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The transcription factor p63 is critical for many biological processes, including development and maintenance of epidermal tissues and tumorigenesis. Here, we report that the TAp63 isoforms regulate cell metabolism through the induction of the mitochondrial glutaminase 2 (GLS2) gene both in primary cells and tumor cell lines. By ChIP analysis and luciferase assay, we confirmed that TAp63 binds directly to the p53/p63 consensus DNA binding sequence within the GLS2 promoter region. Given the critical role of p63 in epidermal differentiation, we have investigated the regulation of GLS2 expression during this process. GLS2 and TAp63 expression increases during the in vitro differentiation of primary human keratinocytes, and depletion of GLS2 inhibits skin differentiation both at molecular and cellular levels. We found that GLS2 and TAp63 expression are concomitantly induced in cancer cells exposed to oxidative stresses. siRNA-mediated depletion of GLS2 sensitizes cells to ROS-induced apoptosis, suggesting that the TAp63/GLS2 axis can be functionally important as a cellular antioxidant pathway in the absence of p53. Accordingly, we found that GLS2 is upregulated in colon adenocarcinoma. Altogether, our findings demonstrate that GLS2 is a bona fide TAp63 target gene, and that the TAp63-dependent regulation of GLS2 is important for both physiological and pathological processes.

Epidemiological studies strongly suggest that chronic psychological stress promotes tumorigenesis. However, its direct link in vivo and the underlying mechanisms that cause this remain unclear. This study provides direct evidence that chronic stress promotes tumorigenesis in vivo; chronic restraint, a well-established mouse model to induce chronic stress, greatly promotes ionizing radiation (IR)-induced tumorigenesis in p53(+/-) mice. The tumor suppressor protein p53 plays a central role in tumor prevention. Loss or attenuation of p53 function contriubutes greatly to tumorigenesis. We found that chronic restraint decreases the levels and function of p53 in mice, and furthermore, promotes the growth of human xenograft tumors in a largely p53-dependent manner. Our results show that glucocorticoids elevated during chronic restraint mediate the effect of chronic restraint on p53 through the induction of serum- and glucocorticoid-induced protein kinase (SGK1), which in turn increases MDM2 activity and decreases p53 function. Taken together, this study demonstrates that chronic stress promotes tumorigenesis in mice, and the attenuation of p53 function is an important part of the underlying mechanism, which can be mediated by glucocortcoids elevated during chronic restraint.

In the present study, retinal Müller cells were cultured in vitro and treated with hydrogen peroxide (oxidative stressor) and cobalt chloride (hypoxic injury). Following 24 hours of culture, compensatory hypertrophy was observed and cellular apoptosis increased. Hypoxia enhanced the migration ability of retinal Müller cells and induced the expression of α-smooth muscle actin. Oxidative stress altered the morphology of Müller cells when compared with hypoxia treatment.

Regulation of energy metabolism is a novel function of p53 in tumor suppression. Parkin (PARK2), a Parkinson disease-associated gene, is a potential tumor suppressor whose expression is frequently diminished in tumors. Here Parkin was identified as a p53 target gene that is an important mediator of p53's function in regulating energy metabolism. The human and mouse Parkin genes contain functional p53 responsive elements, and p53 increases the transcription of Parkin in both humans and mice. Parkin contributes to the function of p53 in glucose metabolism; Parkin deficiency activates glycolysis and reduces mitochondrial respiration, leading to the Warburg effect. Restoration of Parkin expression reverses the Warburg effect in cells. Thus, Parkin deficiency is a novel mechanism for the Warburg effect in tumors. Parkin also contributes to the function of p53 in antioxidant defense. Furthermore, Parkin deficiency sensitizes mice to γ-irradiation-induced tumorigenesis, which provides further direct evidence to support a role of Parkin in tumor suppression. Our results suggest that as a novel component in the p53 pathway, Parkin contributes to the functions of p53 in regulating energy metabolism, especially the Warburg effect, and antioxidant defense, and thus the function of p53 in tumor suppression.

