MBTD1 preserves adult hematopoietic stem cell pool size and function.

Mbtd1 (mbt domain containing 1) encodes a nuclear protein containing a zinc finger domain and four malignant brain tumor (MBT) repeats. We previously generated Mbtd1-deficient mice and found that MBTD1 is highly expressed in fetal hematopoietic stem cells (HSCs) and sustains the number and function of fetal HSCs. However, since Mbtd1-deficient mice die soon after birth possibly due to skeletal abnormalities, its role in adult hematopoiesis remains unclear. To address this issue, we generated Mbtd1 conditional knockout mice and analyzed adult hematopoietic tissues deficient in Mbtd1. We observed that the numbers of HSCs and progenitors increased and Mbtd1-deficient HSCs exhibited hyperactive cell cycle, resulting in a defective response to exogenous stresses. Mechanistically, we found that MBTD1 directly binds to the promoter region of FoxO3a, encoding a forkhead protein essential for HSC quiescence, and interacts with components of TIP60 chromatin remodeling complex and other proteins involved in HSC and other stem cell functions. Restoration of FOXO3a activity in Mbtd1-deficient HSCs in vivo rescued cell cycle and pool size abnormalities. These findings indicate that MBTD1 is a critical regulator for HSC pool size and function, mainly through the maintenance of cell cycle quiescence by FOXO3a.

Necessary and sufficient role for a mitosis skip in senescence induction.

Senescence is a state of permanent growth arrest and is a pivotal part of the antitumorigenic barrier in vivo. Although the tumor suppressor activities of p53 and pRb family proteins are essential for the induction of senescence, molecular mechanisms by which these proteins induce senescence are still not clear. Using time-lapse live-cell imaging, we demonstrate here that normal human diploid fibroblasts (HDFs) exposed to various senescence-inducing stimuli undergo a mitosis skip before entry into permanent cell-cycle arrest. This mitosis skip is mediated by both p53-dependent premature activation of APC/C(Cdh1) and pRb family protein-dependent transcriptional suppression of mitotic regulators. Importantly, mitotic skipping is necessary and sufficient for senescence induction. p16 is only required for maintenance of senescence. Analysis of human nevi also suggested the role of mitosis skip in in vivo senescence. Our findings provide decisive evidence for the molecular basis underlying the induction and maintenance of cellular senescence.

Loss of Bmi1 causes anomalies in retinal development and degeneration of cone photoreceptors.

Retinal development occurs through the sequential but overlapping generation of six types of neuronal cells and one glial cell type. Of these, rod and cone photoreceptors represent the functional unit of light detection and phototransduction and are frequently affected in retinal degenerative diseases. During mouse development, the Polycomb group protein Bmi1 is expressed in immature retinal progenitors and differentiated retinal neurons, including cones. We show here that Bmi1 is required to prevent post natal degeneration of cone photoreceptors and bipolar neurons and that inactivation of Chk2 or p53 could improve but not overcome cone degeneration in Bmi1(-/-) mice. The retinal phenotype of Bmi1(-/-) mice was also characterized by loss of heterochromatin, activation of tandem repeats, oxidative stress and Rip3-associated necroptosis. In the human retina, BMI1 was preferentially expressed in cones at heterochromatic foci. BMI1 inactivation in human embryonic stem cells was compatible with retinal induction but impaired cone terminal differentiation. Despite this developmental arrest, BMI1-deficient cones recapitulated several anomalies observed in Bmi1(-/-) photoreceptors, such as loss of heterochromatin, activation of tandem repeats and induction of p53, revealing partly conserved biological functions between mouse and man.

TGF-beta-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia.

Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO), supporting the proliferation or inhibiting the apoptosis of CML cells. Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy, imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML. Here, using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model, we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a(+/+) and Foxo3a(-/-) mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore, we find that TGF-beta is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-beta inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-beta inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-beta-FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo.

Bmi1 regulates mitochondrial function and the DNA damage response pathway.

Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1(-/-) phenotype. Here we demonstrate that cells derived from Bmi1(-/-) mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1(-/-) mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.

Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo.

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1+/- nor Chk2-/- mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1+/-Chk2-/- and Chk1+/-Chk2+/- mice have a progressive cancer-prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra-S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double-mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53- or Rb-dependent oncogene-induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.

DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1.

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.

