-VDVAD-FMK) did not block the apoptosis, suggesting that caspase-3 might have a critical role in the execution process of apoptosis induced by RGD. RGD peptides have been used extensively to inhibit tumor metastasis. Our results should help in further understanding the RGD peptide-induced apoptosis, which is important since RGD peptides have a potential role in therapies of the future.

Localization of zinc after in vitro mineralization in osteoblastic cells

The present study was designed to investigate the incorporation of zinc (Zn) into cultured UMR-106 osteoblasts in response to mineralization caused by the addition of β-glycerophosphate. As a result of the induced mineralization, the contents of calcium (Ca), phosphorus (P), and Zn in the monolayer increased, whereas the magnesium (Mg) content did not change. The activity of alkaline phosphatase (ALP) also increased during the process.The zinc distribution in the cell monolayer was studied using Zinquin, a fluorescent zinc ion chelator. The double fluorescent labeling with Zinquin and calcein revealed that zinc was localized both as intracellular vesicles and extracellular clusters, whereas calcium was colocalized with extracellular zinc. These results suggest that zinc is involved in the mineralization process of UMR-106 cells.

Chemotactic responses of osteoblastic MC3T3-E1 cells toward zinc chloride.

Although zinc (Zn) is known to participate in bone formation, its exact role in the remodeling of this tissue has not been fully clarified. The present study was designed to investigate whether Zn has a role at the resorptive sites in vitro. We investigated the migration of osteoblastic MC3T3-E1 cells in response to Zn using a Boyden chamber assay. Exposure of MC3T3-E1 cells to Zn stimulated the migration of MC3T3-E1 cells. Checkerboard analysis revealed that the migration of MC3T3-E1 cells toward Zn was a directional (chemotaxis) rather than a random (chemokinesis) motion.Pretreatment of MC3T3-E1 cells with pertussis toxin completely blocked the chemotactic response of cells to Zn, indicating that it is mediated by G-protein-coupled receptors. Because the bone is one of the major Zn storage sites, we suggest that Zn released from bone-resorptive sites plays an important role in the recruitment of osteoblasts and bone renewal.