Acrolein (Acr) is a ubiquitous environmental contaminant; it also can be generated endogenously by lipid peroxidation. Acr contains a carbonyl group and an olefinic double bond; it can react with many cellular molecules including amino acids, proteins and nucleic acids. In this review article we focus on updating information regarding: (i) Acr-induced DNA damage and methods of detection, (ii) repair of Acr-DNA damage, (iii) mutagenicity of Acr-DNA adducts, (iv) sequence specificity and methylation effect on Acr-DNA adduct formation and (v) the role of Acr in human cancer. We have found that Acr can inhibit DNA repair and induces mutagenic Acr-dG adducts and that the binding spectrum of Acr in the p53 gene in normal human bronchial epithelial cells is similar to the p53 mutational spectrum in lung cancer. Since Acr-DNA adduct has been identified in human lung tissue and Acr causes bladder cancer in human and rat models, we conclude that Acr is a major lung and bladder carcinogen, and its carcinogenicity arises via induction of DNA damage and inhibition of DNA repair., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tumor suppressor p53 plays a central role in tumor prevention. As a transcription factor, p53 mainly exerts its function through transcription regulation of its target genes to initiate various cellular responses. To maintain its proper function, p53 is tightly regulated by a wide variety of regulators in cells. Thus, p53, its regulators and regulated genes form a complex p53 network which is composed of hundreds of genes and their products. microRNAs (miRNAs) are a class of endogenously expressed, small non-coding RNA molecules which play a key role in regulation of gene expression at the post-transcriptional level. Recent studies have demonstrated that miRNAs interact with p53 and its network at multiple levels. p53 regulates the transcription expression and the maturation of a group of miRNAs. On the other hand, miRNAs can regulate the activity and function of p53 through direct repression of p53 or its regulators in cells. These findings have demonstrated that miRNAs are important components in the p53 network, and also added another layer of complexity to the p53 network.

Whereas cell cycle arrest, apoptosis, and senescence are traditionally thought of as the major functions of the tumor suppressor p53, recent studies revealed two unique functions for this protein: p53 regulates cellular energy metabolism and antioxidant defense mechanisms. Here, we identify glutaminase 2 (GLS2) as a previously uncharacterized p53 target gene to mediate these two functions of the p53 protein. GLS2 encodes a mitochondrial glutaminase catalyzing the hydrolysis of glutamine to glutamate. p53 increases the GLS2 expression under both nonstressed and stressed conditions. GLS2 regulates cellular energy metabolism by increasing production of glutamate and alpha-ketoglutarate, which in turn results in enhanced mitochondrial respiration and ATP generation. Furthermore, GLS2 regulates antioxidant defense function in cells by increasing reduced glutathione (GSH) levels and decreasing ROS levels, which in turn protects cells from oxidative stress (e.g., H(2)O(2))-induced apoptosis. Consistent with these functions of GLS2, the activation of p53 increases the levels of glutamate and alpha-ketoglutarate, mitochondrial respiration rate, and GSH levels and decreases reactive oxygen species (ROS) levels in cells. Furthermore, GLS2 expression is lost or greatly decreased in hepatocellular carcinomas and the overexpression of GLS2 greatly reduced tumor cell colony formation. These results demonstrated that as a unique p53 target gene, GLS2 is a mediator of p53's role in energy metabolism and antioxidant defense, which can contribute to its role in tumor suppression.

Leukemia stem cells (LSCs) are capable of limitless self-renewal and are responsible for the maintenance of leukemia. Because selective eradication of LSCs could offer substantial therapeutic benefit, there is interest in identifying the signaling pathways that control their development. We studied LSCs in mouse models of acute myelogenous leukemia (AML) induced either by coexpression of the Hoxa9 and Meis1a oncogenes or by the fusion oncoprotein MLL-AF9. We show that the Wnt/beta-catenin signaling pathway is required for self-renewal of LSCs that are derived from either hematopoietic stem cells (HSC) or more differentiated granulocyte-macrophage progenitors (GMP). Because the Wnt/beta-catenin pathway is normally active in HSCs but not in GMP, these results suggest that reactivation of beta-catenin signaling is required for the transformation of progenitor cells by certain oncogenes. beta-catenin is not absolutely required for self-renewal of adult HSCs; thus, targeting the Wnt/beta-catenin pathway may represent a new therapeutic opportunity in AML.

