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Interference with p53 protein inhibits hematopoietic and muscle differentiation
Journal of Cell Biology (JCB)
Abstract
The involvement of p53 protein in cell differentiation has been recently suggested by some observations made with tumor cells and the correlation found between differentiation and increased levels of p53. However, the effect of p53 on differentiation is in apparent contrast with the normal development of p53-null mice. To test directly whether p53 has a function in cell differentiation, we interfered with the endogenous wt-p53 protein of nontransformed cells of two different murine histotypes: 32D myeloid progenitors, and C2C12 myoblasts. A drastic inhibition of terminal differentiation into granulocytes or myotubes, respectively, was observed upon expression of dominant-negative p53 proteins. This inhibition did not alter the cell cycle withdrawal typical of terminal differentiation, nor p21(WAF1/CIP1) upregulation, indicating that interference with endogenous p53 directly affects cell differentiation, independently of the p53 activity on the cell cycle. We also found that the endogenous wt-p53 protein of C2C12 cells becomes transcriptionally active during myogenesis, and this activity is inhibited by p53 dominant-negative expression. Moreover, we found that p53 DNA-binding and transcriptional activities are both required to induce differentiation in p53-negative K562 cells. Taken together, these data strongly indicate that p53 is a regulator of cell differentiation and it exerts this role, at least in part, through its transcriptional activity.
... In fact, p53 stability and its transcriptional activity have been shown to be negatively regulated by HDAC1-mediated deacetylation of lysine residues 373/382 (K373/382) [49]. Moreover, other studies have revealed p53 expression and its transcriptional activity to play a key role in the regulation of myogenic differentiation of embryonic stem cells [7], as well as skeletal muscle precursors [8,21,32]. To this end, p53 acetylation and total protein levels were assessed in CMCs treated with 1 µM entinostat or DMSO diluent control for 5 days. ...
... Congruent with these observations, p53 stability and its transcriptional activity have been shown to be negatively regulated via direct deacetylation of lysine residues 373/382 (K373/382) by HDAC1 [49]. Interestingly, p53 has been identified as an important mediator of myogenic differentiation of embryonic stem cells (via regulation of the mesodermal master genes Brachyury and Mesp1) [7], as well as skeletal muscle precursors [8,21,32]. Thus, we postulate that HDAC1 may indirectly regulate cardiomyogenic transcriptional programs in CMCs by directly modifying p53 acetylation status, and consequently, p53 transcriptional activity. In support of this proposed mechanism, entinostat-induced alterations in CMC cardiogenic program activation was associated with marked increases in K382 p53 protein acetylation-notably recapitulating our previously observed effects with genetic HDAC1 knockdown [23]. ...
Preclinical investigations support the concept that donor cells more oriented towards a cardiovascular phenotype favor repair. In light of this philosophy, we previously identified HDAC1 as a mediator of cardiac mesenchymal cell (CMC) cardiomyogenic lineage commitment and paracrine signaling potency in vitro—suggesting HDAC1 as a potential therapeutically exploitable target to enhance CMC cardiac reparative capacity. In the current study we examined the effects of pharmacologic HDAC1 inhibition, using the benzamide class 1 isoform selective HDAC inhibitor entinostat (MS-275), on CMC cardiomyogenic lineage commitment and CMC-mediated myocardial repair in vivo. Human CMCs pretreated with entinostat or DMSO diluent control were delivered intramyocardially in an athymic nude rat model of chronic ischemic cardiomyopathy 30 days after a reperfused myocardial infarction. Indices of cardiac function were assessed by echocardiography and left ventricular (LV) Millar conductance catheterization 35 days after treatment. Compared with naïve CMCs, entinostat-treated CMCs exhibited heightened capacity for myocyte-like differentiation in vitro and superior ability to attenuate LV remodeling and systolic dysfunction in vivo. The improvement in CMC therapeutic efficacy observed with entinostat pretreatment was not associated with enhanced donor cell engraftment, cardiomyogenesis, or vasculogenesis, but instead with more efficient inhibition of myocardial fibrosis and greater increase in myocyte size. These results suggest that HDAC inhibition enhances the reparative capacity of CMCs, likely via a paracrine mechanism that improves ventricular compliance and contraction and augments myocyte growth and function.
... One of the functions of nucleolin is binding to p53 mRNA to interfere with translation (Jia et al., 2017;Takagi et al., 2005). It has been widely known that the p53 signaling pathway promotes myogenic differentiation Porrello et al., 2000;Soddu et al., 1996). In human myoblasts, iSN04 antagonizes nucleolin and increases p53 protein levels. ...
Myoblasts are myogenic precursors that develop into myotubes during muscle formation. Improving efficiency of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species-specific actions. Seven myoDNs (iSN01–iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)-positive myotubes. The iSN04–berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myotubes and the upregulation of myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets.
... The role of p53 in myoblasts has been intensively studied. An initial study found that the dominant-negative form of p53 inhibits the differentiation of C2C12 cells (Soddu et al., 1996). During myogenic differentiation, p53 cooperates with MyoD to activate transcription of retinoblastoma protein (Porrello et al., 2000), which serves as a cofactor of MyoD to arrest the cell cycle and facilitate muscle cell commitment (Gu et al., 1993;Novitch et al., 1996). ...
Herein we report that the 18-base telomeric oligodeoxynucleotides (ODNs) designed from the Lactobacillus rhamnosus GG genome promote differentiation of skeletal muscle myoblasts which are myogenic precursor cells. We termed these myogenetic ODNs (myoDNs). The activity of one of the myoDNs, iSN04, was independent of Toll-like receptors, but dependent on its conformational state. Molecular simulation and iSN04 mutants revealed stacking of the 13–15th guanines as a core structure for iSN04. The alkaloid berberine bound to the guanine stack and enhanced iSN04 activity, probably by stabilizing and optimizing iSN04 conformation. We further identified nucleolin as an iSN04-binding protein. Results showed that iSN04 antagonizes nucleolin, increases the levels of p53 protein translationally suppressed by nucleolin, and eventually induces myotube formation by modulating the expression of genes involved in myogenic differentiation and cell cycle arrest. This study shows that bacterial-derived myoDNs serve as aptamers and are potential nucleic acid drugs directly targeting myoblasts.
... Our experiments have proved that p53 is acted as a transcription repressor of Malat1 expression to inhibit Malat1 activation. It has been identified that interferes p53 expression inhibits hematopoietic and muscle differentiation [33], reverse function of Malat1 vs. p53 implies Malat1 preserves the ability to enhance the proliferation and inhibit differentiation of hematopoietic and muscle cells. Increased p53 expression causes cell differentiation has been recently suggested by some ob- servations [34][35][36] . ...
