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LIF, a multifunctional cytokine, is frequently overexpressed in many types of solid tumors, including breast cancer, and plays an important role in promoting tumorigenesis. Currently, how LIF promotes tumorigenesis is not well-understood. Metabolic reprogramming is a hallmark of cancer cells and a key contributor to cancer progression. However, the role of LIF in cancer metabolic reprogramming is unclear. In this study, we found that LIF increases glucose uptake and drives glycolysis, contributing to breast tumorigenesis. Blocking glucose uptake largely abolishes the promoting effect of LIF on breast tumorigenesis. Mechanistically, LIF overexpression enhances glucose uptake via activating the AKT/GLUT1 axis to promote glycolysis. Blocking the AKT signaling by shRNA or its inhibitors greatly inhibits glycolysis driven by LIF and largely abolishes the promoting effect of LIF on breast tumorigenesis. These results demonstrate an important role of LIF overexpression in glucose metabolism reprogramming in breast cancers, which contributes to breast tumorigenesis. This study also reveals an important mechanism underlying metabolic reprogramming of breast cancers, and identifies LIF and its downstream signaling as potential therapeutic targets for breast cancers, especially those with LIF overexpression., (© 2022. The Author(s).)

The tumor suppressor p53 gene is mutated in approximately 50% of all human tumors. Many tumor-associated mutant p53 proteins misfold into a common, denatured conformation and accumulate to high levels in human tumors. In such tumors, these mutant forms of p53 provide a "gain of function" to promote tumor progression. Therefore, targeting mutant p53 has become an attractive approach for cancer therapy. In this issue, the study by Lu and colleagues supports the premise that certain forms of mutant p53 are temperature sensitive in conformation; these forms of p53 are mutant in conformation at physiologic temperature, but can refold into a normal, or "wild-type" conformation at lower temperature (32°C to 34°C). Notably, these temperature-sensitive mutants account for up to 7.5% of all human tumors that carry mutant p53, so this fraction of patients is estimated to be quite significant. Results from this study show that employing therapeutic hypothermia to reduce the core temperature of mice bearing tumors with these temperature-sensitive mutant forms of p53 (ts mutant p53) causes ts mutant p53 to switch to a wild-type conformation in tumors, inhibiting tumor growth. Moreover, combining hypothermia with chemotherapy leads to durable remission of such tumors, with no obvious toxicity to normal tissues. See related article by Lu et al., p. 3905 ., (©2021 American Association for Cancer Research.)

Identification of novel functional domains and characterization of detailed regulatory mechanisms in cancer-driving genes is critical for advanced cancer therapy. To date, CRISPR gene editing has primarily been applied to defining the role of individual genes. Recently, high-density mutagenesis via CRISPR tiling of gene-coding exons has been demonstrated to identify functional regions in genes. Furthermore, breakthroughs in combining CRISPR library screens with single-cell droplet RNA sequencing (sc-RNAseq) platforms have revealed the capacity to monitor gene expression changes upon genetic perturbations at single-cell resolution. Here, we present "sc-Tiling," which integrates a CRISPR gene-tiling screen with single-cell transcriptomic and protein structural analyses. Distinct from other reported single-cell CRISPR screens focused on observing gene function and gene-to-gene/enhancer-to-gene regulation, sc-Tiling enables the capacity to identify regulatory mechanisms within a gene-coding region that dictate gene activity and therapeutic response.

The role of p53 in tumor suppression has been extensively studied and well-established. However, the role of p53 in parasitic infections and the intestinal type 2 immunity is unclear. Here, we report that p53 is crucial for intestinal type 2 immunity in response to the infection of parasites, such as Tritrichomonas muris and Nippostrongylus brasiliensis. Mechanistically, p53 plays a critical role in the activation of the tuft cell-IL-25-type 2 innate lymphoid cell circuit, partly via transcriptional regulation of Lrmp in tuft cells. Lrmp modulates Ca 2+ influx and IL-25 release, which are critical triggers of type 2 innate lymphoid cell response. Our results thus reveal a previously unrecognized function of p53 in regulating intestinal type 2 immunity to protect against parasitic infections, highlighting the role of p53 as a guardian of immune integrity.