FoxO3a regulates hematopoietic homeostasis through a negative feedback pathway in conditions of stress or aging.

Stress or aging of tissue-specific stem cells is considered central to the decline of tissue homeostasis in the elderly, although little is known of molecular mechanisms underlying hematopoietic stem cell (HSC) aging and stress resistance. Here, we report that mice lacking the transcription factor forkhead box O3a (FoxO3a) develop neutrophilia associated with inhibition of the up-regulation of negative regulator of cell proliferation, Sprouty-related Ena/VASP homology 1 domain-containing proteins 2 (Spred2) and AKT and ERK activation, in HSCs during hematopoietic recovery following myelosuppressive stress conditions. Compared with aged wild-type mice, more severe neutrophilia was also observed in aged Foxo3a-deficient mice. AKT and ERK activation and inhibition of Spred2 were detected in HSCs from aged FoxO3a-deficient mice. Spred2-deficient mice also developed neutrophilia during hematopoietic recovery following myelosuppressive stress, indicating that FoxO3a plays a pivotal role in maintenance, integrity, and stress resistance of HSCs through negative feedback pathways for proliferation. This will provide new insight into the hematopoietic homeostasis in conditions of aging and stress.

ATM is required for rapid degradation of cyclin D1 in response to gamma-irradiation.

The cellular response to DNA damage induced by gamma-irradiation activates cell-cycle arrest to permit DNA repair and to prevent replication. Cyclin D1 is the key molecule for transition between the G1 and S phases of the cell-cycle, and amplification or overexpression of cyclin D1 plays pivotal roles in the development of several human cancers. To study the regulation of cyclin D1 in the DNA-damaged condition, we analyzed the proteolytic regulation of cyclin D1 expression upon gamma-irradiation. Upon gamma-irradiation, a rapid reduction in cyclin D1 levels was observed prior to p53 stabilization, indicating that the stability of cyclin D1 is controlled in a p53-independent manner. Further analysis revealed that irradiation facilitated ubiquitination of cyclin D1 and that a proteasome inhibitor blocked cyclin D1 degradation under the same conditions. Interestingly, after mutation of threonine residue 286 of cyclin D1, which is reported to be the GSK-3beta phosphorylation site, the mutant protein showed resistance to irradiation-induced proteolysis although inhibitors of GSK-3beta failed to prevent cyclin D1 degradation. Rather, ATM inhibition markedly prevented cyclin D1 degradation induced by gamma-irradiation. Our data indicate that communication between ATM and cyclin D1 may be required for maintenance of genomic integrity achieved by rapid arrest of the cell-cycle, and that disruption of this crosstalk may increase susceptibility to cancer.

DNA damage tumor suppressor genes and genomic instability.

Disruption of the mechanisms that regulate cell-cycle checkpoints, DNA repair, and apoptosis results in genomic instability and the development of cancer in multicellular organisms. The protein kinases ATM and ATR, as well as their downstream substrates Chk1 and Chk2, are central players in checkpoint activation in response to DNA damage. Histone H2AX, ATRIP, as well as the BRCT-motif-containing molecules 53BP1, MDC1, and BRCA1 function as molecular adapters or mediators in the recruitment of ATM or ATR and their targets to sites of DNA damage. The increased chromosomal instability and tumor susceptibility apparent in mutant mice deficient in both p53 and either histone H2AX or proteins that contribute to the nonhomologous end-joining mechanism of DNA repair indicate that DNA damage checkpoints play a pivotal role in tumor suppression.

Bcl-2 and Bcl-XL are indispensable for the late phase of mast cell development from mouse embryonic stem cells.

OBJECTIVE: The aim of this study was to determine the importance of the prosurvival factors Bcl-2 and Bcl-XL for mast cell development and survival. METHODS: bcl-x(-/-) and bcl-2(-/-) mouse embryonic stem cells were maintained in medium supplemented with either interleukin (IL)-3 or IL-3 in combination with stem cell factor (SCF) to favor mast cell development. The development of Bcl-2 family deficient embryonic stem cell-derived mast cells (ESMCs) was monitored and Bcl-2 family gene expression and cell numbers were analyzed. RESULTS: Deficiency in either bcl-x or bcl-2 totally inhibited the development of ESMCs when IL-3 alone was used as a mast cell growth factor. Intriguingly, when IL-3 was used in combination with SCF, the ESMCs developed normally the first 2 weeks but thereafter the cell numbers dropped drastically. The remaining ESMCs express mouse mast cell protease 1, suggesting a mucosal-like phenotype. ESMCs lacking bcl-x or bcl-2 exhibited strong expression of A1, another prosurvival Bcl-2 family member. CONCLUSION: For the first time we provide direct evidence that both bcl-x and bcl-2 are indispensable for mast cell survival during the late phase of their development.