The metastasis-associated lung adenocarcinoma transcription 1 (Malat1) is a highly conserved long non-coding RNA (lncRNA) gene. Previous studies showed that Malat1 is abundantly expressed in many tissues and involves in promoting tumor growth and metastasis by modulating gene expression and target protein activities. However, little is known about the biological function and regulation mechanism of Malat1 in normal cell proliferation.
In this study we conformed that Malat1 is highly conserved across vast evolutionary distances amongst 20 species of mammals in terms of sequence, and found that mouse Malat1 expresses in tissues of liver, kidney, lung, heart, testis, spleen and brain, but not in skeletal muscle. After treating erythroid myeloid lymphoid (EML) cells with All-trans Retinoic Acid (ATRA), we investigated the expression and regulation of Malat1 during hematopoietic differentiation, the results showed that ATRA significantly down regulates Malat1 expression during the differentiation of EML cells. Mouse LRH (Lin-Rhodamine(low) Hoechst(low)) cells that represent the early-stage progenitor cells show a high level of Malat1 expression, while LRB (Lin - Hoechst(Low) Rhodamine(Bright)) cells that represent the late-stage progenitor cells had no detectable expression of Malat1. Knockdown experiment showed that depletion of Malat1 inhibits the EML cell proliferation. Along with the down regulation of Malat1, the tumor suppressor gene p53 was up regulated during the differentiation. Interestingly, we found two p53 binding motifs with help of bioinformatic tools, and the following chromatin immunoprecipitation (ChIP) test conformed that p53 acts as a transcription repressor that binds to Malat1's promoter. Furthermore, we testified that p53 over expression in EML cells causes down regulation of Malat1.
In summary, this study indicates Malat1 plays a critical role in maintaining the proliferation potential of early-stage hematopoietic cells. In addition to its biological function, the study also uncovers the regulation pattern of Malat1 expression mediated by p53 in hematopoietic differentiation. Our research shed a light on exploring the Malat1 biological role including therapeutic significance to inhibit the proliferation potential of malignant cells.
... 10 In this issue, Yang et al. propose a role of p53 in the activation of the myogenic differentiation checkpoint that relies on direct repression of myogenin-a MyoD downstream target gene, whose activation is required for myoblast progression toward terminal differentiation into multinucleated myotubes. 11,12 This finding is of particular interest, when considering that in unperturbed myoblasts p53 rather seems to contribute to muscle differentiation, 13,14 as it suggests that in myoblasts the activity of p53 is biased toward inhibiting differentiation by the DNA damage signaling. The authors first discovered the transcriptional repression of myogenin by p53 in the human rhabdomyosarcoma RD cell line. ...
Cell death and differentiation is a monthly research journal focused on the exciting field of programmed cell death and apoptosis. It provides a single accessible source of information for both scientists and clinicians, keeping them up-to-date with advances in the field. It encompasses programmed cell death, cell death induced by toxic agents, differentiation and the interrelation of these with cell proliferation.
... Our studies on myogenesis provide direct evidence of the role of p53 in cell differentiation in urodeles. We found that both protein and activity levels of p53 increase during myogenesis, consistent with previous reports on the differentiation of mammalian muscle in culture (47,48). Although stabilization of p53 activity during the induction of myogenesis increases the overall efficiency of the process, inhibition of p53 prevents myotube formation, demonstrating that p53 is essential during muscle differentiation in salamander cells. ...
Significance
Our work establishes that endogenous regulation of the activity of the tumor-suppressor p53 is a critical component in vertebrate limb regeneration and is required for the process to occur. We show that down-regulation of p53 is required for cell cycle reentry of postmitotic differentiated cells, the critical step in the formation of the progenitors for the process of regeneration. We propose that the absence of tumor-suppressors that stabilize p53 and the presence of dominant-negative isoforms, permit regeneration by negative regulation of p53. This research sheds light onto previously unknown molecular mechanisms critical for limb regeneration and offers a new perspective on the functions of the tumour suppressor p53.
Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna -mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna -mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.
Skeletal muscle myoblasts are myogenic precursors that develop into myofibers during muscle formation. Improvement of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species-specific actions. Seven myoDNs (iSN01-iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)-positive myocytes. The iSN04-berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myocytes, myotube formation, and myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets.
Skeletal muscle is one of the most abundant and highly plastic tissues. The ubiquitin-proteasome system (UPS) is recognised as a major intracellular protein degradation system, and its function is important for muscle homeostasis and health. Although UPS plays an essential role in protein degradation during muscle atrophy, leading to the loss of muscle mass and strength, its deficit negatively impacts muscle homeostasis and leads to the occurrence of several pathological phenotypes. A growing number of studies have linked UPS impairment not only to matured muscle fibre degeneration and weakness, but also to muscle stem cells and deficiency in regeneration. Emerging evidence suggests possible links between abnormal UPS regulation and several types of muscle diseases. Therefore, understanding of the role of UPS in skeletal muscle may provide novel therapeutic insights to counteract muscle wasting, and various muscle diseases. In this review, we focussed on the role of proteasomes in skeletal muscle and its regeneration, including a brief explanation of the structure of proteasomes. In addition, we summarised the recent findings on several diseases and elaborated on how the UPS is related to their pathological states.
Mechanisms of muscle atrophy are complex and their understanding might help finding therapeutic solutions for pathologies such as amyotrophic lateral sclerosis (ALS). We meta-analyzed transcriptomic experiments of muscles of ALS patients and mouse models, uncovering a p53 deregulation as common denominator. We then characterized the induction of several p53 family members (p53, p63, p73) and a correlation between the levels of p53 family target genes and the severity of muscle atrophy in ALS patients and mice. In particular, we observed increased p63 protein levels in the fibers of atrophic muscles via denervation-dependent and -independent mechanisms. At a functional level, we demonstrated that TAp63 and p53 transactivate the promoter and increased the expression of Trim63 (MuRF1), an effector of muscle atrophy. Altogether, these results suggest a novel function for p63 as a contributor to muscular atrophic processes via the regulation of multiple genes, including the muscle atrophy gene Trim63.
OBJECTIVE:
To evaluate the expression of p53 in the mouse ovary during an artificially induced ovulatory cycle.
STUDY DESIGN:
Ovulation induction was performed using pregnant mares' serum gonadotropin/human chorionic gonadotropin (PMSG/hCG). First, a p53 promoter-chloramphenicol acetyl transferase (CAT) transgenic mouse model was used. Protein samples from ovaries of transgenic mice were assayed for CAT activity as evidence of p53 promoter activation. Next, RNA extracted from CD-1 mouse ovaries was used for reverse transcription/polymerase chain reaction (PCR) and northern blot analysis using a p53-specific probe.
