Sunil Upadhyay

ΔNp63&alpha Overexpression Induces Downregulation of Sirt1 and an Accelerated Aging Phenotype in the Mouse

p63 is highly expressed in the skin and appears to be an early marker of keratinocyte differentiation. To examine the role of p63 in vivo, we generated transgenic mice that overexpress ΔNp63&alpha in the skin. These mice exhibited an accelerated aging phenotype in the skin characterized by striking wound healing defects, decreased skin thickness, decreased subcutaneous fat tissue, hair loss, and decreased cell proliferation. The accelerated skin aging was accompanied by a dramatic decrease in longevity of the mice. We found that aging in ΔNp63&alpha transgenic mice and other mouse models correlated with levels of Sirt1, a mammalian SIR2 orthologue thought to extend the lifespan in lower species. Moreover, increased ΔNp63&alpha expression induced cellular senescence that was rescued by Sirt1. Our data suggest that ΔNp63&alpha levels may affect aging in mammals, at least in part, through regulation of Sirt1.

Mitochondrial Cytochrome B Gene Mutation Promotes Tumor Growth in Bladder Cancer

Mitochondria-encoded Cytochrome B (CYTB) gene mutations were reported in different cancers, but the effect of these mutations on cellular metabolism and growth is unknown. In a murine xenograft and human model of bladder cancer, we show the functional effect of overexpression of a 21-bp deletion mutation (mt) of CYTB. Overexpression of mtCYTB generated increased reactive oxygen species (ROS) accompanied by increased oxygen consumption and lactate production. MtCYTB overexpression induced significant tumor growth in vitro and in vivo by triggering rapid cell cycle progression through up-regulation of the nuclear factor-kappa B2 signaling pathway. Tumor-generated ROS induced in vitro lysis of normal splenocytes. Thus, we present physiologic and functional evidence for the role of a bona fide mitochondrial gene mutation in cancer.

LKB1/STK11 Suppresses Cyclooxygenase-2 Induction and Cellular Invasion through PEA3 in Lung Cancer

We showed that the PEA3 transcriptional factor interacted with LKB1, a serine/threonine kinase, which is somatically mutated in lung cancer. This interaction occurred through the ETS domain of PEA3 and the kinase domain of LKB1. Mutation of LKB1 in lung cancer cells stabilized PEA3. Reintroduction of wild-type (WT) LKB1 into cells induced down-regulation of PEA3 and subsequently resulted in reduced cyclooxygenase-2 RNA and protein expression, whereas germ-line and somatic LKB1 mutants were defective in this activity. LKB1 phosphorylated PEA3 and promoted its degradation through a proteasome-mediated mechanism. Cells expressing mutant LKB1 possessed greater invasive potential compared with cells expressing WT LKB1. Increased invasion of cells with mutant LKB1 was partly due to PEA3 expression, as RNA interference inhibition of PEA3 resulted in dramatic decrease of Matrigel invasion. However, forced expression of PEA3 resulted in down-regulation of epithelial markers and induction of mesenchymal markers. These results suggest that PEA3 stabilization due to LKB1 inactivation could lead to epithelial/mesenchymal transition and greater lung cancer invasion potential.

ATM kinase is a master switch for the ΔNp63α phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of DeltaNp63alpha associated with its increased phosphorylated status. Since DeltaNp63alpha usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of DeltaNp63alpha in HNSCC cells upon cisplatin exposure. We found that DeltaNp63alpha is phosphorylated in the time-dependent fashion at the following positions: S385, T397, and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of DeltaNp63alpha in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of DeltaNp63alpha residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of DeltaNp63alpha degradation. Finally, we demonstrated that the DeltaNp63alpha protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of DeltaNp63alpha following DNA damage.

The effect of p53-RNAi and p53 knockout on human 8-oxoguanine DNA glycosylase (hOgg1) activity

Recent evidence indicates that in vitro p53 augments base excision repair (BER) activities in mammalian cells. To understand the role of p53 in BER, we analyzed the repair activity of hOgg1 in isogenic cell lines HCT116p53+/+ and HCT116p53−/−. We found that hOgg1 activity was significantly decreased in HCT116p53−/− cells as compared with HCT116p53+/+ cells, indicating a functional role for p53 in the regulation of hOGG1. Using gel‐shift assays, we showed that p53 binds to its putative cis‐elements within the hOGG1 promoter. In addition we demonstrated that supplementing p53 in HCT116p53−/− cells enhanced the transcription of hOGG1. To further strengthen our findings, we used p53‐RNAi to study the effects of decreased p53 levels on hOgg1 activity. We observed that p53‐RNAi resulted in decreased hOGG1 expression both at the mRNA and protein levels. This decrease in hOGG1 expression was associated with reduced cell viability upon oxidative damage and reduced hOgg1 activity as evidenced by the 8‐oxoG incision assay. Taken together, our results indicate that loss of p53 function can lead to decreased hOgg1 repair activity.

Forced Cytochrome B gene mutation expression induces mitochondrial proliferation and prevents apoptosis in human uroepithelial SV-HUC-1 cells

Mitochondria encoded Cytochrome B (CYTB) gene mutations were reported in tumors of different anatomic origin but the functional significance of these mutations are not well studied. Earlier, we found a 7‐amino acid deletion mutation in the CYTB gene in a primary bladder cancer patient. In the present study, we overexpressed this 7‐amino acid deletion mutation of CYTB gene in SV‐40 transformed human uroepithelial HUC‐1 cells. The nuclear transcribed mitochondrial CYTB (mtCYTB) was targeted into the mitochondria and generated increased copies of mitochondria and mitochondrial COX‐I protein in the transfected HUC‐1 cells. The proapoptotic protein Bax largely remained confined to the cytoplasm of the mtCYTB transfected HUC‐1 cells without release of Cytochrome C. The downstream apoptotic proteins PARP also remained uncleaved along with increased Lamin B1 in the mtCYTB transfected cells. Our results demonstrate that forced overexpression of mtCYTB in transformed human uroepithelial HUC‐1 cells triggered mitochondrial proliferation and induction of an antiapoptotic signaling cascade favoring sustained cellular growth. Coding mitochondrial DNA mutations appear to have significant functional contribution in tumor progression. Published 2009 UICC.

Targeting human 8-oxoguanine DNA glycosylase to mitochondria protects cells from 2-methoxyestradiol-induced-mitochondria-dependent apoptosis

2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite of 17β-estradiol, is known to induce mitochondria-mediated apoptosis through several mechanisms. We sought to study the effect of mitochondrialy targeted hOGG1 (MTS-hOGG1) on HeLa cells exposed to 2-ME. MTS-hOGG1-expressing cells exposed to 2-ME showed increased cellular survival and had significantly less G2/M cell cycle arrest compared to vector-only-transfected cells. In addition, 2-ME exposure resulted in an increase in mitochondrial membrane potential, increased apoptosis, accompanied by higher activation of caspase-3, -9, cleavage of Bid to tBid and protein poly(ADP-ribose) polymerase (PARP) cleavage in HeLa cells lacking MTS-hOGG1. Fas inhibitors cerulenin or C75 inhibited 2-ME-induced caspase activation, PARP cleavage, apoptosis and reversed mitochondrial membrane hyperpolarization, thereby recapitulating the increased expression of MTS-hOGG1. Hence, MTS-hOGG1 plays an important protective role against 2-ME-mediated mitochondrial damage by blocking apoptosis induced through the Fas pathway.