This project was aimed to develop a simple, convenient and reliable computer image processing software for the measurement of lens dimensions, including the radius of curvature of anterior lens surface (RCALS), the radius of curvature of posterior lens surface (RCPLS) and the lens volume (LV). On the basis of lens images captured by B ultrasound, our computer software was designed to calculate the three parameters of lens in accordance to geometry principle. This software comprises Program I and Program II, and they both possess different calculation methods. Then they were used in a group of normal volunteers who were recruited via randomization and the outcomes were compared. The results showed that the two programs were developed successfully, and the outcomes of RCALS, RCPLS and LV calculated by the two programs were similar. The standard deviation of Program II is smaller than that of Program I. The computer software for calculating RCALS, RCPLS and LV was characterized by scientific design and easy-to-use. In respect of calculating repetition, Program II was better than Program I, and it possesses prosperous prospect in terms of clinical research and application.

In response to various stress signals, which introduce infidelity into the processes of cell growth and division, p53 initiates cell-cycle arrest, apoptosis, or senescence to maintain fidelity throughout the cell cycle. Although these functions are traditionally thought of as the major functions of the p53 protein for tumor suppression, recent studies have revealed some additional novel functions of the p53 pathway. These include the down-regulation of two central cell-growth pathways, the IGF/AKT-1 and mTOR pathways, and the up-regulation of the activities of the endosomal compartment. The IGF-1/AKT and mTOR pathways are two evolutionarily conserved pathways that play critical roles in regulation of cell proliferation, survival, and energy metabolism. In response to stress, p53 transcribes a group of critical negative regulators in these two pathways, including IGF-BP3, PTEN, TSC2, AMPK beta1, and Sestrin1/2, which leads to the reduction in the activities of these two pathways. Furthermore, p53 transcribes several critical genes regulating the endosomal compartment, including TSAP6, Chmp4C, Caveolin-1, and DRAM, and increases exosome secretion, the rate of endosomal removal of growth factor receptors (e.g., EGFR) from cell surface, and enhances autophagy. These activities all function to slow down cell growth and division, conserve and recycle cellular resources, communicate with adjacent cells and dendritic cells of the immune system, and inform other tissues of the stress signals. This coordinated regulation of IGF-1/AKT/mTOR pathways and the endosomal compartment by the p53 pathway integrates the molecular, cellular, and systemic levels of activities and prevents the accumulations of errors in response to stress and restores cellular homeostasis after stress.

Leukemias are composed of a hierarchy of cells, only a fraction of which have stem cell-like properties and are capable of self-renewal. Mixed lineage leukemia (MLL) fusion proteins produced by translocations involving the MLL gene on chromosome 11q23 confer stem cell-like properties on committed hematopoietic progenitors. This provides an opportunity to assess changes in immunophenotype, gene expression, and epigenetic programs during the transition from a hematopoietic cell with minimal inherent self-renewal capability to cells capable of leukemic self-renewal.

The tumor suppressor protein p53 plays an important role in maternal reproduction in mice through transcriptional regulation of leukemia inhibitory factor (LIF), a cytokine crucial for blastocyst implantation. To determine whether these observations could be extended to humans, a list of single-nucleotide polymorphisms (SNPs) in the p53 pathway that can modify the function of p53 was assembled and used to study their impact on human fertility. The p53 allele encoding proline at codon 72 (P72) was found to be significantly enriched over the allele encoding arginine (R72) among in vitro fertilization (IVF) patients. The P72 allele serves as a risk factor for implantation failure. LIF levels are significantly lower in cells with the P72 allele than in cells with the R72 allele, which may contribute to the decreased implantation and fertility associated with the P72 allele. Selected alleles in SNPs in LIF, Mdm2, Mdm4, and Hausp genes, each of which regulates p53 levels in cells, are also enriched in IVF patients. Interestingly, the role of these SNPs on fertility was much reduced or absent in patients older than 35 years of age, indicating that other functions may play a more important role in infertility in older women. The association of SNPs in the p53 pathway with human fertility suggests that p53 regulates the efficiency of human reproduction. These results also provide a plausible explanation for the evolutionary positive selection of some alleles in the p53 pathway and demonstrate the alleles in the p53 pathway as a good example of antagonistic pleiotropy.