RESULTS:
Increased CAT activity was noted in transgenic mice treated with PMSG/hCG as compared with controls. PCR studies on transgenic mice using primers for CAT and on CD-1 mice using primers for wild type p53 substantiated this observation. Furthermore, CAT assay and northern analysis, performed on samples obtained at serial time intervals from induction, indicated that maximal p53 expression occurs around the time of ovulation, beginning 48 hours after PMSG and peaking 6-12 hours after hCG administration.
CONCLUSION:
The temporal expression of p53 in the ovary during a PMSG/hCG artificially induced ovulatory cycle may indicate a role for p53 in processes of differentiation of granulosa cells into luteal cells.
Previously, we had shown that high magnitude stretch (HMS), rather than low magnitude stretch (LMS), induced significant apoptosis of skeletal muscle C2C12 myoblasts. However, the molecular mechanism remains obscure. In this study, we found that p53 protein accumulated in the nucleus of LMS-loaded cells, whereas it translocated into mitochondria of HMS-loaded cells. Knocking down endogenous p53 by shRNA abrogated HMS-induced apoptosis. Furthermore, we demonstrated that overaccumulation of reactive oxygen species (ROS) during HMS-inactivated AKT that was activated in LMS-treated cells, which accounted for the distinct p53 subcellular localizations under HMS and LMS. Blocking ROS generation by N-acetylcysteine (NAC) or overexpressing constitutively active AKT vector (CA-AKT) inhibited HMS-incurred p53 mitochondrial translocation and promoted its nuclear targeting. Moreover, both NAC and CA-AKT significantly attenuated HMS-induced C2C12 apoptosis. Finally, we found that Ser389 phosphorylation of p53 was a downstream event of ROS-inactivated AKT pathway, which was critical to p53 mitochondrial trafficking during HMS stimuli. Transfecting p53-shRNA C2C12s with the mutant p53 (S389A) that was unable to target p53 to mitochondria underwent significantly lower apoptosis than transfection with wild-type p53. Altogether, our study uncovered that mitochondrial localization of p53, resulting from p53 Ser389 phosphorylation through ROS-inactivated AKT pathway, prompted C2C12 myoblast apoptosis during HMS stimulation.
Purpose:
The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Furthermore, we have recently demonstrated that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. In addition, we have also shown that p53 controls both transcription factors, C-Maf and Prox-1 as well as lens crystallin genes, αA, β- and γ-crystallins. Here, we have examined whether p53 also regulates other known target genes during its modulation of lens differentiation. The human and mouse lens epithelial cells, FHL124 and αTN4-1 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin.
Methods:
Mice used in this study were handled in compliance with the "Protocol for the Care and Use of Laboratory Animals" (Sun Yat-sen University). Adult mice were used for the collection of lens cells. These samples were used for extraction of total proteins. A total of 32 embryonic mice {8 at 14.5 ED, 8 at 17.5 ED and 8 newborns for wild type} were used for immunohistochemistry, which were used for co-localization study. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J.
Results:
Immunohistochemistry revealed that both the cell cycle checking genes, p21 and Gadd45α and the apoptotic genes, Bcl-2 and PUMA, display developmental changes associated with p53 during mouse lens development. Knockdown of p53 in the mouse lens epithelial cells caused inhibition of lens differentiation. Associated with this inhibition, the cell cycle genes displayed significant downreglation, the apoptotic genes was also attenuated but to a much less degree. In addition, we found that bFGF can induce dose-dependent upregulation of the upstream kinases, CHK1/2 and ERK1/2, both known to phosphorylate p53 and activate the later. Furthermore, We showed that in both developing lens and human lens epithelial cells, p53 can be co-localized with the catalytic subunit of the protein phoshphatase-1 (PP-1), suggesting that PP-1 regulates p53 phosphorylation status both in vivo and in vitro.
Conclusion:
Taken together, our results suggest that during mouse lens development, p53 activity is regulated by ERK and CHK kinases-mediated activation, and by PP-1-mediated inactivation. p53 can regulate multiple groups of genes to mediate lens differentiation.
Satellite cells are adult muscle stem cells residing in a specialized niche that regulates their homeostasis. How niche-generated signals integrate to regulate gene expression in satellite cell-derived myoblasts is poorly understood. We undertook an unbiased approach to study the effect of the satellite cell niche on satellite cell-derived myoblast transcriptional regulation and identified the tumor suppressor p53 as a key player in the regulation of myoblast quiescence. After activation and proliferation, a subpopulation of myoblasts cultured in the presence of the niche upregulates p53 and fails to differentiate. When satellite cell self-renewal is modeled ex vivo in a reserve cell assay, myoblasts treated with Nutlin-3, which increases p53 levels in the cell, fail to differentiate and instead become quiescent. Since both these Nutlin-3 effects are rescued by small interfering RNA-mediated p53 knockdown, we conclude that a tight control of p53 levels in myoblasts regulates the balance between differentiation and return to quiescence.
The insulin receptor susbtrate-3 (IRS-3) is a member of a family of intermediate adapter proteins that function as major intracellular targets for phosphorylation by the activated insulin and IGF-I receptors. Among the four IRS proteins identified so far, IRS-3 exhibits a rather peculiar expression pattern during both the embryonic development and adult life, suggesting a different mechanism of regulation of its expression. In this study, we cloned the 5' flanking region of the mIRS-3 gene and analyzed its promoter activity. The mIRS-3 promoter is inhibited by wild-type p53, and this effect is completely abolished by cotransfection of a dominant negative p53. Tumor-derived p53 mutants show variable, but lower suppressing capability than wt p53. In addition, treatment with doxorubicin inhibits endogenous expression of mIRS-3 mRNA in C2C12 and 3T3-L1 cells. The DNA region spanning from nucleotides -287 and -178 in the mIRS-3 promoter is responsible for a 32.2% reduction of the mouse double minute 2 (MDM2) promoter activity, suggesting its involvement in the p53-mediated inhibitory effect. In conclusion, our study demonstrates that the mIRS-3 promoter is regulated by p53 at the transcriptional level. The inhibition of mIRS-3 promoter by wild-type p53, and its de-repression by tumor-derived p53 mutants, appears to be similar to that previously reported for the IGF-I receptor promoter, suggesting a common role of these two genes in p53-mediated cell growth and differentiation.
