Maintenance of genomic integrity in hematopoietic stem cells

Hematopoietic stem cells (HSCs) maintain hematopoietic homeostasis throughout a mammal’s lifespan through self-renewal and differentiation into mature blood cells. Within a bone marrow niche, HSCs adopt a quiescent state and remain in the non-dividing, G0 phase of the cell cycle. It was recently shown that maintenance of genomic integrity is crucial for the preservation of self-renewal capacity of HSCs. In this review, we focus on progress in elucidating the roles of reactive oxygen species (ROS) and DNA damage responses (DDR) in maintaining genomic integrity, and thus HSC function. Several studies have demonstrated that inappropriate ROS levels arising from disruption of the Atm, PI3K-Akt, or Mdm2-p53 pathways impair HSC function in vivo. Intriguing evidence that stem cells use specific DDR mechanisms is also accumulating. Although murine HSCs are more resistant than progenitor cells to mild DNA damage in vivo, the surviving HSCs frequently acquire genetic aberrations that can lead to leukemogenesis. Indeed, non-dividing HSCs employ the error-prone non-homologous end-joining pathway of DNA repair to fix DNA breaks, whereas progenitors undergo apoptosis; proliferating HSCs employ the high-fidelity homologous recombination mechanism. Dissection of HSC-specific mechanisms for the maintenance of genomic integrity may provide valuable insights into the biology of both HSCs and leukemia stem cells.     

ATM promotes apoptosis and suppresses tumorigenesis in response to Myc

Overexpression of the c-myc oncogene contributes to the development of a significant number of human cancers. In response to deregulated Myc activity, the p53 tumor suppressor is activated to promote apoptosis and inhibit tumor formation. Here we demonstrate that p53 induction in response to Myc overexpression requires the ataxia-telangiectasia mutated (ATM) kinase, a major regulator of the cellular response to DNA double-strand breaks. In a transgenic mouse model overexpressing Myc in squamous epithelial tissues, inactivation of Atm suppresses apoptosis and accelerates tumorigenesis. Deregulated Myc expression induces DNA damage in primary transgenic keratinocytes and the formation of gammaH2AX and phospho-SMC1 foci in transgenic tissue. These findings suggest that Myc overexpression causes DNA damage in vivo and that the ATM-dependent response to this damage is critical for p53 activation, apoptosis, and the suppression of tumor development.     

The catalytic subunit of DNA-dependent protein kinase selectively regulates p53-dependent apoptosis but not cell-cycle arrest

DNA damage induced by ionizing radiation (IR) activates p53, leading to the regulation of downstream pathways that control cell-cycle progression and apoptosis. However, the mechanisms for the IR-induced p53 activation and the differential activation of pathways downstream of p53 are unclear. Here we provide evidence that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) serves as an upstream effector for p53 activation in response to IR, linking DNA damage to apoptosis. DNA-PKcs knockout (DNA-PKcs-/-) mice were exposed to whole-body IR, and the cell-cycle and apoptotic responses were examined in their thymuses. Our data show that IR induction of apoptosis and Bax expression, both mediated via p53, was significantly suppressed in the thymocytes of DNA-PKcs-/- mice. In contrast, IR-induced cell-cycle arrest and p21 expression were normal. Thus, DNA-PKcs deficiency selectively disrupts p53-dependent apoptosis but not cell-cycle arrest. We also confirmed previous findings that p21 induction was attenuated and cell-cycle arrest was defective in the thymoctyes of whole body-irradiated Atm-/- mice, but the apoptotic response was unperturbed. Taken together, our results support a model in which the upstream effectors DNA-PKcs and Atm selectively activate p53 to differentially regulate cell-cycle and apoptotic responses. Whereas Atm selects for cell-cycle arrest but not apoptosis, DNA-PKcs selects for apoptosis but not cell-cycle arrest.     

Activation of p53 by oncogenes

p53 is activated by a variety of cellular stresses, including DNA damage, hypoxia, and mitogenic oncogenes, but the extent to which each signal engages p53 as a tumour suppressor remains unknown. In non-immortal cells, the adenovirus E1A oncogene activates p53 to promote apoptosis, whereas oncogenic ras activates p53 to promote cellular senescence. Inactivation of p53 prevents E1A-induced apoptosis or Ras-induced senescence, allowing proliferation to continue unabated. In each instance, the ability of the oncogene to activate p53 involves the same functions as are required for their transforming potential, implying that p53 activation acts as a fail-safe mechanism to counter hyperproliferative signals. Furthermore, p19(ARF) is strictly required for oncogene signalling to p53. The fact that ARF--itself a tumour suppressor--acts as an intermediary in this response argues that the tumour suppressor activity of p53 can arise from its ability to eliminate oncogene-expressing cells.     