Characterization of gene expression programs and pathways important for normal and cancer stem cells has become an active area of investigation. Microarray analysis of various cell populations provides an opportunity to assess genomewide expression programs to define cellular identity and to potentially identify pathways activated in various stem cells. Here we describe methods to isolate a leukemia stem cell population, amplify RNA, and perform microarray analyses.

We created a mouse model wherein conditional expression of an Mll-AF4 fusion oncogene induces B precursor acute lymphoblastic (ALL) or acute myeloid leukemias (AML). Gene expression profile analysis of the ALL cells demonstrated significant overlap with human MLL-rearranged ALL. ChIP-chip analysis demonstrated histone H3 lysine 79 (H3K79) methylation profiles that correlated with Mll-AF4-associated gene expression profiles in murine ALLs and in human MLL-rearranged leukemias. Human MLL-rearranged ALLs could be distinguished from other ALLs by their H3K79 profiles, and suppression of the H3K79 methyltransferase DOT1L inhibited expression of critical MLL-AF4 target genes. We thus demonstrate that ectopic H3K79 methylation is a distinguishing feature of murine and human MLL-AF4 ALLs and is important for maintenance of MLL-AF4-driven gene expression.

Extensive studies have shown that p53 is important in tumour prevention. However, little is known about its normal physiological function. Here we show that p53 is important in reproduction, in a gender-specific manner. Significant decreases in embryonic implantation, pregnancy rate and litter size were observed in matings with p53-/- female mice but not with p53-/- male mice. The gene encoding leukaemia inhibitory factor (LIF), a cytokine critical for implantation, was identified as a p53-regulated gene that functions as the downstream mediator of this effect. p53 can regulate both basal and inducible transcription of LIF. Loss of p53 decreased both the level and function of LIF in uteri. Lower LIF levels were observed in the uteri of p53-/- mice than in those of p53+/+ mice, particularly at day 4 of pregnancy, when transiently induced high levels of LIF were crucial for embryonic implantation. This observation probably accounts for the impaired implantation of embryos in p53-/- female mice. Administration of LIF to pregnant p53-/- mice restored maternal reproduction by improving implantation. These results demonstrate a function for p53 in maternal reproduction through the regulation of LIF. Evidence is accumulating that p53 may have a similar function in humans.

Cancer is a disease of aging. The accumulation of mutations in individual cells over a lifetime is thought to be the reason. In this work, we explored an additional hypothesis: could p53 function decline with age, which would contribute to an enhanced mutation frequency and tumorigenesis in the aging process? The efficiency of the p53 response to gamma-irradiation was found to decline significantly in various tissues of aging mice from several inbred strains, including lower p53 transcriptional activity and p53-dependent apoptosis. This decline resulted from a decreased stabilization of the p53 protein after stress. The function of the Ataxia-telangiectasia mutated (ATM) kinase declined significantly with age, which may then be responsible for the decline of the p53 response to radiation. Declining p53 responses to other stresses were also observed in the cultured splenocytes from aging mice. Interestingly, the time of onset of this decreased p53 response correlated with the life span of mice; mice that live longer delay their onset of decreased p53 activity with time. These results suggest an enhanced fixation of mutations in older individuals because of the declining fidelity of p53-mediated apoptosis or senescence in response to stress, and they suggest a plausible explanation for the correlation between tumorigenesis and the aging process.