Histone deacetylase (HDAC) regulation is an essential process in myogenic differentiation. Inhibitors targeting the activity of specific HDAC family members have been shown to enhance the cardiogenic differentiation capacity of discrete progenitor cell types; a key property of donor cell populations contributing to their afforded benefits in cardiac cell therapy applications. The influence of HDAC inhibition on cardiac-derived mesenchymal stromal cell (CMC) transdifferentiation or the role of specific HDAC family members in dictating cardiovascular cell lineage specification has not been investigated. In the current study, the consequences of HDAC inhibition on patient-derived CMC proliferation, cardiogenic program activation, and cardiovascular differentiation/cell lineage specification were investigated using pharmacologic and genetic targeting approaches. Here, CMCs exposed to the pan-HDAC inhibitor sodium butyrate (NaBu) exhibited induction of a cardiogenic transcriptional program and heightened expression of myocyte and endothelial lineage-specific markers when coaxed to differentiate in vitro. Further, shRNA knockdown screens revealed CMCs depleted of HDAC1 to promote the induction of a cardiogenic transcriptional program characterized by enhanced expression of cardiomyogenic- and vasculogenic-specific markers, a finding which depended upon and correlated with enhanced acetylation and stabilization of p53. Cardiogenic gene activation and elevated p53 expression levels observed in HDAC1-depleted CMCs were associated with improved aptitude to assume a cardiomyogenic/vasculogenic cell-like fate in vitro. These results suggest that HDAC1 depletion-induced p53 expression alters CMC cell fate decisions and identify HDAC1 as a potential exploitable target to facilitate CMC-mediated myocardial repair in ischemic cardiomyopathy. This article is protected by copyright. All rights reserved.
The plethora of biochemical and genetic data on p53 is chiefly based on in vitro and cell culture systems or the analysis of tumors late in their life history. Translating this data into truly physiological systems reveals many levels of complexity, and draws into question many of the dogmas and assumptions that litter the field. To advance knowledge of the p53 response pathway and utilize the information rationally in the clinic will require a concerted attempt to broaden understanding of the physiological biochemistry of the p53 family of proteins in vivo.
The p53 gene family members p53, p73, and p63 display several isoforms derived from the presence of internal promoters and alternative splicing events. They are structural homologs but hold peculiar functional properties. p53, p73, and p63 are tumor suppressor genes that promote differentiation, senescence, and apoptosis. p53, unlike p73 and p63, is frequently mutated in cancer often displaying oncogenic “gain of function” activities correlated with the induction of proliferation, invasion, chemoresistance, and genomic instability in cancer cells. These oncogenic functions are promoted either by the aberrant transcriptional cooperation of mutant p53 (mutp53) with transcription cofactors (e.g., NF-Y, E2F1, Vitamin D Receptor, Ets-1, NF-kB and YAP) or by the interaction with the p53 family members, p73 and p63, determining their functional inactivation. The instauration of these aberrant transcriptional networks leads to increased cell growth, low activation of DNA damage response pathways (DNA damage response and DNA double-strand breaks response), enhanced invasion, and high chemoresistance to different conventional chemotherapeutic treatments. Several studies have clearly shown that different cancers harboring mutant p53 proteins exhibit a poor prognosis when compared to those carrying wild-type p53 (wt-p53) protein. The interference of mutantp53/p73 and/or mutantp53/p63 interactions, thereby restoring p53, p73, and p63 tumor suppression functions, could be among the potential therapeutic strategies for the treatment of mutant p53 human cancers.
It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cell cycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates c-Maf and Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the present study, we present further evidence to show that p53 can regulate lens differentiation by controlling expression of the differentiation genes coding for the lens crystallins. First, the alpha A and beta A3/A1 gene promoters or introns all contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin gene promoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferase reporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53 causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds to crystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of various crystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together, our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.
It has been shown that p53 has a critical role in the differentiation and functionality of various multipotent progenitor cells. P53 mutations can lead to genome instability and subsequent functional alterations and aberrant transformation of mesenchymal stem cells (MSCs). The significance of p53 in safeguarding our body from developing osteosarcoma (OS) is well recognized. During bone remodeling, p53 has a key role in negatively regulating key factors orchestrating the early stages of osteogenic differentiation of MSCs. Interestingly, changes in the p53 status can compromise bone homeostasis and affect the tumor microenvironment. This review aims to provide a unique opportunity to study the p53 function in MSCs and OS. In the context of loss of function of p53, we provide a model for two sources of OS: MSCs as progenitor cells of osteoblasts and bone tumor microenvironment components. Standing at the bone remodeling point of view, in this review we will first explain the determinant function of p53 in OS development. We will then summarize the role of p53 in monitoring MSC fidelity and in regulating MSC differentiation programs during osteogenesis. Finally, we will discuss the importance of loss of p53 function in tissue microenvironment. We expect that the information provided herein could lead to better understanding and treatment of OS.
The ability to repeatedly regenerate limbs during the entire lifespan of an animal is restricted to certain salamander species among vertebrates. This ability involves dedifferentiation of post-mitotic cells into progenitors that in turn form new structures. A long-term enigma has been how injury leads to dedifferentiation. Here we show that skeletal muscle dedifferentiation during newt limb regeneration depends on a programmed cell death response by myofibres. We find that programmed cell death-induced muscle fragmentation produces a population of 'undead' intermediate cells, which have the capacity to resume proliferation and contribute to muscle regeneration. We demonstrate the derivation of proliferating progeny from differentiated, multinucleated muscle cells by first inducing and subsequently intercepting a programmed cell death response. We conclude that cell survival may be manifested by the production of a dedifferentiated cell with broader potential and that the diversion of a programmed cell death response is an instrument to achieve dedifferentiation.
BACKGROUND—While loss of p53 function is a key oncogenic step in human tumorigenesis, mutations of p53 are generally viewed as late events in the metaplasia-dysplasia-adenocarcinoma sequence of Barrett's oesophagus. Recent reports of a series of genes (p63, p73, and others) exhibiting close homology to p53 raise the possibility that abnormalities of these p53 family members may exert their influence earlier in the sequence.
AIM—Following recent characterisation of expression of p63 and a major isoform ΔNp63 by generation of an antiserum that recognises p63 isoforms, but not p53, our aim was a comparative study of expression of p63 protein and p53 protein in a morphologically well defined biopsy series representative of all stages of the metaplasia-dysplasia-carcinoma sequence in Barrett's oesophagus.
METHODS—A series of 60 biopsy cases representing normal oesophagus through to invasive adenocarcinoma were stained, using immunohistochemistry, with antibodies to p63 and p53. All biopsies derived from patients with endoscopic and histopathological substantiation of a diagnosis of traditional/classical Barrett's oesophagus.
RESULTS—There was exact concordance in p53 and p63 expression in more advanced forms of neoplasia, high grade dysplasia, and invasive adenocarcinoma, while p63, but not p53, was detected in the proliferative compartment of some non-neoplastic oesophageal tissue, in both squamous mucosa and in the non-neoplastic metaplastic glandular epithelium.
CONCLUSIONS—In neoplastic Barrett's oesophagus there is upregulation of both p63 and p53 while p63 isoforms may well have an important role in epithelial biology in both non-metaplastic and metaplastic mucosa of the oesophagus. While abnormalities of p53 function represent an indisputable and critical element of neoplastic transformation, other closely linked genes and their proteins have a role in both the physiology and pathophysiology of the oesophageal mucosa.
