Androgen receptor auto-regulates its expression by a negative feedback loop through upregulation of IFI16 protein

Expression of androgen receptor (AR) in prostate epithelial cells is thought to regulate cell proliferation, differentiation, and survival. However, the molecular mechanisms remain unclear. We report that re-expression of AR in PC-3 human prostate cancer cell line resulted in upregulation of IFI16 protein, a negative regulator of cell growth. We found that the IFI16 protein bound to AR in a ligand-dependent manner and the DNA-binding domain (DBD) of the AR was sufficient to bind IFI16. Furthermore, re-expression of IFI16 protein in LNCaP prostate cancer cells, which do not express IFI16 protein, resulted in downregulation of AR expression and an inhibition of the expression of AR target genes. Our observations identify a role for IFI16 protein in AR-mediated functions.     

Molecular characterization and localization of Plasmodium falciparum choline kinase

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K_m values for choline and ATP are found to be 145 ± 20 μM and 2.5 ± 0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the α and β content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.     

Lansoprazole protects and heals gastric mucosa from non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy by inhibiting mitochondrial as well as Fas-mediated death pathways with concurrent induction of mucosal cell renewal

We have investigated the mechanism of antiapoptotic and cell renewal effects of lansoprazole, a proton pump inhibitor, to protect and heal gastric mucosal injury in vivo induced by indomethacin, a non-steroidal anti-inflammatory drug (NSAID). Lansoprazole prevents indomethacin-induced gastric damage by blocking activation of mitochondrial and Fas pathways of apoptosis. Lansoprazole prevents indomethacin-induced up-regulation of proapoptotic Bax and Bak and down-regulation of antiapoptotic Bcl-2 and Bcl(xL) to maintain the normal proapoptotic/antiapoptotic ratio and thereby arrests indomethacin-induced mitochondrial translocation of Bax and collapse of mitochondrial membrane potential followed by cytochrome c release and caspase-9 activation. Lansoprazole also inhibits indomethacin-induced Fas-mediated mucosal cell death by down-regulating Fas or FasL expression and inhibiting caspase-8 activation. Lansoprazole favors mucosal cell renewal simultaneously by stimulating gene expression of prosurvival proliferating cell nuclear antigen, survivin, epidermal growth factor, and basic fibroblast growth factor. The up-regulation of Flt-1 further indicates that lansoprazole activates vascular epidermal growth factor-mediated controlled angiogenesis to repair gastric mucosa. Lansoprazole also stimulates the healing of already formed ulcers induced by indomethacin. Time course study of healing indicates that it switches off the mitochondrial death pathway completely but not the Fas pathway. However, lansoprazole heals mucosal lesions almost completely after overcoming the persisting Fas pathway, probably by favoring the prosurvival genes expression. This study thus provides the detailed mechanism of antiapoptotic and prosurvival effects of lansoprazole for offering gastroprotection against indomethacin-induced gastropathy.     

Differential induction of the 200-family proteins in Daudi Burkitt's lymphoma cells by interferon-alpha

Interferon (IFN)-inducible "effector" proteins mediate the biological activities of the IFNs. Therefore, the identification of the functional role(s) of IFN inducible proteins in IFN action is important to elucidate the molecular mechanisms by which IFNs inhibit cell growth. One family (the "200-family") of IFN-inducible proteins includes structurally related murine (p202a, p202b, p203, p204 and D3) and human (MNDA, IFI-16 and AIM2) proteins. However, their role in IFN action remains to be established. Here we report that IFN-alpha treatment of Daudi Burkitt's lymphoma cells resulted in differential induction of MNDA, IFI 16, and a p202-related protein (p202RP). Interestingly, IFN induction of p202RP preceded the induction of MNDA and IFI 16 proteins and the growth inhibition by IFN. Additionally, the induction of these proteins by IFN was accompanied by: (i) a transient increase in p21(WAF1/CIP1) levels; (ii) an increase in the functional form of pRb and p130; (iii) an inhibition of the sequence-specific DNA binding activity of E2F complexes; and (iv) a marked decrease in c-Myc levels. Our observations reported herein provide support to the hypothesis that IFN-inducible p202RP and MNDA proteins from the 200-family contribute to the growth inhibitory activities of the IFNs.     

Apoptosis in proliferating, senescent, and immortalized keratinocytes.

Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-kappaB were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-kappaB is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-kappaB, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-kappaB but not early differentiation.     

Mutant A53T alpha-synuclein induces neuronal death by increasing mitochondrial autophagy

Parkinson disease is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. α-Synuclein accumulation in turn leads to compensatory effects that may include the up-regulation of autophagy. Another common feature of Parkinson disease (PD) is mitochondrial dysfunction. Here, we provide evidence that the overactivation of autophagy may be a link that connects the intracellular accumulation of α-synuclein with mitochondrial dysfunction. We found that the activation of macroautophagy in primary cortical neurons that overexpress mutant A53T α-synuclein leads to massive mitochondrial destruction and loss, which is associated with a bioenergetic deficit and neuronal degeneration. No mitochondrial removal or net loss was observed when we suppressed the targeting of mitochondria to autophagosomes by silencing Parkin, overexpressing wild-type Mitofusin 2 and dominant negative Dynamin-related protein 1 or blocking autophagy by silencing autophagy-related genes. The inhibition of targeting mitochondria to autophagosomes or autophagy was also partially protective against mutant A53T α-synuclein-induced neuronal cell death. These data suggest that overactivated mitochondrial removal could be one of the contributing factors that leads to the mitochondrial loss observed in PD models.