p53 is the most commonly mutated gene in human cancer. After activation by cellular stresses such as DNA damage or oncogene activation, p53, a sequence-specific DNA-binding protein, induces the expression of target genes which mediate tumor suppression. Two recently identified p53 homologues, p63 and p73, appear to function similarly to p53, that is, they both activate target gene expression and suppress cell growth when overexpressed; however, the p63 and p73 genes are rarely mutated in human cancer and do not adhere to Knudson's classical model of a tumor suppressor gene. Recently, exciting observations suggest nonoverlapping functions for the family members. Herein, we outline the recent literatures identifying and characterizing both the common and distinct target genes of the p53 family transcription factors in relation to their signaling pathways.

Abstract-Estrogen has antiinflammatory and vasoprotective effects when administered to young women or experimental animals that appear to be converted to proinflammatory and vasotoxic effects in older subjects, particularly those that have been hormone free for long periods. Clinical studies have raised many important questions about the vascular effects of estrogen that cannot easily be answered in human subjects. Here we review cellular/molecular mechanisms by which estrogen modulates injury-induced inflammation, growth factor expression, and oxidative stress in arteries and isolated vascular smooth muscle cells, with emphasis on the role of estrogen receptors and the nuclear factor-B (NFB) signaling pathway, as well as evidence that these protective mechanisms are lost in aging subjects. Ovarian Hormones and Cardiovascular Disease in WomenCardiovascular disease is the leading cause of death among women in the United States, and coronary heart disease (CHD) develops in women on average 10 years later than in men. This lag has been attributed, at least in part, to the protective effects of female sex hormones, particularly estrogens (defined as naturally occurring activators of estrogen receptors) before menopause. 1-3 Mechanistic studies carried out in in vitro preparations and in laboratory animals have shown that both natural and synthetic estrogens have antiinflammatory and vasoprotective effects. 4 -18 Further, the natural endogenous estrogen 17␤-estradiol has been shown to cause rapid endotheliumindependent dilation of coronary arteries of men and women, to augment endothelium-dependent relaxation of human coronary arteries ex vivo, and to improve endothelial function as assessed by the brachial artery flow-mediated dilation response in postmenopausal women. 19 Importantly, the latter vasoprotective effects of estrogen have been observed in the early postmenopausal years in both healthy women and those with CHD, but not in older (Ն60 years) postmenopausal women, regardless of the presence or absence of CHD. 19,20 See accompanying article on page 277Observational studies have shown substantial benefit (Ϸ50% reduction in CHD) of hormone therapy in women who choose to use menopausal hormones (and usually begin taking them in the perimenopausal or early postmenopausal period). 21 Randomized controlled trials of menopausal hormone therapy, which typically enroll women 10 years or longer after menopause, after many years of estrogen deprivation, have shown increases in CHD events with hormone treatment (usually conjugated equine estrogenϮa progestin) in this older (60 to 79 years) age group. [22][23][24] In contrast, subgroup analyses of the Women's Health Initiative have shown that women in whom hormone therapy was initiated at a younger age (50 to 59 years), and earlier post menopause tended to have reduced risk of CHD and total mortality. 25,26 Use of unopposed conjugated estrogen was associated with lower risk of CHD than combined estrogenϩprogestin (medroxyprogesterone acetate), and an ancillary study sh...

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.

Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. Here, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in glial cells. We show that ER stress is present in the central nervous system (CNS) concomitant with inflammation and astrogliosis in the multiple sclerosis (MS) mouse model of experimental autoimmune encephalomyelitis (EAE). Astrocytes do not easily succumb to ER stress but rather activate an inflammatory program involving activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of interleukin 6 (IL-6) and several chemokines. Moreover, the activation of STAT3 signaling is dependent on PERK, a central component of the ER stress response, which we show is phosphorylated by JAK1. Disruption of PERK abrogates ER stress-induced activation of STAT3 and subsequent gene expression. Additionally, ER-stressed astrocytes, via paracrine signaling, can stimulate activation of microglia, leading to production of IL-6 and oncostatin M (OSM). These IL-6 cytokines can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK/JAK1/STAT3 signaling pathway that elicits a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation, which may be relevant in diseases such as MS.