Keywords: p53; p63; immunohistochemistry; Barrett's oesophagus; dysplasia; adenocarcinoma
Ankrd2 may be a link between the sarcomere and the nucleus; a similar role has recently been proposed for CARP that has a high level of structural and functional conservation with Ankrd2. Both Ankrd2 and CARP are involved in striated muscle hypertrophy. The mechanism by which muscle stretch is sensed and signals are transduced is still unknown; however, Ankrd2 and CARP could play similar roles in pathways leading to hypertrophy, the triggering mechanisms being heart pressure overload monitored by CARP and mechanical stretch in skeletal muscle monitored by Ankrd2. Recently Ankrd2 and CARP have been proposed as members of a family of muscle ankyrin repeat proteins (MARPs) that form a complex with titin, myopalladin and calpain protease p94, involved in signaling and regulation of gene expression in response to muscle stress. Here, we show that Ankrd2 is able to interact with the Z-disc protein telethonin as well as being able to interact with three transcription factors: YB-1, PML and p53. Ankrd2 binding to the ubiquitous transcription factor YB-1 can be demonstrated both in vitro and in vivo; this is not very surprising, since a similar interaction was previously described for CARP. However, the interactions with PML and p53 are unexpected new findings, with interesting implications in the Ankrd2 signaling cascade. Ankrd2 co-localizes with the transcriptional co-activator and co-repressor PML in nuclear bodies (NBs) in human myoblasts as detected by confocal immunofluorescence. Interestingly, we show that Ankrd2 not only binds the tumor suppressor protein p53 both in vitro and in vivo but also enhances the up-regulation of the p21WAFI/CIPI promoter by p53. Therefore, our findings strengthen the hypothesis that Ankrd2 may be involved in sensing stress signals and linking these to muscle gene regulation.
Acute muscle injury and physiological stress from chronic muscle diseases and aging lead to impairment of skeletal muscle function. This raises the question of whether p53, a cellular stress sensor, regulates muscle tissue repair under stress conditions. By investigating muscle differentiation in the presence of genotoxic stress, we discovered that p53 binds directly to the myogenin promoter and represses transcription of myogenin, a member of the MyoD family of transcription factors that plays a critical role in driving terminal muscle differentiation. This reduction of myogenin protein is observed in G1-arrested cells and leads to decreased expression of late but not early differentiation markers. In response to acute genotoxic stress, p53-mediated repression of myogenin reduces post-mitotic nuclear abnormalities in terminally differentiated cells. This study reveals a mechanistic link previously unknown between p53 and muscle differentiation, and suggests new avenues for managing p53-mediated stress responses in chronic muscle diseases or during muscle aging.Cell Death and Differentiation advance online publication, 12 December 2014; doi:10.1038/cdd.2014.189.
The tumor suppressor, p53 regulates a large number of target genes to control cell proliferation and apoptosis. In addition, it is also implicated in the regulation of cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates several genes including c-Maf and Prox1, two important transcription factors for lens differentiation, and αA and βA3/A1, the lens differentiation markers. In the present study, we present evidence to show that the γA-crystallin gene distal promoter and the first intron also contain p53 binding sites and are capable of mediating p53 control during mouse lens development. First, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from human lens epithelial cells (HLE) directly binds to the p53 binding sites present in the γA-crystallin gene. Second, the exogenous wild type p53 induces the dose-dependent expression of the luciferase reporter gene driven by the basic promoter containing the γA-crystallin gene p53 binding site. In contrast, the exogenous dominant negative mutant p53 causes a dose-dependent inhibition of the same promoter. Third, ChIP assays revealed that p53 binds to the γA-crystallin gene promoter in vivo. Finally, in the p53 knockout mouse lenses, the expression level of the γAcrystallin gene was found attenuated in comparison with that in the wild type mouse lenses. Together, our results reveal that p53 regulates γA-crystallin gene expression during mouse lens development. Thus, p53 directly regulates all 3 types of crystallin genes to control lens differentiation.
P53 plays an important role in several aspects of the cell physiology : cell cycle controle, response to genotoxic agents, apoptosis induction... Numerous data concerning a possible involvement of P53 during the differentiation process and during embryogenesis have been collected but are still much debated. The analysis of data dealing with the role of P53 during the differenciation of cell lines or primary cells are discussed and compared to the data obtained in normal versus p53 knock-out mice.
Neuroblastoma-derived tumor cells, unlike cells from other tumor types, characteristically express a wild- type but cytoplasmically sequestered p53 protein. To ascertain whether the p53 in these cells retained any physiological activity, we inactivated it in SK-N-SH cells, a neuroblastoma-derived cell line, by introducing the human papilloma virus type 16 E6 expression plasmid. Parent SK-N-SH cell cultures are composed of two cell types exhibiting characteristic morphologies designated neuroblastic (N-type) or substrate-adherent fibroblastic (S-type) cells, both of which have been shown to spontaneously transdifferentiate or interconvert. We report here that down-regulation of p53 resulted in conversion of SK-N-SH cells to the substrate-adherent fibroblast-like S-type cells. The morphologic conversion was accompanied by a loss of neurofilament expression, a marker for the neuronal N-type cells, an increase in the expression of vimentin, and a lack of responsiveness to retinoic acid-induced neuronal differentiation. Importantly, we did not observe N-type cells in the E6-transfected cell population, suggesting that they were incapable of transdifferentiating to the N-type morphology. We also tested the ability of these E6-transfected S-type cells to form colonies in soft agar and observed a markedly reduced capacity of these cells to do so when compared with the parent and mutant E6-transfected cells. These results suggest that p53 is required for the maintenance of the neuroblastic tumorigenic phenotype.
It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cell cycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates c-Maf and Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the present study, we present further evidence to show that p53 can regulate lens differentiation by controlling expression of the differentiation genes coding for the lens crystallins. First, the aA and bA3/A1 gene promoters or introns all contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin gene promoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferase reporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53 causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds to crystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of various crystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together, our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.
Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis.
Paired-like homeodomain transcription factor 1 (PITX1) was specifically up-regulated in patients with facioscapulohumeral muscular dystrophy (FSHD) by comparing the genome-wide mRNA expression profiles of 12 neuromuscular disorders. In addition, it is the only known direct transcriptional target of the double homeobox protein 4 (DUX4) of which aberrant expression has been shown to be the cause of FSHD. To test the hypothesis that up-regulation of PITX1 contributes to the skeletal muscle atrophy seen in patients with FSHD, we generated a tet-repressible muscle-specific Pitx1 transgenic mouse model in which expression of PITX1 in skeletal muscle can be controlled by oral administration of doxycycline. After PITX1 was over-expressed in the skeletal muscle for 5 weeks, the mice exhibited significant loss of body weight and muscle mass, decreased muscle strength, and reduction of muscle fiber diameters. Among the muscles examined, the tibialis anterior, gastrocnemius, quadricep, bicep, tricep and deltoid showed significant reduction of muscle mass, while the soleus, masseter and diaphragm muscles were not affected. The most prominent pathological change was the development of atrophic muscle fibers with mild necrosis and inflammatory infiltration. The affected myofibers stained heavily with NADH-TR with the strongest staining in angular-shaped atrophic fibers. Some of the atrophic fibers were also positive for embryonic myosin heavy chain using immunohistochemistry. Immunoblotting showed that the p53 was up-regulated in the muscles over-expressing PITX1. The results suggest that the up-regulation of PITX1 followed by activation of p53-dependent pathways may play a major role in the muscle atrophy developed in the mouse model.