The insulin-like growth factor 1 (IGF-1)-AKT-mTOR pathways sense the availability of nutrients and mitogens and respond by signaling for cell growth and division. The p53 pathway senses a variety of stress signals which will reduce the fidelity of cell growth and division, and responds by initiating cell cycle arrest, senescence, or apoptosis. This study explores four p53-regulated gene products, the beta1 and beta2 subunits of the AMPK, which are shown for the first time to be regulated by the p53 protein, TSC2, PTEN, and IGF-BP3, each of which negatively regulates the IGF-1-AKT-mTOR pathways after stress. These gene products are shown to be expressed under p53 control in a cell type and tissue-specific fashion with the TSC2 and PTEN proteins being coordinately regulated in those tissues that use insulin-dependent energy metabolism (skeletal muscle, heart, white fat, liver, and kidney). In addition, these genes are regulated by p53 in a stress signal-specific fashion. The mTOR pathway also communicates with the p53 pathway. After glucose starvation of mouse embryo fibroblasts, AMPK phosphorylates the p53 protein but does not activate any of the p53 responses. Upon glucose starvation of E1A-transformed mouse embryo fibroblasts, a p53-mediated apoptosis ensues. Thus, there is a great deal of communication between the p53 pathway and the IGF-1-AKT and mTOR pathways.

The tumor suppressor gene p53 is frequently mutated in cigarette smoke (CS)-related lung cancer. The p53 binding pattern of carcinogenic polycyclic aromatic hydrocarbons (PAHs) found in CS coincides with the p53 mutational pattern found in lung cancer, and PAHs have thus been considered to be major culprits for lung cancer. However, compared with other carcinogenic compounds, such as aldehydes, the amount of PAHs in CS is minute. Acrolein (Acr) is abundant in CS, and it can directly adduct DNA. Acr-DNA adducts, similar to PAH-DNA adducts, induce predominantly G-to-T transversions in human cells. These findings raise the question of whether Acr-DNA adducts are responsible for p53 mutations in CS-related lung cancer. To determine the role of Acr-DNA adducts in p53 mutagenesis in CS-related lung cancer we mapped the distribution of Acr-DNA adducts at the sequence level in the p53 gene of lung cells using the UvrABC incision method in combination with ligation-mediated PCR. We found that the Acr-DNA binding pattern is similar to the p53 mutational pattern in human lung cancer. Acr preferentially binds at CpG sites, and this enhancement of binding is due to cytosine methylation at these sequences. Furthermore, we found that Acr can greatly reduce the DNA repair capacity for damage induced by benzo[a]pyrene diol epoxide. Together these results suggest that Acr is a major etiological agent for CS-related lung cancer and that it contributes to lung carcinogenesis through two detrimental effects: DNA damage and inhibition of DNA repair.

Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed. However, it remains unclear whether cancer stem cells must be phenotypically similar to normal tissue stem cells or whether they can retain the identity of committed progenitors. Here we show that leukaemia stem cells (LSC) can maintain the global identity of the progenitor from which they arose while activating a limited stem-cell- or self-renewal-associated programme. We isolated LSC from leukaemias initiated in committed granulocyte macrophage progenitors through introduction of the MLL-AF9 fusion protein encoded by the t(9;11)(p22;q23). The LSC were capable of transferring leukaemia to secondary recipient mice when only four cells were transferred, and possessed an immunophenotype and global gene expression profile very similar to that of normal granulocyte macrophage progenitors. However, a subset of genes highly expressed in normal haematopoietic stem cells was re-activated in LSC. LSC can thus be generated from committed progenitors without widespread reprogramming of gene expression, and a leukaemia self-renewal-associated signature is activated in the process. Our findings define progression from normal progenitor to cancer stem cell, and suggest that targeting a self-renewal programme expressed in an abnormal context may be possible.

Over the past 10 years the signal transduction networks for p53, IGF-1-AKT, and TOR pathways have been assembled in worms, flies, and mammals, and their functions elucidated. In the past 1-2 years a number of genes and their proteins have been identified that permit extensive communication and coordination between these pathways. These three pathways are involved in sensing and integrating signals arising from nutrient and growth factor availability, signals from sensory and sexual organs, and intrinsic and extrinsic stress signals. In turn these pathways regulate cell growth, proliferation, and death. These networks are central to our understanding of a variety of physiological and pathological conditions, including cancer, diabetes, and longevity.