Leukemia inhibitory factor (LIF) is a cytokine essential for maintaining pluripotency of mouse embryonic stem cells. However, its role in adult intestinal stem cells (ISCs) is unclear. The adult intestinal epithelium has a high self-renewal rate driven by ISCs in crypts. Here, we find that LIF is present in the ISC niche in crypts and critical for the function of ISCs in maintaining the intestinal epithelial homeostasis and regeneration. Mechanistically, LIF maintains β-catenin activity through the AKT/GSK3β signaling to regulate ISC functions. LIF deficiency in mice impairs the renewal of the intestinal epithelium under the physiological condition. Further, LIF deficiency in mice impairs the regeneration of intestinal epithelium in response to radiation and shortens the lifespan of mice after high doses of radiation due to gastrointestinal (GI) syndrome, which can be rescued by administering recombinant LIF (rLIF). Importantly, LIF exhibits a radioprotective role in wild-type (WT) mice by protecting mice from lethal radiation-induced GI syndrome; administering rLIF promotes intestinal epithelial regeneration and prolongs survival in WT mice after radiation. These results reveal a previously unidentified and a crucial role of LIF in ensuring ISC function, promoting regeneration of the intestinal epithelium in response to radiation and protecting against radiation-induced GI syndrome.

p53 is a key tumor suppressor. As a transcription factor, p53 accumulates in cells in response to various stress signals and selectively transcribes its target genes to regulate a wide variety of cellular stress responses to exert its function in tumor suppression. In addition to tumor suppression, p53 is also involved in many other physiological and pathological processes, e.g. anti-infection, immune response, development, reproduction, neurodegeneration and aging. To maintain its proper function, p53 is under tight and delicate regulation through different mechanisms, particularly the posttranslational modifications. The tripartite motif (TRIM) family proteins are a large group of proteins characterized by the RING, B-Box and coiled-coil (RBCC) domains at the N-terminus. TRIM proteins play important roles in regulation of many fundamental biological processes, including cell proliferation and death, DNA repair, transcription, and immune response. Alterations of TRIM proteins have been linked to many diseases including cancer, infectious diseases, developmental disorders, and neurodegeneration. Interestingly, recent studies have revealed that many TRIM proteins are involved in the regulation of p53, and at the same time, many TRIM proteins are also regulated by p53. Here, we review the cross-talk between p53 and TRIM proteins, and its impact upon cellular biological processes as well as cancer and other diseases., (© 2020 Chongqing Medical University. Production and hosting by Elsevier B.V.)

Purpose: To examine associations of KRAS mutation with tumor deposit status and overall survival in colorectal cancer (CRC) patients., Methods: This retrospective cohort study included patients with incidental CRC diagnosed during 2010-2014 and recorded statuses of KRAS and tumor deposit in the National Cancer Database of the USA. Multivariable logistic regression and time-varying Cox regression analyses were used., Results: We included 45,761 CRC patients with KRAS status (24,027 [52.5%] men, 24,240 [53.0%] < 65 years old, 17,338 [37.9%] with KRAS mutation). Adjusted for microsatellite instability, age, pathologic stage and tumor grade, KRAS mutation (versus wild type) was associated with tumor deposit presence (odds ratio = 1.11, 95% CI 1.02-1.20). KRAS mutation was also linked to worse overall survival of CRC patients regardless of tumor deposit status (adjusted Hazard ratio [HR] = 1.20, 95% CI 1.07-1.33 for CRC with tumor deposits, and adjusted HR = 1.24, 95% CI 1.14-1.35 or CRC without) or tumor stage (adjusted HR = 1.32, 95% CI 1.14-1.54 for early-stage and adjusted HR = 1.18, 95% CI 1.10-1.27 for late-stage). Microsatellite instability was associated with better overall survival in CRC without tumor deposit (adjusted HR = 0.89, 95% CI 0.79-0.99), but not in CRC with tumor deposit (adjusted HR = 1.12, 95% CI 0.97-1.30)., Conclusion: KRAS mutation is independently associated with tumor deposit presence and a worse overall survival in CRC patients.