Development of colon cancer is a multistep process frequently involving mutations in both theAPC and p53 tumor suppressor genes. In this study we treated the HCT-116 colon cancer cell line with alkylating agents includingN-methyl-N′-nitro-N-nitrosoguanidine (MNNG),which is known to cause colon cancer in animals, and examined the expression of both APC and p53genes. Exposure of cells with MNNG caused an 8–12-fold increase in the level of APC mRNA and protein. APC induction was shown to result from increased nuclear transcription of the APCgene and correlated with a concomitant increase in the p53 protein level after MNNG treatment. A necessary role for p53 in
APCgene regulation is supported by the failure of MNNG to induceAPC expression in cell lines either expressing very low levels of p53 (HeLa cells) or no p53 (K562 erythroleukemia cells). The
overexpression of wild-type p53 gene into HCT-116 cells mimicked the effect of MNNG-induced expression of APCmRNA. A direct causal role for p53 in APC gene regulation was further evaluated by transfecting the wild-typep53 gene into K562 cells and observing a 5-fold increase in the APC gene expression. These results support a model featuring a direct link between p53 and APC in response to alkylation-induced
DNA damage and suggest a novel role for p53 in a stress-response pathway involving APC.
The interaction of integrins with extracellular matrix is known to promote cell survival by inhibiting apoptotic signaling.
In contrast, we demonstrate here that the α6β4 integrin induces apoptosis in carcinoma cells by stimulating p53 function. Specifically, we show that expression of α6β4 in carcinoma cells that lack this integrin stimulates an increase in the transactivating function of p53 as demonstrated
by the ability of this integrin to up-regulate the expression of a p53-sensitive reporter gene as well as the endogenous p53
response gene, bax. In addition, we report that α6β4 triggers apoptosis in carcinoma cells that express wild-type but not mutant p53 and that these α6β4 functions are inhibited by a dominant negative p53 construct. Importantly, we provide a link between integrin signaling and
p53 activation by demonstrating that the clustering of α6β4 with a β4integrin-specific antibody promotes p53-dependent apoptosis in cells that express both α6β4 and wild-type p53. These studies are the first to demonstrate that a specific integrin can promote apoptosis by activating
p53. Moreover, given the ability of α6β4 to stimulate invasion (Shaw, L. M., Rabinovitz, I., Wang, H. F., Toker, A., and Mercurio, A. M. (1997) Cell 91, 949–960), these studies suggest that the ability of α6β4 to promote carcinoma progression will be enhanced in tumor cells that express mutant, inactive forms of p53.
Skeletal myogenesis comprises myoblast replication and differentiation into striated multinucleated myotubes. Agents that interfere with myoblast replication are important tools for the understanding of myogenesis. Recently, we showed that cholesterol depletion by methyl-β-cyclodextrin (MCD) enhances the differentiation step in chick-cultured myogenic cells, involving the activation of the Wnt/β-catenin signaling pathway. However, the effects of cholesterol depletion on myoblast replication have not been carefully studied. Here we show that MCD treatment increases cell proliferation in primary chick myogenic cell cultures. Treatment of myogenic cells with the anti-mitotic reagent cytosine arabinoside, immediately following cholesterol depletion, blocks the MCD-induced effects on proliferation. Cholesterol depletion induced an increase in the number of desmin-positive mononucleated cells, and an increase in desmin expression. MCD induces an increase in the expression of the cell cycle regulator p53 and the master switch gene MyoD1. Treatment with BIO, a specific inhibitor of GSK3β, induced effects similar to MCD on cell proliferation; while treatment with Dkk1, a specific inhibitor of the Wnt/β-catenin pathway, neutralized the effects of MCD. These findings indicate that rapid changes in the cholesterol content in cell membranes of myoblasts can induce cell proliferation, possibly by the activation of the Wnt/β-catenin signaling pathway.
The tumor suppressor p53 plays a key role in regulating apoptosis and cell cycle progression. In addition, p53 is implicated in control of cell differentiation in muscle, the circulatory system, ocular lens and various carcinoma tissues. However, the mechanisms by which p53 controls cell differentiation are not fully understood. Here we present evidence that p53 directly regulates c-Maf and Prox1, two important transcription factors controlling differentiation in the ocular lens. First, human and murine c-Maf and Prox1 gene promoters contain authentic p53 DNA binding sites. Second, p53 directly binds to the p53 binding sites found in the promoter regions. Third, exogenous p53 induces dose-dependent expression of the luciferase report gene driven by both c-Maf and Prox1 promoters, and p53 binds to both promoters in the ChIP assays. Fourth, in the in vitro differentiation model, knockdown of p53 significantly inhibits lens differentiation which is associated with downregulated expression of c-Maf and Prox1. Finally, in p53 knockout mice, the expression of c-Maf and Prox1 are significantly altered. Together, our results reveal that p53 regulates lens differentiation through modulation of two important transcription factors, c-Maf and Prox1, and through them p53 thus controls expression of various differentiation-related downstream crystallin genes.
Introduction The Usage of Large-Scale Transcriptome Analysis to Identify Transcripts from ES Cells Modulation of Cell Cycle and P53 during Differentiation in ES Cells Effects of the Retinoblastoma (RB)/E2F Pathway on Differentiation in ES Cells Role of the PI3K/AKT Pathway in the Maintenance of ES Cell Pluripotency and Viability Conclusions References
DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity.
Approximately half of human tumors bear p53 mutations (Hollestein et al., 1997). The most prevalent type consists of missense
mutations that are frequently accompanied by loss of the remaining wild-type p53 (wt-p53) allele (Hainaut et al., 1997; Levine,
1997). The major site of the p53 mutations is the highly conserved DNA binding core domain (Hussain et al., 1998; Prives et
al., 1999). Thus, mutant p53 (mt-p53) proteins are unable to specifically bind DNA and to activate specific wt-p53 target
genes. Unlike wt-p53, whose half-life is short, mutant p53 proteins are quite stable and abundantly present in cancer cells.