Cell growth and proliferation requires an intricate coordination between the stimulatory signals arising from nutrients and growth factors and the inhibitory signals arising from intracellular and extracellular stresses. Alteration of the coordination often causes cancer. In mammals, the mTOR (mammalian target of rapamycin) protein kinase is the central node in nutrient and growth factor signaling, and p53 plays a critical role in sensing genotoxic and other stresses. The results presented here demonstrate that activation of p53 inhibits mTOR activity and regulates its downstream targets, including autophagy, a tumor suppression process. Moreover, the mechanisms by which p53 regulates mTOR involves AMP kinase activation and requires the tuberous sclerosis (TSC) 1/TSC2 complex, both of which respond to energy deprivation in cells. In addition, glucose starvation not only signals to shut down mTOR, but also results in the transient phosphorylation of the p53 protein. Thus, p53 and mTOR signaling machineries can cross-talk and coordinately regulate cell growth, proliferation, and death.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens. PAHs are classified into bay and fjord region compounds according to structural differences in the molecule region where enzymatic epoxidation occurs. Dibenzo[a,l]pyrene (DB[a,l]P), one of the fjord region compounds, has been demonstrated to be the most carcinogenic PAH known to date. DB[a,l]P is activated to fjord region (+)-syn and (-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) metabolites. In this study, we analyzed mutagenesis induced by (+)-syn- and (-)-anti-DB[a,l]PDE at the cII transgene in Big-Blue mouse cells. The mutant frequency of untreated cells (background level) was 6.53 x 10(-5). This level increased 3.7-fold for 20 nmol/L, 5.3-fold for 50 nmol/L, and 7.9-fold for 100 nmol/L (+)-syn-DB[a,l]PDE, respectively. In the case of (-)-anti-DB[a,l]PDE it increased 4.5-fold for 20 nmol/L, 6.7-fold for 50 nmol/L, and 10.6-fold for 100 nmol/L, respectively, indicating that (-)-anti-DB[a,l]PDE is slightly more mutagenic than (+)-syn-DB[a,l]PDE. The mutational spectra of (+)-syn- and (-)-anti-DB[a,l]PDE were quite similar except for several hotspots, specific for either (+)-syn-DB[a,l]PDE or (-)-anti-DB[a,l]PDE. The most frequently induced mutations were A to T transversions, which were 43.9% for (+)-syn- and 38.8% for (-)-anti-DB[a,l]PDE. In addition, G to T transversions were induced significantly, at frequencies of 18.5% by (+)-syn- and 18.1% by (-)-anti-DB[a,l]PDE. Using UvrABC cleavage and ligation-mediated PCR or the terminal transferase-dependent PCR method, we have determined DB[a,l]PDE-DNA adduct formation sites and repair rates in carcinogen-exposed cells. The mutation hotspots coincided with sites of strong adduct formation, but not all of the adduct hotspots were mutational hotspots. Slow adduct removal occurred for both (+)-syn- and (-)-anti-DB[a,l]PDE adducts over a time period of up to 72 hours. The data suggest that, although the (-)-anti-isomer is slightly more mutagenic, DNA adducts of both DB[a,l]PDE stereoisomers may have similar biological properties. We discuss the implications of these findings for human cancer mutagenesis.

Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect in V(D)J recombination. In addition, Artemis mutants are radiosensitive and chromosomally unstable, which has been attributed to a defect in nonhomologous end joining (NHEJ). We show here, however, that Artemis-depleted cell extracts are not defective in NHEJ and that Artemis-deficient cells have normal repair kinetics of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however, to interact with known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also show that Artemis is required for the maintenance of a normal DNA damage-induced G2/M cell cycle arrest. Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or Chk2. These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and chromosomal instability of Artemis-deficient cells may be due to defects in cell cycle responses after DNA damage.