Phosphoglycerate dehydrogenase (PHGDH), the first rate-limiting enzyme of serine synthesis, is frequently overexpressed in human cancer. PHGDH overexpression activates serine synthesis to promote cancer progression. Currently, PHGDH regulation in normal cells and cancer is not well understood. Parkin, an E3 ubiquitin ligase involved in Parkinson's disease, is a tumor suppressor. Parkin expression is frequently downregulated in many types of cancer, and its tumor-suppressive mechanism is poorly defined. Here, we show that PHGDH is a substrate for Parkin-mediated ubiquitination and degradation. Parkin interacted with PHGDH and ubiquitinated PHGDH at lysine 330, leading to PHGDH degradation to suppress serine synthesis. Parkin deficiency in cancer cells stabilized PHGDH and activated serine synthesis to promote cell proliferation and tumorigenesis, which was largely abolished by targeting PHGDH with RNA interference, CRISPR/Cas9 KO, or small-molecule PHGDH inhibitors. Furthermore, Parkin expression was inversely correlated with PHGDH expression in human breast cancer and lung cancer. Our results revealed PHGDH ubiquitination by Parkin as a crucial mechanism for PHGDH regulation that contributes to the tumor-suppressive function of Parkin and identified Parkin downregulation as a critical mechanism underlying PHGDH overexpression in cancer.

Repair of DNA double-stranded breaks (DSBs) during lymphocyte development is essential for V(D)J recombination and forms the basis of immunoglobulin variable region diversity. Understanding of this process in lymphogenesis has historically been centered on the study of RAG1/2 recombinases and a set of classical non-homologous end-joining factors. Much less has been reported regarding the role of chromatin modifications on this process. Here, we show a role for the non-redundant histone H3 lysine methyltransferase, Setd2, and its modification of lysine-36 trimethylation (H3K36me3), in the processing and joining of DNA ends during V(D)J recombination. Loss leads to mis-repair of Rag-induced DNA DSBs, especially when combined with loss of Atm kinase activity. Furthermore, loss reduces immune repertoire and a severe block in lymphogenesis as well as causes post-mitotic neuronal apoptosis. Together, these studies are suggestive of an important role of Setd2/H3K36me3 in these two mammalian developmental processes that are influenced by double-stranded break repair., Competing Interests: Declaration of Interests S.A.A. has been a consultant and/or shareholder for Epizyme Inc, Imago Biosciences, Cyteir Therapeutics, C4 Therapeutics, Syros Pharmaceuticals, OxStem Oncology, Accent Therapeutics, and Mana Therapeutics. S.A.A. has received research support from Janssen, Novartis, and AstraZeneca. S.H.C. is currently an employee at Beam Therapeutics. The authors have no additional financial interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Infant B-cell acute lymphoblastic leukemias (B-ALLs) that harbor MLL-AF4 rearrangements are associated with a poor prognosis. One important obstacle to progress for this patient population is the lack of immunocompetent models that faithfully recapitulate the short latency and aggressiveness of this disease. Recent whole-genome sequencing of MLL-AF4 B-ALL samples revealed a high frequency of activating RAS mutations; however, single-agent targeting of downstream effectors of the RAS pathway in these mutated MLL- r B-ALLs has demonstrated limited and nondurable antileukemic effects. Here, we demonstrate that the expression of activating mutant N-Ras G12D cooperates with Mll-Af4 to generate a highly aggressive serially transplantable B-ALL in mice. We used our novel mouse model to test the sensitivity of Mll-Af4/N-Ras G12D leukemia to small molecule inhibitors and found potent and synergistic preclinical efficacy of dual targeting of the Mek and Atr pathways in mouse- and patient-derived xenografts with both mutations in vivo, suggesting this combination as an attractive therapeutic opportunity that might be used to treat patients with these mutations. Our studies indicate that this mouse model of Mll-Af4/N-Ras B-ALL is a powerful tool to explore the molecular and genetic pathogenesis of this disease subtype, as well as a preclinical discovery platform for novel therapeutic strategies.

The mutation of the Parkin gene is a cause of familial Parkinson's disease. A growing body of evidence suggests that Parkin also functions as a tumor suppressor. Parkin is an ubiquitin E3 ligase, and plays important roles in a variety of cellular processes implicated in tumorigenesis, including cell cycle, cell proliferation, apoptosis, metastasis, mitophagy and metabolic reprogramming. Here we review the role and mechanism of Parkin in cancer.