One certain outcome of p53 mutations is the loss of wild type activities such as growth arrest, apoptosis, and differentiation
(Michalovitz et al., 1990; Yonish-Rouach et al., 1991; Soddu et al., 1996; Almog et al., 1997). However, at variance with
other tumor suppressor genes, cells with p53 mutations maintain expression of the fulllength protein. This may suggest that,
at least certain mutant forms of p53 can gain additional functions through which actively contribute to cancer progression
(Prives et al., 1999; Sigal et al., 2000; Strano et al., 2001; Bullock et al., 2001). Such evidence is provided by several
in vitro and in vivo studies (Haley et al., 1990; Dittmer et al., 1993; Gualberto et al., 1998; Frazier et al., 1998; Li et al., 1998; Blandino
et al., 1999; Aas et al., 1996; Irwin et al., 2003; Strano et al., 2003)
Mutations in the TP53 gene or the p53 pathway are required for the formation of many human cancers, including brain tumors. The p53 protein is
a multi-modal tumor suppressor that controls many processes that are disrupted during tumor formation, in large part by functioning
as a transcription factor. p53 regulates the expression of many target genes, including those encoding anti-proliferative,
pro-apoptotic, and anti-angiogenic proteins that mediate many of its intracellular tumor-suppressive activities. The p53 protein
can also directly induce apoptosis by associating with mitochondrial proteins in a transcription-independent fashion. Furthermore,
p53 exerts oncosuppressive effects through cell-extrinsic mechanisms that rely heavily on its ability to control exosome-mediated
protein secretion. Here we provide an overview of p53 functions in tumor suppression and review its involvement in the formation
of brain tumors.
KeywordsCancer–p53–p14ARF–MDM2–MDM4–Brain tumor–Tumor suppressor–Gliomas–Signaling–Apoptosis–Cell growth
The myelodysplastic syndromes [MDS] and acute myelogenous leukemia [AML] occurring subsequent to toxic exposure constitute
the clinically apparent secondary hematologic disorders [sHD]. The incidence of these disorders has significantly increased
during the past 40 years. This increase is most likely to be due to at least two phenomena: 1) increased exposure of individuals
to hemopoietic toxins as part of treatment for malignant or autoimmune diseases; and 2) the increasing age of our population.
It is generally assumed that “spontaneous” myelodysplastic syndromes (no history of toxic exposure) in the elderly are actually
the result of a lifetime accumulation of genetic damage resulting from repeated low level toxic exposures in the environment.
Despite there being some differences in the frequency of specific chromosomal abnormalities between secondary MDS [sMDS] and
spontaneous MDS, these two entities are similar in terms of stem cell involvement, the characteristics of the marrow and peripheral
blood, and the clinical courses of these two syndromes. In this chapter these two “types” of MDS, will be considered as one.
The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells (Qin et al. 2008. Cancer Research. 68(11):4150) and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Here, we demonstrate that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. First, during mouse lens development, p53 is expressed and differentially phosphorylated at different residues. Associated with p53 expression, Bak is also significantly expressed during mouse lens development. Second, human p53 directly regulates Bak promoter and Bak expression in p53 knockout mice (p53-/-) was significantly downregulated. Third, during in vitro bFGF-induced lens cell differentiation, knockdown of p53 or Bak leads to significant inhibition of lens cell differentiation. Fourth, besides the major distribution of Bak in cytoplasm, it is also localized in the nucleus in normal lens or bFGF-induced differentiating lens cells. Finally, p53 and Bak are co-localized in both cytoplasm and nucleus, and their interaction regulates the stability of p53. Together, these results demonstrate for the first time that the p53-Bak apoptotic signaling axis plays an essential role in regulating lens differentiation.
Single mutations in the DNA binding domain of p53 cause a radical shift in function from tumor suppressor to oncogene. The
mutated proteins lose the negative feedback regulation mediated by MDM2. Their oncogenic activity consists of a dominant negative
inhibition of the remaining wild-type p53 protein, and a gain of function (GOF) activity independent of wild-type p53 inhibition.
An understanding of the properties of these very common oncogenes is yielding promising therapeutic approaches, and is predicted
to offer more clinical applications as the field develops.
Differentiation of endometrial stromal cells into decidual cells is crucial for optimal endometrial receptivity. Data from our previous microarray study implied that expression of many cell cycle regulators are changed during decidualization and inhibition of DNA methylation in vitro. In this study, we hypothesized that both the classic progestin treatment and DNA methylation inhibition would inhibit stromal cell proliferation and cell cycle transition.
The human endometrial stromal cell line (HESC) was treated from 2 days to 18 days with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (AZA), a mixture of estradiol/progestin/cyclic adenosine monophosphate ([cAMP]; medroxy-progesterone acetate [MPA mix]) or both. Cell growth was measured by cell counting, cell cycle transition and apoptosis were analyzed by flow cytometry, expression of cell cycle regulators were analyzed by quantitative polymerase chain reaction (qPCR) and Western blotting, and change in DNA methylation profiles were detected by methylation-specific PCR.
Both AZA and MPA mix inhibited the proliferation of HESC for at least 7 days. Treatment with MPA mix resulted in an early G0/G1 inhibition followed by G2/M phase inhibition at 18 days. In contrast, AZA treatment inhibited cell cycle progression at the G2/M phase throughout. The protein levels of p21(Cip1)and 14-3-3σ were increased with both AZA and MPA mix treatments without any change in the DNA methylation profiles of the genes.
Our data imply that the decidualization of HESC is associated with cell cycle arrest at G0/G1 phase initially and G2/M phase at later stages. Our results also suggest that p53 pathway members play a role in the cell cycle regulation of endometrial stromal cells.
Cells from multicellular organisms self-destroy when no longer needed or when damaged. They do this by activating genetically controlled machineries that lead to apoptosis. Skeletal muscles in adult animals are fully differentiated syncytial cells. Apoptosis has been described in developing and, recently, in adult skeletal muscle. The cellular and molecular aspects of myoblast and myofibre apoptosis and their role in disease are analysed in this review. Alterations in the pathways that regulate myoblasts proliferation/differentiation lead to induction of apoptosis during myogenesis both in vivo and in vitro. In adult muscle myofibres apoptosis seems to start from segmental areas of myofibres often producing loss of a single myonucleus. The bcl2/bax system is active in muscle when apoptosis occurs. On the other hand conflicting results are reported on the role played by FasL/Fas system. These findings are confirmed by in vitro results on myotubes and on their susceptibility to apoptosis. Though apoptosis has been shown to occur in the skeletal muscle, the role played in diseases and the pattern followed in myogenic cells are far from being clear.