Lipid peroxidation (LPO) is a cellular process that commonly takes place under normal physiological conditions. Under excessive oxidative stress, the level of LPO becomes very significant, and a growing body of evidence has shown that excessive LPO may be involved in carcinogenesis. Trans-4-hydroxy-2-nonenal (4-HNE) is a major product of LPO, and its level becomes relatively high in cells under oxidative stress. 4-HNE is able to react readily with various cellular components, including DNA and proteins. We previously found that the 4-HNE-DNA adduct is a potent mutagen in human cells and is preferentially formed at codon 249 of the p53 gene, a mutational hotspot in human cancers. To further understand the role of 4-HNE in carcinogenesis, we addressed the question of whether 4-HNE affects DNA repair in human cells. We found that the repair capacity for benzo[a]pyrene diol epoxide and UV light-induced DNA damage was greatly compromised in human cells or human cell extracts treated with 4-HNE, which is mainly through interaction of 4-HNE with cellular repair proteins. We also found that 4-HNE greatly sensitizes cells to benzo[a]pyrene diol epoxide- and UV-induced killing. Together these results strongly suggest that this LPO metabolite damages not only DNA but also DNA repair mechanisms in human cells. We propose that these two detrimental effects of LPO may contribute synergistically to human carcinogenesis.

Polycyclic aromatic hydrocarbons (PAHs) and their derivatives, such as benzo[a]pyrene (B[a]P), (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), and 5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE), are complete carcinogens. However, the tumor promotion effects of PAHs remain unclear. We therefore investigated the possible activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NFkappaB) in mouse epidermal Cl41 cells after different PAHs treatments, including B[a]P, B[a]PDE, chrysene-1,2-diol-3,4-epoxid (CDE), and 5-MCDE. The results showed that B[a]PDE and 5-MCDE were able to activate AP-1 and NF-kappaB, whereas B[a]P showed only marginal effect on AP-1 activation, and B[a]P and CDE had no effect on NF-kappaB activation. Treatment with either B[a]PDE or 5-MCDE also resulted in mitogen-activated protein kinases (MAPKs) activation as well as inhibitory subunit kappa-B (IkappaBalpha) phosphorylation and degradation, whereas B[a]P and CDE had no effect. Pretreatment with PD98059, a specific inhibitor for extracellular signal-regulated protein kinases (ERKs) upstream kinase MEK1/2, or SB202190, a p38 kinase inhibitor, resulted in a dramatic inhibition of B[a]PDE-induced AP-1 transactivation. In addition, B[a]PDE-induced AP-1 activation was also inhibited by overexpressing a dominant negative mutant of JNK1 in the cells. All these suggest ERKs, c-jun N-terminal kinases (JNKs), and p38 kinase signal transduction pathways are required for AP-1 induction by B[a]PDE. Taken together, B[a]PDE and 5-MCDE are the active compounds of PAHs to initiate signaling pathways. Considering the important roles of AP-1 and NF-kappaB in tumor promotion, we speculated the activation of AP-1 and NF-kappaB by B[a]PDE and 5-MCDE may involve in their or their parent compounds' tumor promotion effects. This study may help in better understanding the tumor promotion effects of PAHs., (Copyright 2004 Wiley-Liss, Inc.)

Nickel (II), a ubiquitous environmental and industrial contaminant, is a well-known human carcinogen, particularly in human lung cancer. Although by itself it is a weak mutagen, nickel (II) is able to significantly enhance the genotoxicity of other mutagens and carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and ultraviolet light. Certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs. To understand the interplay of nickel (II) and PAHs in mutagenesis and human carcinogenesis, we used a shuttle vector mutagenicity assay to examine the effect of nickel (II) on (+/-) anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE)-induced mutagenesis in human cells. BPDE is an activated metabolite of benzo[a]pyrene (BP), a major carcinogen in cigarette smoke. The shuttle vector pSP189 modified with BPDE was transfected into human cells with and without nickel (II) exposure. We found that nickel (II) exposure significantly enhanced BPDE-induced mutation frequency, but did not change BPDE-induced mutational spectrum in the supF gene of pSP189 plasmids replicated in nucleotide excision repair (NER)-proficient human cells. However, the enhancing effect of nickel (II) on BPDE-induced mutation frequency was not observed in NER-deficient human XPA cells. We also found that nickel (II) exposure of human cells did not change the spontaneous mutation frequency of the supF gene in NER-proficient or NER-deficient human cells, indicating that nickel (II) did not affect the replication fidelity in human cells. Using a plasmid containing a luciferase reporter gene and a host cell reactivation assay, we have found that nickel (II) exposure greatly inhibited the repair of BPDE-DNA adducts in NER-proficient but not in NER-deficient cells. Together these results strongly suggest that nickel (II) can greatly enhance the mutagenicity and genotoxicity of PAHs by inhibiting the NER pathway in human cells, and this may constitute an important mechanism for nickel (II)-induced human carcinogenesis.