Differential Roles of Rad18 and Chk2 in Genome Maintenance and Skin Carcinogenesis Following UV Exposure.

Defects in DNA polymerase Eta (Polη) cause the sunlight-sensitivity and skin cancer-propensity disorder xeroderma pigmentosum variant. The extent to which Polη function depends on the upstream E3 ubiquitin ligase Rad18 is controversial and has not been investigated using mouse models. Therefore, we tested the role of Rad18 in UV-inducible skin tumorigenesis. Because Rad18 deficiency leads to compensatory DNA damage signaling by Chk2, we also investigated genetic interactions between Rad18 and Chk2 in vivo. Chk2-/-Rad18-/- mice were prone to spontaneous lymphomagenesis. Both Chk2-/- and Chk2-/-Rad18-/- mice were prone to UV-B irradiation-induced skin tumorigenesis when compared with wild-type (WT) animals, but unexpectedly Rad18-/- mice did not recapitulate the skin tumor propensity of Polη mutants. UV-irradiated Rad18-/- cells were more susceptible to G1/S arrest and apoptosis than WT cultures. Chk2 deficiency alleviated both UV-induced G1/S phase arrest and apoptosis of WT and Rad18-/- cells, but led to increased genomic instability. Taken together, our results demonstrate that the tumor-suppressive role of Polη in UV-treated skin is Rad18 independent. We also define a role for Chk2 in suppressing UV-induced skin carcinogenesis in vivo. This study identifies Chk2 dysfunction as a potential risk factor for sunlight-induced skin tumorigenesis in humans.

DNase II and the Chk2 DNA damage pathway form a genetic barrier blocking replication of horizontally transferred DNA.

We have previously shown that DNA from dying tumor cells may be transferred to living cells via the uptake of apoptotic bodies and may contribute to tumor progression. DNA encoding H-ras(V12) and c-myc oncogenes may be transferred to the nucleus of the phagocyte but will only integrate and propagate in p53- and p21-deficient mouse embryonic fibroblasts, whereas normal cells are resistant to transformation. Here, we show that this protective mechanism (activation of p53 and p21 after uptake of apoptotic bodies) is dependent on DNA fragmentation, where inhibition of the caspase-activated DNase in the apoptotic cells, in conjunction with genetic ablation of lysosomal DNase II in the phagocytes, completely blocks p53 activation and consequently allows DNA replication of transferred DNA. We, therefore, suggest that there is a causal relationship between DNA degradation during apoptosis and p53 activation. In addition, we could further show that Chk2-/- cells were capable of replicating the hyg(R) gene taken up from engulfed apoptotic cells, suggesting involvement of the DNA damage response. These data show that the phagocytosing cell is sensing the degraded DNA within the apoptotic cell, hence preventing these genes from being replicated, probably through activation of the DNA damage response. We, therefore, hypothesize that DNase II together with the Chk2, p53, and p21 pathway form a genetic barrier blocking the replication of potentially harmful DNA introduced via apoptotic bodies, thereby preventing transformation and malignant development.

Hydrocephalus, situs inversus, chronic sinusitis, and male infertility in DNA polymerase lambda-deficient mice: possible implication for the pathogenesis of immotile cilia syndrome.