The NF-B family mediates immune and inflammatory responses. In many cancers, NF-B is constitutively activated and induces the expression of genes that facilitate tumorigenesis. ING4 is a tumor suppressor that is absent or mutated in several cancers. Herein, we demonstrate that in human gliomas, NF-B is constitutively activated, ING4 expression is negligible, and NF-B-regulated gene expression is elevated. We demonstrate that an ING4 and NF-B interaction exists but does not prevent NF-B activation, nuclear translocation, or DNA binding. Instead, ING4 and NF-B bind simultaneously at NF-B-regulated promoters, and this binding correlates with reductions in p65 phosphorylation, p300, and the levels of acetylated histones and H3-Me3K4, while enhancing the levels of HDAC-1 at these promoters. Using a knockdown approach, we correlate reductions in ING4 protein levels with increased basal and inducible NF-B target gene expression. Collectively, these data suggest that ING4 may specifically regulate the activity of NF-B molecules that are bound to target gene promoters.

p73, a p53 family protein, shares significant sequence homolog and functional similarity with p53. However, unlike p53, p73 has at least seven alternatively spliced isoforms with different carboxyl termini (p73␣-). Moreover, the p73 gene can be transcribed from a cryptic promoter located in intron 3, producing seven more proteins (⌬Np73␣-). ⌬Np73, which does not contain the N-terminal activation domain in p73, has been thought to be transcriptionally inactive and dominant negative over p53 or p73. To systemically analyze the activity of the ⌬N variant, we generated stable cell lines, which inducibly express ⌬Np73␣, ⌬Np73␤, and various ⌬Np73␤ mutants by using the tetracycline-inducible expression system. Surprisingly, we found that ⌬Np73␤ is indeed active in inducing cell cycle arrest and apoptosis. Importantly, we found that, when ⌬Np73␤ is expressed at a physiologically relevant level, it is capable of suppressing cell growth. We then demonstrated that these ⌬Np73␤ activities are not cell type specific. We showed that the 13 unique residues at the N terminus are required for ⌬Np73␤ to suppress cell growth. We also found that, among the 13 residues, residues 6 to 10 are critical to ⌬Np73␤ function. Furthermore, we found that ⌬Np73␤ is capable of inducing some p53 target genes, albeit to a lesser extent than does p73␤. Finally, we found that the 13 unique residues, together with the N-terminal PXXP motifs, constitute a novel activation domain. Like ⌬Np73␤, ⌬Np73␥ is active in transactivation. However, unlike ⌬Np73␤, ⌬Np73␣ is inactive in suppressing cell growth. Our data, together with others' previous findings, suggest that ⌬Np73␤ may have distinct functions under certain cellular circumstances.p73, along with p53 and p63, constitutes the p53 family. p73 shares 63% identity in amino acids with p53 in the DNAbinding domain, including all the DNA contact residues, 38% identity in the tetramerization domain, and 29% identity in the transactivation domain (31,37,55). In contrast to the human p53 gene, which is found to only encode one protein, human TP73 produces at least seven alternatively spliced isoforms with different carboxyl termini (p73␣-), termed the TA variant (10,28,38,53). For example, p73␣ is the longest form of the p73 protein, which contains a sterile ␣ motif (SAM domain) and an extreme C-terminal region, whereas p73␤ is a smaller polypeptide, missing the extreme C-terminal region and most of the SAM domain in p73␣ (8,29,31,50). In addition to the alternative splicing in the C terminus, TP73 is also transcribed from a cryptic promoter located in intron 3, which gives rise to at least another seven isoforms (⌬Np73␣-), termed the ⌬N variant (28,55,56,58). The ⌬N variant does not contain the activation domain in p73 due to lack of sequences encoded by exon 2 (45, 56). However, the ⌬N variant acquires 13 unique residues at the N terminus compared with the TA variant (45, 56). Similar to TP73, TP63 encodes both .In addition to the significant sequence homology, p53 and p73 share a lot of functional similar...