Individuals can often use conceptual models to learn about the business domain to be supported by an information system. We investigate the extent to which such models can help novices (i.e., individuals who lack knowledge in the business domain and in conceptual modeling) to obtain an understanding of the domain codified in the model. We focus on two factors that we predict will influence novices’ understanding: (1) decomposition quality: whether the conceptual model manifests a good decomposition of the domain, and (2) multiple forms of information: whether the conceptual model is accompanied by information in another form (e.g., a textual narrative). We hypothesize that both factors will have positive effects on understanding and that these effects depend on whether the individual seeks a surface or deep understanding. Our results are largely in line with our predictions. Moreover, our results suggest that while novices are generally aware that having multiple forms of information affects their understanding, they are unaware that decomposition quality affects their understanding. Based on these results, we recommend that practitioners include complementary forms of information (such as a textual narrative) along with conceptual models and be careful to ensure that their conceptual models manifest a good decomposition of the domain.
Remodeling is a stringently controlled process that enables adequate response of muscle cells to constant physical stresses. In this process, different kinds of stimuli have to be sensed and converted into biochemical signals that ultimately lead to alterations of muscle phenotype. Several multiprotein complexes located in the sarcomere and organized on the titin molecular spring have been identified as stress sensors and signal transducers. In this review, we focus on Ankrd1/CARP and Ankrd2/Arpp proteins,which belong to the muscle ankyrin repeat protein family (MARP) involved in a mechano-signaling pathway that links myofibrillar stress response to muscle gene expression. Apart from the mechanosensory function, they have an important role in transcriptional regulation, myofibrillar assembly, cardiogenesis and myogenesis. Their altered expression has been demonstrated in neuromuscular disorders, cardiovascular diseases, as well as in tumors, suggesting a role in pathological processes. Although analyzed in a limited number of patients, there is a considerable body of evidence that MARP proteins could be suitable candidates for prognostic and diagnostic biomarkers.
During myogenesis, precursor cells irreversibly withdraw from the cell cycle as they differentiate into mature myotubes. The state of myocyte differentiation also influences the propensity of these cells to undergo apoptosis. Proliferative precursor cells are far more susceptible to apoptotic cell death than are terminally differentiated myotubes. The upregulation of the cdk inhibitor p21 and the dephosphorylation of pRb are critical regulatory events that establish both the post-mitotic and apoptosis-resistant states. The coordinate regulation of cell proliferation and death provides the organism with a mechanism to control the deposition of muscle mass during embryonic development.
Neural crest development involves epithelial-mesenchymal transition (EMT), during which epithelial cells are converted into individual migratory cells. Notably, the same signaling pathways regulate EMT function during both development and tumor metastasis. p53 plays multiple roles in the prevention of tumor development; however, its precise roles during embryogenesis are less clear. We have investigated the role of p53 in early cranial neural crest (CNC) development in chick and mouse embryos. In the mouse, p53 knockout embryos displayed broad craniofacial defects in skeletal, neuronal and muscle tissues. In the chick, p53 is expressed in CNC progenitors and its expression decreases with their delamination from the neural tube. Stabilization of p53 protein using a pharmacological inhibitor of its negative regulator, MDM2, resulted in reduced SNAIL2 (SLUG) and ETS1 expression, fewer migrating CNC cells and in craniofacial defects. By contrast, electroporation of a dominant-negative p53 construct increased PAX7(+) SOX9(+) CNC progenitors and EMT/delamination of CNC from the neural tube, although the migration of these cells to the periphery was impaired. Investigating the underlying molecular mechanisms revealed that p53 coordinates CNC cell growth and EMT/delamination processes by affecting cell cycle gene expression and proliferation at discrete developmental stages; disruption of these processes can lead to craniofacial defects.
In skeletal muscle cells, the PC4 (Tis7/Ifrd1) protein is known to function as a coactivator of MyoD by promoting the transcriptional
activity of myocyte enhancer factor 2C (MEF2C). In this study, we show that up-regulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that
PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that
such an effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions
as a negative regulator of NF-κB, known to inhibit MyoD expression post-transcriptionally. In fact, up-regulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-κB p65, in concomitance
with induction of MyoD expression. On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We
also observe that PC4 potentiates the inhibition of NF-κB transcriptional activity mediated by histone deacetylases and that
PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring
the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling
the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis.
During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca(2+) channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca(2+)-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca(2+) release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca(2+)-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without alpha1((S))DHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in alpha1((S))DHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or alpha1((S))DHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca(2+) content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.
The muscle ankyrin repeat protein (MARP) family member Ankrd1/CARP is a part of the titin-mechanosensory signaling complex in the sarcomere and in response to stretch it translocates to the nucleus where it participates in the regulation of cardiac genes as a transcriptional co-repressor. Several studies have focused on its structural role in muscle, but its regulatory role is still poorly understood. To gain more insight into the regulatory function of Ankrd1/CARP we searched for transcription factors that could interact and modulate its activity. Using protein array methodology we identified the tumor suppressor protein p53 as an Ankrd1/CARP interacting partner and confirmed their interaction both in vivo and in vitro. We demonstrate a novel role for Ankrd1/CARP as a transcriptional co-activator, moderately up regulating p53 activity. Furthermore, we show that p53 operates as an upstream effector of Ankrd1/CARP, by up regulating the proximal ANKRD1 promoter. Our findings suggest that, besides acting as a transcriptional co-repressor, Ankrd1/CARP could have a stimulatory effect on gene expression in cultured skeletal muscle cells. It is probable that Ankrd1/CARP has a role in the propagation of signals initiated by myogenic regulatory factors (MRFs) during myogenesis.
32D C13(G) is an interleukin 3(IL3)-dependent non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocytic colony stimulating factor (G-CSF). Infections of 32D C13(G) cells with either Kirsten rat sarcoma virus or Balb murine sarcoma virus, both containing a v-ras oncogene, generates clones that can permanently grow in G-CSF without differentiation. 32D-Ki-ras cells show a heterogeneous morphology ranging from the promyelocytic to the myelocytic stage of differentiation, and express high levels of both myeloperoxidase (MPO) and lactoferrin (LF) mRNA. 32D-Ha-ras cells show a more immature phenotype and express MPO but no LF mRNA. The apparent differentiation block of both 32D Ki-ras and 32D Ha ras can be reversed by treatment with the chemical inducers retinoic acid, sodium butyrate or dimethylsulphoxide, which leads to terminal differentiation into granulocytes. When 32D-Ki-ras and 32D-Ha-ras cells are cultured in medium containing IL-3 they become adherent and express some monocyte-macrophage markers. Upon prolonged exposure to IL3, 32D-Ki-ras, but not 32D-Ha-ras, resume suspension growth. Both 32D-Ki-ras and 32D-Ha-ras rapidly die if grown in chemically defined medium in the absence of any growth factor and are non-tumorigenic in immunosuppressed mice. These findings indicate that ras activation may interfere with the normal response to growth and differentiation factors in cells of the granulocytic lineage. These alterations may represent a critical, although non-sufficient, step in leukemogenesis.