A growing number of DNA polymerases have been identified, although their physiological function and relation to human disease remain mostly unknown. DNA polymerase lambda (Pol lambda; also known as Pol beta2) has recently been identified as a member of the X family of DNA polymerases and shares 32% amino acid sequence identity with DNA Pol beta within the polymerase domain. With the use of homologous recombination, we generated Pol lambda(-/-) mice. Pol lambda(-/-) mice develop hydrocephalus with marked dilation of the lateral ventricles and exhibit a high rate of mortality after birth, although embryonic development appears normal. Pol lambda(-/-) mice also show situs inversus totalis and chronic suppurative sinusitis. The surviving male, but not female, Pol lambda(-/-) mice are sterile as a result of spermatozoal immobility. Microinjection of sperm from male Pol lambda(-/-) mice into oocytes gives rise to normal offspring, suggesting that the meiotic process is not impaired. Ultrastructural analysis reveals that inner dynein arms of cilia from both the ependymal cell layer and respiratory epithelium are defective, which may underlie the pathogenesis of hydrocephalus, situs inversus totalis, chronic sinusitis, and male infertility. Sensitivity of Pol lambda(-/-) cells to various kinds of DNA damage is indistinguishable from that of Pol lambda(+/+) cells. Collectively, Pol lambda(-/-) mice may provide a useful model for clarifying the pathogenesis of immotile cilia syndrome.

CHK2 is involved in the p53-independent radiosensitizing effects of valproic acid.

Radiotherapy is an effective treatment for the majority of types of localized solid cancer. However, the risk of side effects to the surrounding normal tissues limits radiotherapeutic approaches. Whilst the mechanism of action of valproic acid, an inhibitor of histone deacetylase, remains unknown, the inhibitor is a potential antineoplastic radiosensitizer. The present study demonstrated the in vitro radiosensitizing effects of valproic acid on the human breast cancer MCF7 cell line, and revealed that valproic acid increased the level of DNA breakage, apoptosis and senescence. In addition, western blot analyses revealed that valproic acid induced tumor suppressor protein (p)53 and p21 expression, and activated checkpoint kinase 2 (CHK2) in MCF7 cells and primary mouse embryonic fibroblasts. Notably, treatment with valproic acid also induced increases in the level of p21 protein levels and CHK2 activity in p53-null colon cancer HCT116 cells. Furthermore, the present study demonstrated that valproic acid-induced radiosensitization was largely dependent on the activity of CHK2. The results of the present study reveal that valproic acid may exhibit clinical utility with respect to increasing the anticancer efficacy of radiotherapy by affecting the level of p53.

Targeting of Chk2 as a countermeasure to dose-limiting toxicity triggered by topoisomerase-II (TOP2) poisons.

The DNA damage response (DDR) gene cell cycle checkpoint kinase 2 (Chk2) triggers programmed cell death and lethal radiation-induced toxicity in mice in vivo. However, it is not well established to what extent targeting of Chk2 may protect from dose-limiting toxicities (DLT) inflicted by mainstay cancer chemotherapy. We screened different classes of chemotherapy in wild type and Chk2-deficient cells. Here we show that loss of Chk2 protect from cell death in vitro and lethal toxicity in vivo following treatment with topoisomerase II (TOP2)-inhibitors whereas no such protection was observed following treatment with topoisomerase I (TOP1) inhibitors. Furthermore, through combined in silico and functional screens of the Diversity Set II (NCI/NTP) chemical library we identified the carbanilide-derivative NSC105171, also known as ptu-23, as a novel Chk2 inhibitor (Chk2i). Indeed, NSC105171 can be administered safely to mice to countermeasure etoposide-induced toxicity. Incorporation of Chk2i into chemotherapy protocols employing TOP2-inhibitors may be an effective strategy to prevent DLT's without interfering with treatment.

Agonists of the TRAIL Death Receptor DR5 Sensitize Intestinal Stem Cells to Chemotherapy-Induced Cell Death and Trigger Gastrointestinal Toxicity.

The combination of TRAIL death receptor agonists and radiochemotherapy to treat advanced cancers continues to be investigated in clinical trials. We previously showed that normal cells with a functional DNA damage response (DDR) upregulate the expression of death-inducing receptor DR5/TRAILR2/TNFRSF10B in a p53-dependent manner that sensitizes them to treatment with DR5 agonists. However, it is unclear if targeting DR5 selectively sensitizes cancer cells to agonist treatment following exposure to DNA-damaging chemotherapy, and to what extent normal tissues are targeted. Here, we show that the combined administration of the DR5 agonistic monoclonal antibody (mAb) and chemotherapy to wild-type mice triggered synergistic gastrointestinal toxicities (GIT) that were associated with the death of Lgr5(+) crypt base columnar stem cells in a p53- and DR5-dependent manner. Furthermore, we confirmed that normal human epithelial cells treated with the human DR5-agonistic mAb and chemotherapeutic agents were also greatly sensitized to cell death. Interestingly, our data also indicated that genetic or pharmacologic targeting of Chk2 may counteract GIT without negatively affecting the antitumor responses of combined DR5 agonist/chemotherapy treatment, further linking the DDR to TRAIL death receptor signaling in normal cells. In conclusion, the combination of DR5-targeting agonistic mAbs with DNA damaging chemotherapy may pose a risk of developing toxicity-induced conditions, and the effects of mAb-based strategies on the dose-limiting toxicity of chemotherapy must be considered when establishing new combination therapies.