Glioblastoma (GBM) is the most aggressive, neurologically destructive and deadly tumor of the central nervous system (CNS). In GBM, the transcription factors NF-κB and STAT3 are aberrantly activated and associated with tumor cell proliferation, survival, invasion and chemoresistance. In addition, common activators of NF-κB and STAT3, including TNF-α and IL-6, respectively, are abundantly expressed in GBM tumors. Herein, we sought to elucidate the signaling crosstalk that occurs between the NF-κB and STAT3 pathways in GBM tumors. Using cultured GBM cell lines as well as primary human GBM xenografts, we elucidated the signaling crosstalk between the NF-κB and STAT3 pathways utilizing approaches that either a) reduce NF-κB p65 expression, b) inhibit NF-κB activation, c) interfere with IL-6 signaling, or d) inhibit STAT3 activation. Using the clinically relevant human GBM xenograft model, we assessed the efficacy of inhibiting NF-κB and/or STAT3 alone or in combination in mice bearing intracranial xenograft tumors in vivo. We demonstrate that TNF-α-induced activation of NF-κB is sufficient to induce IL-6 expression, activate STAT3, and elevate STAT3 target gene expression in GBM cell lines and human GBM xenografts in vitro. Moreover, the combined inhibition of NF-κB and STAT3 signaling significantly increases survival of mice bearing intracranial tumors. We propose that in GBM, the activation of NF-κB ensures subsequent STAT3 activation through the expression of IL-6. These data verify that pharmacological interventions to effectively inhibit the activity of both NF-κB and STAT3 transcription factors must be used in order to reduce glioma size and aggressiveness.

BackgroundWe have shown that glucosamine (GlcN) or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) treatment augments O-linked-N-acetylglucosamine (O-GlcNAc) protein modification and attenuates inflammatory mediator expression, leukocyte infiltration and neointima formation in balloon injured rat carotid arteries and have identified the arterial smooth muscle cell (SMC) as the target cell in the injury response. NFκB signaling has been shown to mediate the expression of inflammatory genes and neointima formation in injured arteries. Phosphorylation of the p65 subunit of NFκB is required for the transcriptional activation of NFκB. This study tested the hypothesis that GlcN or PUGNAc treatment protects vascular SMCs against tumor necrosis factor (TNF)-α induced inflammatory stress by enhancing O-GlcNAcylation and inhibiting TNF-α induced phosphorylation of NFκB p65, thus inhibiting NFκB signaling.Methodology/Principal FindingsQuiescent rat aortic SMCs were pretreated with GlcN (5 mM), PUGNAc (10−4 M) or vehicle and then stimulated with TNF-α (10 ng/ml). Both treatments inhibited TNF-α-induced expression of chemokines [cytokine-induced neutrophil chemoattractant (CINC)-2β and monocyte chemotactic protein (MCP)-1] and adhesion molecules [vascular cell adhesion molecule (VCAM)-1 and P-Selectin]. Both treatments inhibited TNF-α induced NFκB p65 activation and promoter activity, increased NFκB p65 O-GlcNAcylation and inhibited NFκB p65 phosphorylation at Serine 536, thus promoting IκBα binding to NFκB p65.ConclusionsThere is a reciprocal relationship between O-GlcNAcylation and phosphorylation of NFκB p65, such that increased NFκB p65 O-GlcNAc modification inhibits TNF-α-Induced expression of inflammatory mediators through inhibition of NFκB p65 signaling. These findings provide a mechanistic basis for our previous observations that GlcN and PUGNAc treatments inhibit inflammation and remodeling induced by acute endoluminal arterial injury.