We examined the influence of ROS on the phosphorylation and complex formation of Bcl-2 family proteins in Mn-superoxide dismutase (SOD) antisense-transfected squamous cell carcinoma cells, OSC-4 cells. The increase of intracellular ROS level induced by cis-diamminedichloroplatinum (CDDP) and γ γ γ γ-ray treatment was greater in antisense-transfected cells than in control vector-transfected cells, and apoptosis was more extensively induced in the former. Antisense-transfected cells expressed high levels of Bax and Bak, but low levels of Bcl-2 and Bcl-X L when treated with CDDP, peplomycin, 5-fluorouracil or γ γ γ γ-rays. After treatment with these agents, the phosphorylation of protein kinase A, Bcl-2 (Thr56) and Bad (Ser155) was increased, especially in antioxidant (N-acetylcysteine and pyrrolidine dithiocarbamate)-pretreated control cells, but the phosphorylation levels were very low in the antisense-transfected cells. Bcl-2 ubiquitination was increased, but ubiquitination of Bad and Bax was decreased in the antisense-transfected cells, although their ubiquitination was increased by the antioxidants. These results reveal that ROS induce apoptosis by regulating the phosphorylation and ubiquitination of Bcl-2 family proteins, resulting in increased proapoptotic protein levels and decreased antiapoptotic protein expression. here are multiple signal pathways to induce apoptosis, including those originating from mitochondria and Fas. 1-4)The signal originating from Fas and Fas-associated proteins passes to caspase 3 through caspase 8 and other caspases. 5-7)The mitochondrial signal is also transduced to caspase 3 via cytochrome c, apoptosis protease-activating factor-1, ATP, and caspase 9. 8) These two pathways exhibit cross-talk with a proapoptotic Bcl-2 family protein, Bid. 9) Bcl-2 family proteins including Bid control the release of cytochrome c from mitochondria regulating VDAC. 10)Bcl-2 family proteins are divided into two groups, proapoptotic and antiapoptotic, according to their chemical structure, that is, BH3 and BH4.11, 12) Proapoptotic and antiapoptotic proteins form heterodimers and inhibit each other's activity. [13][14][15] The dimerization is influenced by phosphorylation of the amino acid residues of the proapoptotic members, Bax, Bak, and Bik.14-17) The expression level of each Bcl-2 family protein is controlled by transcription, heterodimerization, and ubiquitination.13-26) Phosphorylation of antiapoptotic Bcl-2 family proteins inhibits the binding of these proteins and polyubiquitin. [27][28][29][30] Apoptosis is therefore deeply associated with the phosphorylation of Bcl-2 family proteins.Recently, it has been established that some growth factors induce survival signal-activating kinases such as PKA and PKB, the MAP family, ERK1/2, and AP-1. [31][32][33][34] The activation of these kinases finally results in an increase in antiapoptotic Bcl-2 expression. 29,35,36) Suppression of these kinase activities is, therefore, required for the induction of apoptosis.ROS possess both cell-impairing and cel...

Dental pulp/dentine complex regeneration is indispensable to the construction of biotissue-engineered tooth roots and represents a promising approach to therapy for irreversible pulpitis. We used a tissue-engineering method based on odontogenic stem cells to design a three-dimensional (3D) and scaffold-free stem-cell sheet-derived pellet (CSDP) with the necessary physical and biological properties. Stem cells were isolated and identified and stem cells from root apical papilla (SCAPs)-based CSDPs were then fabricated and examined. Compact cell aggregates containing a high proportion of extracellular matrix (ECM) components were observed, and the CSDP culture time was prolonged. The expression of alkaline phosphatase (ALP), dentine sialoprotein (DSPP), bone sialoprotein (BSP) and runt-related gene 2 (RUNX2) mRNA was higher in CSDPs than in cell sheets (CSs), indicating that CSDPs have greater odonto/osteogenic potential. To further investigate this hypothesis, CSDPs and CSs were inserted into human treated dentine matrix fragments (hTDMFs) and transplanted into the subcutaneous space in the backs of immunodeficient mice, where they were cultured in vivo for 6 weeks. The root space with CSDPs was filled entirely with a dental pulp-like tissue with well-established vascularity, and a continuous layer of dentine-like tissue was deposited onto the existing dentine. A layer of odontoblast-like cells was found to express DSPP, ALP and BSP, and human mitochondria lined the surface of the newly formed dentine-like tissue. These results clearly indicate that SCAP-CSDPs with a mount of endogenous ECM have a strong capacity to form a heterotopic dental pulp/dentine complex in empty root canals; this method can be used in the fabrication of bioengineered dental roots and also provides an alternative treatment approach for pulp disease.