Chk2 is dispensable for p53-mediated G1 arrest but is required for a latent p53-mediated apoptotic response

In response to genotoxic stress, mammalian cells can activate cell cycle checkpoint pathways to arrest the cell for repair of DNA damage or induce apoptosis to eliminate damaged cells. The checkpoint kinase, Chk2, has been implicated in both of these responses and is believed to function in an ataxia telangiectasia (Atm)-dependent manner. We show here that Chk2-/- mouse embryo fibroblasts (MEFs), unlike Atm-/- or p53-/- MEFs, behaved like normal MEFs in manifesting p21 induction and G(1) arrest upon exposure to gamma-irradiation. Therefore, Chk2 is not involved in p53-mediated G(1) arrest. To examine the role of Chk2 in p53-dependent apoptotic response, we used adenovirus E1A-expressing MEFs. We show that Chk2-/- cells, like p53-/- cells, did not undergo DNA damage-induced apoptosis, whereas Atm-/- cells behaved like normal cells in invoking an apoptotic response. Furthermore, this apoptosis could occur in the absence of protein synthesis, suggesting that it is preexisting, or "latent," p53 that mediates this response. We conclude that Chk2 is not involved in Atm- and p53-dependent G(1) arrest, but is involved in the activation of latent p53, independently of Atm, in triggering DNA damage-induced apoptosis.     

Restoration of p53 function: a new therapeutic strategy to induce tumor regression?

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.     

p53 signaling in response to increased DNA damage sensitizes AML1-ETO cells to stress-induced death

Chromosomal translocation (8;21) is present in 10% to 15% of patients with acute myeloid leukemia. Expression of the AML1-ETO (AE) fusion protein alone is not sufficient to induce leukemia, but the nature of the additional genetic alterations is unknown. It is unclear whether AE facilitates acquisition of these cooperating events. We show that AE down-regulates genes involved in multiple DNA repair pathways, potentially through a mechanism involving direct binding at promoter elements, and increases the mutation frequency in vivo. AE cells display increased DNA damage in vitro and have an activated p53 pathway. This results in increased basal apoptosis and enhanced sensitivity to DNA damaging agents. Intriguingly, microarray data indicate that t(8;21) patient samples exhibit decreased expression of DNA repair genes and increased expression of p53 response genes compared with other acute myeloid leukemia (AML) patient samples. Inhibition of the p53 pathway by RNAi increases the resistance of AE cells to DNA damage. We thus speculate that AML1-ETO may facilitate accumulation of genetic alterations by suppressing endogenous DNA repair. It is possible that the superior outcome of t(8;21) patients is partly due to an activated p53 pathway, and that loss of the p53 response pathway is associated with disease progression.     

Analysis of the apoptotic and therapeutic activities of histone deacetylase inhibitors by using a mouse model of B cell lymphoma

Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival, including inhibition of cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis, and modulation of immune responses, and show promising activity against hematological malignancies in clinical trials. Using the Emu-myc model of B cell lymphoma, we screened tumors with defined genetic alterations in apoptotic pathways for therapeutic responsiveness to the HDACi vorinostat. We demonstrated a direct correlation between induction of tumor cell apoptosis in vivo and therapeutic efficacy. Vorinostat did not require p53 activity or a functional death receptor pathway to kill Emu-myc lymphomas and mediate a therapeutic response but depended on activation of the intrinsic apoptotic pathway with the proapoptotic BH3-only proteins Bid and Bim playing an important role. Our studies provide important information regarding the mechanisms of action of HDACi that have broad implications regarding stratification of patients receiving HDACi therapy alone or in combination with other anticancer agents.     

Mitochondrial oxidative stress caused by Sod2 deficiency promotes cellular senescence and aging phenotypes in the skin

Cellular senescence arrests the proliferation of mammalian cells at risk for neoplastic transformation, and is also associated with aging. However, the factors that cause cellular senescence during aging are unclear. Excessive reactive oxygen species (ROS) have been shown to cause cellular senescence in culture, and accumulated molecular damage due to mitochondrial ROS has long been thought to drive aging phenotypesin vivo. Here, we test the hypothesis that mitochondrial oxidative stress can promote cellular senescence in vivo and contribute to aging phenotypes in vivo, specifically in the skin. We show that the number of senescent cells, as well as impaired mitochondrial (complex II) activity increase in naturally aged mouse skin. Using a mouse model of genetic Sod2 deficiency, we show that failure to express this important mitochondrial anti-oxidant enzyme also impairs mitochondrial complex II activity, causes nuclear DNA damage, and induces cellular senescence but not apoptosis in the epidermis. Sod2 deficiency also reduced the number of cells and thickness of the epidermis, while increasing terminal differentiation. Our results support the idea that mitochondrial oxidative stress and cellular senescence contribute to aging skin phenotypes in vivo.     