It has been long recognized that in mammalian cells, DNA damage is preferentially repaired in the transcribed strand of transcriptionally active genes. However, recently, we found that in Chinese hamster ovary (CHO) cells, UV-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in both the transcribed and the non-transcribed strand of exon 1 of the dihydrofolate reductase (DHFR) gene. We mapped CPD repair at the nucleotide level in the transcriptionally active DHFR gene and the adjacent upstream OST gene, both of which have been translocated to two chromosomal positions that differ from their normal endogeneous positions. This allowed us to study the role of transcription, genomic context and chromatin structure on repair. We found that CPD repair in the transcribed strand is the same for endogenous and translocated DHFR genes, and the order of repair efficiency is exon 1 > exon 2 > exon 5. However, unlike the endogenous DHFR gene, efficient repair of CPDs in the non-transcribed strand of exon 1 is not observed in the translocated DHFR gene. CPDs are efficiently repaired in the transcribed strand in endogenous and translocated OST genes, which indicates that efficient repair in exon 1 of the non-transcribed strand of the endogenous DHFR gene is not due to the extension of transcription-coupled repair of the OST gene. Using micrococcal nuclease digestion, we probed the chromatin structure in the DHFR gene and found that chromatin structure in the exon 1 region of endogenous DHFR is much more open than at translocated loci. These results suggest that while transcription-coupled repair is transcription dependent, global genomic repair is greatly affected by chromatin structure.

The enzyme carnitine palmitoyltransferase-1 (CPT1) regulates long-chain fatty acid (LCFA) entry into mitochondria, where the LCFAs undergo beta-oxidation. To investigate the mechanism(s) by which central metabolism of lipids can modulate energy balance, we selectively reduced lipid oxidation in the hypothalamus. We decreased the activity of CPT1 by administering to rats a ribozyme-containing plasmid designed specifically to decrease the expression of this enzyme or by infusing pharmacological inhibitors of its activity into the third cerebral ventricle. Either genetic or biochemical inhibition of hypothalamic CPT1 activity was sufficient to substantially diminish food intake and endogenous glucose production. These results indicated that changes in the rate of lipid oxidation in selective hypothalamic neurons signaled nutrient availability to the hypothalamus, which in turn modulated the exogenous and endogenous inputs of nutrients into the circulation.

4-Aminobiphenyl (4-ABP) is a major etiological agent for human bladder cancer. Metabolically activated 4-ABP is able to interact with DNA to form adducts that may induce mutations and initiate carcinogenesis. Thirty to sixty percent of bladder cancer has a mutation in the tumor suppressor p53 gene, and the mutational spectrum bears unique features. To date the DNA binding spectrum of 4-ABP in the p53 gene is not known due to the lack of methodology to detect 4-ABP-DNA adducts at nucleotide sequence level. We have found that UvrABC nuclease, a nucleotide excision repair complex isolated from Escherichia coli, is able to incise specifically and quantitatively DNA fragments modified with N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), an activated intermediate of 4-ABP. Using the UvrABC nuclease incision method, we mapped the binding spectrum of N-OH-4-ABP in DNA fragments containing exons 5, 7, and 8 of the human p53 gene and also determined the effect of C5 cytosine methylation on N-OH-4-ABP-DNA binding. We found that codon 285, a mutational hotspot at a non-CpG site in bladder cancer, is the preferential binding site for N-OH-4-ABP. We also found that C5 cytosine methylation greatly enhanced N-OH-4-ABP binding at CpG sites, and that two mutational hotspots at CpG sites, codons 175 and 248, became preferential binding sites for N-OH-4-ABP only after being methylated. These results suggest that both the unique DNA binding specificity of 4-ABP and cytosine methylation contribute to the mutational spectrum of the p53 gene in human bladder cancer.