The transcriptional factor hypoxia-inducible factor-1 (HIF-1) plays an important role in solid tumor cell growth and survival. Overexpression of HIF-1α α α α has been demonstrated in many human tumors and predicts a poor response to chemoradiotherapy. We examined the HIF-1α α α α-induced survival pathways in human oral squamous cell carcinoma cell (OSCC) lines. The results showed that forced expression of HIF-1α α α α suppressed hypoxia-induced apoptosis of OSCC lines by inhibiting cytochrome c release from mitochondria. Overexpression of HIF-1α α α α inhibited the generation of reactive oxygen species (ROS), elevation of intracellular Ca 2+ concentration, reduction of mitochondrial membrane potential, and cytosolic accumulation of cytochrome c, which resulted in the inactivation of caspase-9 and caspase-3. In addition, antiapoptotic Bcl-2 and Bcl-X L levels were increased and pro-apoptotic Bax and Bak levels were decreased in the HIF-1α α α α-overexpressing OSCC line. T he transcriptional factor hypoxia-inducible factor-1 (HIF-1) is one of the key regulators of oxygen homeostasis. HIF-1 is a heterodimer composed of HIF-1α and HIF-1β subunits, both belonging to the basic-helix-loop-helix PER-ARNT-SIM family of transcriptional factors. HIF-1β, an aryl hydrocarbon receptor nuclear translocator, is constitutively present in normoxic cells, whereas expression levels of HIF-1α are dependent on intracellular oxygen concentration. Under normoxic conditions, HIF-1α protein is rapidly degraded through the ubiquitin-proteasome pathway.(1,2) Degradation of HIF-1α is mediated by the ubiquitin E3 ligase complex, in which the von Hippel Lindau protein (pVHL) binds to the oxygen-dependent degradation domain in the HIF-1α subunits. (3,4) However, under hypoxic conditions, HIF-1α degradation is suppressed, and stabilized HIF-1α activates the transcription of a repertoire of target genes such as glucose transporters, glycolytic enzymes and angiogenic factors.(5-8) Solid tumors often have an insufficient blood supply because tumor cells grow faster than endothelial cells, so the newly formed vascular supply is frequently disorganized, which results in hypoxic regions in tumor tissues. (9)(10)(11) HIF-1α is therefore a resistive molecule against cancer therapy.The transcriptional factor HIF-1α is an important mediator in solid tumor development in vivo because of its promotion of angiogenesis and anaerobic metabolism and inhibition of apoptosis induction in tumor cells. (12)(13)(14) In fact, overexpression of HIF-1α protein coupled with a poor prognosis has been observed in human cancers. (15)(16)(17)(18)(19)(20) In addition, HIF-1α inhibits the induction of apoptosis in tumor cells. (21,22) Apoptosis is mediated by two distinct pathways: the mitochondrial apoptotic pathway and the death receptor (Fas-FasL)-mediated pathway. Decrease of the mitochondrial membrane potential (∆ψ m ) and release of cytochrome c from mitochondria are important apoptotic processes and members of the Bcl-2 family, including Bax, Bcl-2 and Bcl-X L , are c...

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The transcription factor hypoxia-inducible factor-1a (HIF-1a) is the key regulator that controls the hypoxic response of mammalian cells. The overexpression of HIF-1a has been demonstrated in many human tumors. However, the role of HIF-1a in the therapeutic efficacy of chemotherapy and radiotherapy in cancer cells is poorly understood. In this study, we investigated the influence of HIF-1a expression on the susceptibility of oral squamous cell carcinoma (OSCC) cells to chemotherapeutic drugs (cis-diamminedichloroplatinum and 5-fluorouracil) and c-rays. Treatment with chemotherapeutic drugs and c-rays enhanced the expression and nuclear translocation of HIF-1a, and the susceptibility of OSCC cells to the drugs and c-rays was negatively correlated with the expression level of HIF-1a protein. The overexpression of HIF-1a induced OSCC cells to become more resistant to the anticancer agents, and down-regulation of HIF-1a expression by small interfering RNA enhanced the susceptibility of OSCC cells to them. In the HIF-1a-knockdown OSCC cells, the expression of P-glycoprotein, heme oxygenase-1, manganese-superoxide dismutase and ceruloplasmin were downregulated and the intracellular levels of chemotherapeutic drugs and reactive oxygen species were sustained at higher levels after the treatment with the anticancer agents. These results suggest that enhanced HIF-1a expression is related to the resistance of tumor cells to chemo-and radio-therapy and that HIF-1a is an effective therapeutic target for cancer treatment. ' 2006 Wiley-Liss, Inc.Key words: hypoxia-inducible factor-1a; chemotherapeutic drugs; g-rays; P-glycoprotein; heme oxygenase-1 Solid tumors generally possess hypoxic areas in their central portion because of decreased vascular supply associated with the effects of treatment and the originally increased energy demand of cancer cells, and the hypoxic tissue is one of the serious matters for consideration in the control of malignant tumors. Most tumor cells possess the ability to undergo apoptosis in response to hypoxic conditions. However, tumor cells can adapt to hypoxic conditions by employing a variety of survival tools, which result in the promotion of cancer cell growth and metastasis. [1][2][3] This adaptation of cancer cells to hypoxia is mainly mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). 4 HIF-1 is a heterodimeric transcription factor consisting of an oxygen-regulated a subunit (HIF1a) and a stable nuclear factor, HIF-1b/aryl hydrocarbon receptor nuclear translocator (ARNT). Under normoxic conditions, HIF-1a is rapidly degraded by the proteosome after being targeted for ubiquitination. HIF-1a translocates to the nucleus under hypoxic conditions and forms an active complex with HIF-1b; the complex binds to the hypoxia-response element (HRE) in the target genes, which results in the transactivation of these genes. 5 Proteins encoded by such genes contribute to the blood supply, energy production, growth/survival, invasion/metastasis and resistance.It has been frequently rep...