BH3-only proteins Puma and Bim are rate-limiting for gamma-radiation- and glucocorticoid-induced apoptosis of lymphoid cells in vivo

Numerous p53 target genes have been implicated in DNA damage-induced apoptosis signaling, but proapoptotic Bcl-2 (B-cell leukemia 2) family members of the BH3 (Bcl-2 homolog region [BH] 3)-only subgroup appear to play the critical initiating role. In various types of cultured cells, 3 BH3-only proteins, namely Puma (p53 up-regulated modulator of apoptosis), Noxa, and Bim (Bcl-2 interacting mediator of cell death), have been shown to initiate p53-dependent as well as p53-independent apoptosis in response to DNA damage and treatment with anticancer drugs or glucocorticoids. In particular, the absence of Puma or Bim renders thymocytes and mature lymphocytes refractory to varying degrees to death induced in vitro by growth factor withdrawal, DNA damage, or glucocorticoids. To assess the in vivo relevance of these findings, we subjected mice lacking Puma, Noxa, or Bim to whole-body gamma-radiation or the glucocorticoid dexamethasone and compared lymphocyte survival with that in wild-type and BCL2-transgenic mice. Absence of Puma or Bcl-2 overexpression efficiently protected diverse types of lymphocytes from the effects of gamma-radiation in vivo, and loss of Bim provided lower but significant protection in most lymphocytes, whereas Noxa deficiency had no impact. Furthermore, both Puma and Bim were found to contribute significantly to glucocorticoid-induced killing. Our results thus establish that Puma and Bim are key initiators of gamma-radiation- and glucocorticoid-induced apoptosis in lymphoid cells in vivo.     

Angiotensin II-mediated oxidative DNA damage accelerates cellular senescence in cultured human vascular smooth muscle cells via telomere-dependent and independent pathways

Angiotensin II (Ang II) induces reactive oxygen species (ROS) production by human vascular smooth muscle cells (hVSMCs). ROS have been implicated in the development of both acute stress-induced premature senescence (SIPS) and chronic replicative senescence. Global oxidative DNA damage triggers SIPS and telomere DNA damage accelerates replicative senescence, both mediated via p53. This study tests the hypothesis that DNA is an important target for Ang II-induced ROS leading to senescence via telomere-dependent and independent pathways. DNA damage was quantified using the Comet assay, telomere DNA length by Southern blotting and hVSMC senescence by senescence-associated beta-galactosidase staining. Exposure to Ang II increased DNA damage in hVSMCs within 4 hours. Inhibition by an AT1 receptor antagonist (losartan metabolite: E3174) or catalase, confirmed that Ang II-induced DNA damage was AT1 receptor-mediated, via the induction of ROS. Acute exposure to Ang II resulted in SIPS within 24 hours that was prevented by coincubation with E3174 or catalase. SIPS was associated with increased p53 expression but was not dependent on telomere attrition because overexpression of human telomerase did not prevent Ang II-induced SIPS. Exposure to Ang II over several population doublings accelerated the rate of telomere attrition (by >2-fold) and induced premature replicative senescence of hVSMCs--an effect that was also attenuated by E3174 or catalase. These data demonstrate that Ang II-induced ROS-mediated DNA damage results in accelerated biological aging of hVSMCs via 2 mechanisms: (1) Acute SIPS, which is telomere independent, and (2) accelerated replicative senescence which is associated with accelerated telomere attrition.     

Chronic doxorubicin cardiotoxicity is mediated by oxidative DNA damage-ATM-p53-apoptosis pathway and attenuated by pitavastatin through the inhibition of Rac1 activity

