Characterization of Fasting-Induced p21 Expression and Protection of Intestinal Stem Cells

The cyclin dependent kinase inhibitor, p21, plays a key role in cell cycle. Additionally, the p21 gene, Cdkn1a (hereafter p21) is often used as a marker of cellular stress. To investigate p21 promoter activity under basal conditions and in response to various forms of stress, we generated knock-in imaging reporter mice that express firefly luciferase (FLuc) under the control of the endogenous p21 promoter. I demonstrated that FLuc expression and bioluminescence detection mirrored endogenous p21 protein levels and promoter activity in vivo. Contrary to previously known roles for p53-mediate expression of p21, imaging of reporter cells demonstrated that p53 prevented the ERK/MAPK pathway from activating p21 expression when quiescent cells were stimulated with serum to re-enter the cell cycle. In addition, low light bioluminescence imaging identified p21 expression in specific regions of individual organs that were not previously observed including the paraventricular, arcuate and dorsomedial nuclei of the hypothalamus - regions that detect nutrient levels in the blood stream and regulate metabolism throughout the body. These results suggested a link between p21 expression and metabolic regulation. I found that food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation, including liver, pancreas, and hypothalamic nuclei. The ability of fasting to induce p21 expression was found to be independent of p53, but dependent on the transcription factor FOXO1, which was bound to the p21 promoter region only in fasted mice. Previous work has shown that short-term fasting protects mice from what would normally be lethal doses of etoposide. I hypothesized that p21 may be involved in this protection, as p21 expression increased in response to both fasting and DNA damage. I demonstrated that fasting prior to a high dose of etoposide treatment enhanced survival by protecting small intestinal stem cells, but that p21 was not required for this protection. While high dose etoposide treatment caused complete destruction of the crypt-villus architecture and near complete loss of small intestinal stem cells in free-feeding mice, fasting prior to etoposide treatment enabled stem cell survival and subsequent reconstitution of the small intestinal crypts and villi. Using LacZ reporter mice and lineage tracing, I found that both crypt base columnar (CBC) and +4 stem cells contributed to crypt restoration in the fasted mice. Though etoposide in fed and fasted mice induced similar amounts of DNA double strand breaks (DSBs) immediately following treatment, resolution of DNA DSBs, as measured by loss of γH2AX staining, occurred more quickly in stem cells of fasted mice compared to those of fed mice

Sex differences in brain tumor glutamine metabolism reveal sex-specific vulnerabilities to treatment

BACKGROUND: Brain cancer incidence and mortality rates are greater in males. Understanding the molecular mechanisms that underlie those sex differences could improve treatment strategies. Although sex differences in normal metabolism are well described, it is currently unknown whether they persist in cancerous tissue. METHODS: Using positron emission tomography (PET) imaging and mass spectrometry, we assessed sex differences in glioma metabolism in samples from affected individuals. We assessed the role of glutamine metabolism in male and female murine transformed astrocytes using isotope labeling, metabolic rescue experiments, and pharmacological and genetic perturbations to modulate pathway activity. FINDINGS: We found that male glioblastoma surgical specimens are enriched for amino acid metabolites, including glutamine. Fluoroglutamine PET imaging analyses showed that gliomas in affected male individuals exhibit significantly higher glutamine uptake. These sex differences were well modeled in murine transformed astrocytes, in which male cells imported and metabolized more glutamine and were more sensitive to glutaminase 1 (GLS1) inhibition. The sensitivity to GLS1 inhibition in males was driven by their dependence on glutamine-derived glutamate for α-ketoglutarate synthesis and tricarboxylic acid (TCA) cycle replenishment. Females were resistant to GLS1 inhibition through greater pyruvate carboxylase (PC)-mediated TCA cycle replenishment, and knockdown of PC sensitized females to GLS1 inhibition. CONCLUSION: Our results show that clinically important sex differences exist in targetable elements of metabolism. Recognition of sex-biased metabolism may improve treatments through further laboratory and clinical research. FUNDING: This work was supported by NIH grants, Joshua\u27s Great Things, the Siteman Investment Program, and the Barnard Research Fund

Effects of Dietary Restriction on Cancer Development and Progression

The effects of caloric restriction on tumor growth and progression are known for over a century. Indeed, fasting has been practiced for millennia, but just recently has emerged the protective role that it may exert toward cells. Fasting cycles are able to reprogram the cellular metabolism, by inducing protection against oxidative stress and prolonging cellular longevity. The reduction of calorie intake as well as short- or long-term fasting has been shown to protect against chronic and degenerative diseases, such as diabetes, cardiovascular pathologies, and cancer. In vitro and in vivo preclinical models showed that different restriction dietary regimens may be effective against cancer onset and progression, by enhancing therapy response and reducing its toxic side effects. Fasting-mediated beneficial effects seem to be due to the reduction of inflammatory response and downregulation of nutrient-related signaling pathways able to modulate cell proliferation and apoptosis. In this chapter, we will discuss the most significant studies present in literature regarding the molecular mechanisms by which dietary restriction may contribute to prevent cancer onset, reduce its progression, and positively affect the response to the treatments

Forkhead box O1 (FOXO1) protein, but not p53, contributes to robust induction of p21 expression in fasted mice

Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body. These results suggested a link between p21 expression and metabolic regulation. We found that short-term food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei. Conditional reporter mice were generated that enabled hepatocyte-specific expression of p21 to be monitored in vivo. Bioluminescence imaging demonstrated that fasting induced a 7-fold increase in p21 expression in livers of reporter mice and Western blotting demonstrated an increase in protein levels as well. The ability of fasting to induce p21 expression was found to be independent of p53 but dependent on FOXO1. Finally, occupancy of the endogenous p21 promoter by FOXO1 was observed in the livers of fasted but not fed mice. Thus, fasting promotes loading of FOXO1 onto the p21 promoter to induce p21 expression in hepatocytes.Fil: Tinkum, Kelsey L.. BRIGHT Institute; Estados Unidos. Mallinckrodt Institute of Radiology; Estados UnidosFil: White, Lynn S.. Mallinckrodt Institute of Radiology; Estados Unidos. BRIGHT Institute; Estados UnidosFil: Marpegan, Luciano. University of Washington; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Herzog, Erik. University of Washington; Estados UnidosFil: Piwnica Worms, David. Mallinckrodt Institute of Radiology; Estados Unidos. BRIGHT Institute; Estados UnidosFil: Piwnica Worms, Helen. Mallinckrodt Institute of Radiology; Estados Unidos. BRIGHT Institute; Estados Unido

Bioluminescence Imaging Captures the Expression and Dynamics of Endogenous p21 Promoter Activity in Living Mice and Intact Cells▿

To interrogate endogenous p21WAF1/CIP1 (p21) promoter activity under basal conditions and in response to various forms of stress, knock-in imaging reporter mice in which expression of firefly luciferase (FLuc) was placed under the control of the endogenous p21 promoter within the Cdkn1a gene locus were generated. Bioluminescence imaging (BLI) of p21 promoter activity was performed noninvasively and repetitively in mice and in cells derived from these mice. We demonstrated that expression of FLuc accurately reported endogenous p21 expression at baseline and under conditions of genotoxic stress and that photon flux correlated with mRNA abundance and, therefore, bioluminescence provided a direct readout of p21 promoter activity in vivo. BLI confirmed that p53 was required for activation of the p21 promoter in vivo in response to ionizing radiation. Interestingly, imaging of reporter cells demonstrated that p53 prevents the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway from activating p21 expression when quiescent cells are stimulated with serum to reenter the cell cycle. In addition, low-light BLI identified p21 expression in specific regions of individual organs that had not been observed previously. This inducible p21FLuc knock-in reporter strain will facilitate imaging studies of p53-dependent and -independent stress responses within the physiological context of the whole animal

Fine-tuning p53 activity through C-terminal modification significantly contributes to HSC homeostasis and mouse radiosensitivity

Cell cycle regulation in hematopoietic stem cells (HSCs) is tightly controlled during homeostasis and in response to extrinsic stress. p53, a well-known tumor suppressor and transducer of diverse stress signals, has been implicated in maintaining HSC quiescence and self-renewal. However, the mechanisms that control its activity in HSCs, and how p53 activity contributes to HSC cell cycle control, are poorly understood. Here, we use a genetically engineered mouse to show that p53 C-terminal modification is critical for controlling HSC abundance during homeostasis and HSC and progenitor proliferation after irradiation. Preventing p53 C-terminal modification renders mice exquisitely radiosensitive due to defects in HSC/progenitor proliferation, a critical determinant for restoring hematopoiesis after irradiation. We show that fine-tuning the expression levels of the cyclin-dependent kinase inhibitor p21, a p53 target gene, contributes significantly to p53-mediated effects on the hematopoietic system. These results have implications for understanding cell competition in response to stresses involved in stem cell transplantation, recovery from adverse hematologic effects of DNA-damaging cancer therapies, and development of radioprotection strategies

Cooperative p16 and p21 action protects female astrocytes from transformation

Abstract Mechanisms underlying sex differences in cancer incidence are not defined but likely involve dimorphism (s) in tumor suppressor function at the cellular and organismal levels. As an example, sexual dimorphism in retinoblastoma protein (Rb) activity was shown to block transformation of female, but not male, murine astrocytes in which neurofibromin and p53 function was abrogated (GBM astrocytes). Correlated sex differences in gene expression in the murine GBM astrocytes were found to be highly concordant with sex differences in gene expression in male and female GBM patients, including in the expression of components of the Rb and p53 pathways. To define the basis of this phenomenon, we examined the functions of the cyclin dependent kinase (CDK) inhibitors, p16, p21 and p27 in murine GBM astrocytes under conditions that promote Rb-dependent growth arrest. We found that upon serum deprivation or etoposide-induced DNA damage, female, but not male GBM astrocytes, respond with increased p16 and p21 activity, and cell cycle arrest. In contrast, male GBM astrocytes continue to proliferate, accumulate chromosomal aberrations, exhibit enhanced clonogenic cell activity and in vivo tumorigenesis; all manifestations of broad sex differences in cell cycle regulation and DNA repair. Differences in tumorigenesis disappeared when female GBM astrocytes are also rendered null for p16 and p21. These data elucidate mechanisms underlying sex differences in cancer incidence and demonstrate sex-specific effects of cytotoxic and targeted therapeutics. This has critical implications for lab and clinical research