MLL/SET methyltransferases catalyze methylation of histone 3 lysine 4 and play critical roles in development and cancer. We assessed MLL/SET proteins and found that SETD1A is required for survival of acute myeloid leukemia (AML) cells. Mutagenesis studies and CRISPR-Cas9 domain screening show the enzymatic SET domain is not necessary for AML cell survival but that a newly identified region termed the "FLOS" (functional location on SETD1A) domain is indispensable. FLOS disruption suppresses DNA damage response genes and induces p53-dependent apoptosis. The FLOS domain acts as a cyclin-K-binding site that is required for chromosomal recruitment of cyclin K and for DNA-repair-associated gene expression in S phase. These data identify a connection between the chromatin regulator SETD1A and the DNA damage response that is independent of histone methylation and suggests that targeting SETD1A and cyclin K complexes may represent a therapeutic opportunity for AML and, potentially, for other cancers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this study, total mercury (THg) and methylmercury (MeHg) concentrations in muscles (leg and breast), organs (intestine, heart, stomach, liver) and blood were investigated for backyard chickens, ducks and geese of the Wanshan Mercury Mine, China. THg in poultry meat products range from 7.9 to 3917.1 ng/g, most of which exceeded the Chinese national standard limit for THg in meat (50 ng/g). Elevated MeHg concentrations (0.4-62.8 ng/g) were also observed in meat products, suggesting that poultry meat can be an important human MeHg exposure source. Ducks and geese showed higher Hg levels than chickens. For all poultry species, the highest Hg concentrations were observed in liver (THg: 23.2-3917.1 ng/g; MeHg: 7.1-62.8 ng/g) and blood (THg: 12.3-338.0 ng/g; MeHg: 1.4-17.6 ng/g). We estimated the Hg burdens in chickens (THg: 15.3-238.1 μg; MeHg: 2.2-15.6 μg), ducks (THg: 15.3-238.1 μg; MeHg: 3.5-14.7 μg) and geese (THg: 83.8-93.4 μg; MeHg: 15.4-29.7 μg). To not exceed the daily intake limit for THg (34.2 μg/day) and MeHg (6 μg/day), we suggested that the maximum amount (g) for chicken leg, breast, heart, stomach, intestine, liver, and blood should be 1384, 1498, 2315, 1214, 1081, 257, and 717, respectively; the maximum amount (g) for duck leg, breast, heart, stomach, intestine, liver, and blood should be 750, 1041, 986, 858, 752, 134, and 573, respectively; and the maximum amount (g) for goose leg, breast, heart, stomach, intestine, liver, and blood should be 941, 1051, 1040, 1131, 964, 137, and 562, respectively., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tumor suppressor p53 and its signaling pathway play a central role in tumor prevention. The E3 ubiquitin ligase MDM2, which is a direct p53 transcriptional target and also the most critical negative regulator of p53, forms an autoregulatory negative feedback loop with p53 in the cell to tightly regulate the levels and activity of p53. MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that play a critical role in the post-translational regulation of gene expression. Recent studies have revealed that miRNAs directly regulate the levels of p53 or MDM2 to modulate the p53 function in tumor suppression. Recently, we identified miR-339-5p as a new miRNA that directly represses MDM2 to activate p53 and enhance p53 function in tumor suppression. Thus, miRNAs have become a new but important component of the p53 signaling pathway through regulating the p53/MDM2 feedback loop.

Background: Variants in the emerin gene (EMD) were implicated in X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD), characterized by early-onset contractures of tendons, progressive muscular weakness and cardiomyopathy. To date, 223 mutations have been reported in EMD gene and the majority of them caused a predominant skeletal muscular phenotype. In this study, we identified a novel deletion mutation in EMD exon 1, which results in almost a complete loss of emerin protein in a large Chinese family. However, the patients suffered severe dilated cardiomyopathy (DCM) but very mild skeletal muscle disorder., Case Presentation: Whole exome sequencing (WES) and linkage analysis were performed to identify the underlying mutation in a Chinese DCM family spanning five generations. A missense variation in the GPR50 gene was found co-segregated with the disease phenotype, whereas no functional alteration was detected in the variant GPR50 protein. When analyzing the failure sequences in the exome sequencing data, a novel deletion mutation (c.26&#95;39delATACCGAGCTGACC) in EMD exon 1, was identified in this family. Different from the typical clinical features caused by most reported EMD mutations, patients in our study presented very mild skeletal muscle degeneration that had not been diagnosed until the mutation was found., Conclusion: We described a family with rare clinical presentations caused by a novel EMD deletion mutation. Our findings broaden the heterogeneous spectrum of phenotypes attributed to EMD mutations and provide new insight to explain the genotype-phenotype correlations between EMD mutations and EDMD symptoms.

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.

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.

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.

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.

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.

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.)