FOXO transcription factors in cell-cycle regulation and the response to oxidative stress.

Mammalian forkhead members of the class O (FOXO) transcription factors, including FOXO1, FOXO3a, and FOXO4, are implicated in the regulation of a variety of cellular processes, including the cell cycle, apoptosis, DNA repair, stress resistance, and metabolism. FOXO proteins are negatively regulated by the phosphatidylinositol 3-kinase-Akt signaling pathway, which is activated by growth factors and cytokines. Recent studies indicate that the activities of FOXO proteins are also regulated by oxidative stress, which induces their phosphorylation, translocation to the nucleus, and acetylation-deacetylation. Similar to the tumor suppressor p53, FOXO is activated by stress and induces the expression of genes that contribute to cell-cycle arrest, suggesting that it also functions as a tumor suppressor.

Ataxia telangiectasia mutated (Atm) knockout mice as a model of osteopenia due to impaired bone formation.

ATM is a member of the PI-3 kinase protein family, encoded by the gene, ATM, responsible for ataxia telangiectasia (AT). AT is recognized as a genomic instability syndrome, sharing accelerated senescence symptoms in human and mouse. Here, we present evidence that the bone phenotype of Atm knockout (AtmKO) mice is similar to that observed in disuse and/or aging syndromes. A significant decrease in 3-dimensional bone volume fraction (BV/TV) of the fifth lumbar vertebra was observed in AtmKO mice by microCT, compared with heterozygous control mice at 10 weeks of age. Bone histomorphometry revealed that both BFR/BS and Oc.S/BS were significantly decreased in KO mice. To determine the cellular basis of this bone phenotype, we employed in vitro osteoclastogenesis and colony formation assays using bone marrow cells derived from KO and control mice. There was no difference in osteoclast formation in ex vivo cultures. CFU-F was markedly reduced in AtmKO-derived cultures compared with control mice, whereas differentiation of calvaria-derived osteoblasts did not differ between the genotypes. Furthermore, expression levels of IGF1R were significantly decreased, and p38 was aberrantly phosphorylated in marrow stromal cells from AtmKO mice. These results indicate that the pathogenesis of the osteopenic phenotype in AtmKO mice is similar to that of disuse and/or aging syndromes and is caused, at least in part, by a stem cell defect due to lack of IGF signaling.

In vivo cooperation between Bcl-xL and the phosphoinositide 3-kinase-Akt signaling pathway for the protection of epidermal keratinocytes from apoptosis.

To investigate the function of Bcl-xL in the skin, we established keratinocyte-specific Bcl-x gene-targeted mice under the keratin 5 promoter (K5). K5.Bcl-xL-/- mice were viable, devoid of alteration in the development of skin or appendages. However, they harbored spontaneous apoptotic keratinocytes in the epidermis. Bcl-xL-deficient keratinocytes cultured in vitro readily underwent apoptosis in the absence of growth factors, but the addition of HGF or EGF resulted in restoration of cell survival, which was reversed by treatment with wortmannin, an inhibitor of phosphoinositide-3 kinase (PI3K). Topical treatment of K5.Bcl-xL-/- mice with wortmannin sensitized the skin for apoptosis induced by UV (UV) B, although wild-type epidermis was only marginally affected by this treatment, suggesting that the resistance to UVB largely depended on PI3K-Akt signaling in Bcl-xL-deficient mice but not in wild-type mice. Furthermore, UVB irradiation resulted in redistribution of phosphorylated Akt from the basal layer to the suprabasal layer, indicating that Akt could spatially cooperate with Bcl-xL upon UVB exposure in the upper epidermis where Bcl-xL is normally localized. These results suggest that Bcl-xL and the PI3K-Akt pathway form a cooperative, intercompensatory axis for the protection of epidermal keratinocytes from apoptosis in vivo.