Doxorubicin is known to have cumulative dose-dependent cardiotoxicity, and a tumor suppressor protein p53 has been implicated in the pathogenesis of doxorubicin cardiotoxicity. However, how p53 is induced by doxorubicin and mediates the cardiotoxic effects of doxorubicin remains elusive. In cultured cardiac myocytes, doxorubicin induced oxidative stress, DNA damage, ATM activation, and p53 induction. A free radical scavenger NAC attenuated all of these events, whereas an ATM kinase inhibitor wortmannin attenuated doxorubicin-induced ATM activation and p53 induction but not oxidative stress. Doxorubicin treatment in vivo also induced oxidative stress, DNA damage, ATM activation, and p53 accumulation. These observations suggest that p53 induction by doxorubicin is mediated by oxidative DNA damage-ATM pathway. Doxorubicin-induced contractile dysfunction and myocyte apoptosis in vivo were attenuated in heterozygous p53 deficient mice and cardiac-restricted Bcl-2 transgenic mice, suggesting that myocyte apoptosis plays a central role downstream of p53 in doxorubicin cardiotoxicity. We also tested whether pitavastatin exerts protective effects on doxorubicin cardiotoxicity. Pitavastatin attenuated doxorubicin-induced oxidative stress, DNA damage, ATM activation, p53 accumulation, and apoptosis in vitro. Pitavastatin also attenuated myocyte apoptosis and contractile dysfunction in vivo. The beneficial effects of pitavastatin were reversed by intermediate products of the mevalonate pathway that are required for the activation of Rac1, and Rac1 inhibitor exhibited cardioprotective effects comparable to those of pitavastatin. These data collectively suggest that doxorubicin-induced cardiotoxicity is mediated by oxidative DNA damage-ATM-p53-apoptosis pathway, and is attenuated by pitavastatin through its antioxidant effect involving Rac1 inhibition.     

Atm and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system

Ataxia-telangiectasia is a hereditary multisystemic disease resulting from mutations of ataxia telangiectasia, mutated (ATM) and is characterized by neurodegeneration, cancer, immune defects, and hypersensitivity to ionizing radiation. The molecular details of ATM function in the nervous system are unclear, although the neurological lesion in ataxia-telangiectasia becomes apparent early in life, suggesting a developmental origin. The central nervous system (CNS) of Atm-null mice shows a pronounced defect in apoptosis induced by genotoxic stress, suggesting ATM functions to eliminate neurons with excessive genomic damage. Here, we report that the death effector Bax is required for a large proportion of Atm-dependent apoptosis in the developing CNS after ionizing radiation (IR). Although many of the same regions of the CNS in both Bax-/- and Atm-/- mice were radioresistant, mice nullizygous for both Bax and Atm showed additional reduction in IR-induced apoptosis in the CNS. Therefore, although the major IR-induced apoptotic pathway in the CNS requires Atm and Bax, a p53-dependent collateral pathway exists that has both Atm- and Bax-independent branches. Further, Atm- and Bax-dependent apoptosis in the CNS also required caspase-3 activation. These data implicate Bax and caspase-3 as death effectors in neurodegenerative pathways.     

DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions

DNA damage leads to a halt in proliferation owing to apoptosis or senescence, which prevents transmission of DNA alterations. This cellular response depends on the tumor suppressor p53 and functions as a powerful barrier to tumor development. Adult stem cells are resistant to DNA damage-induced apoptosis or senescence, however, and how they execute this response and suppress tumorigenesis is unknown. We show that irradiation of hematopoietic and mammary stem cells up-regulates the cell cycle inhibitor p21, a known target of p53, which prevents p53 activation and inhibits p53 basal activity, impeding apoptosis and leading to cell cycle entry and symmetric self-renewing divisions. p21 also activates DNA repair, limiting DNA damage accumulation and self-renewal exhaustion. Stem cells with moderate DNA damage and diminished self-renewal persist after irradiation, however. These findings suggest that stem cells have evolved a unique, p21-dependent response to DNA damage that leads to their immediate expansion and limits their long-term survival.Adult stem cells (SCs) are thought to be resistant to DNA damage (DD)-induced apoptosis or senescence owing to the activation of unique pro-survival and DD repair (DDR) responses (1–3). Genetic alterations that decrease DNA repair activities lead to increased DD and reduced self-renewal in SCs, suggesting that DDR is critical to preservation of SC function (1, 4, 5). DDR decreases during physiological aging, a phenomenon correlated with the accumulation of endogenous DD and decreased self-renewal in aged SCs (6–9).In differentiated cells, DD triggers a checkpoint response that leads to apoptosis or senescence and depends on activation of the tumor suppressor p53 (10). This is considered a powerful tumor-suppressor mechanism, as demonstrated by the finding that p53 is invariably inactivated in spontaneous tumors (11). After irradiation, p53 is up-regulated in populations enriched for hematopoietic, hair follicle bulge, and colon SCs (5, 12–15). Whether this is critical for activation of the DDR response and maintenance of self-renewal, why p53 induction does not result in SC apoptosis or senescence, and how tumor suppression is executed in SCs remain unclear, however. Indirect evidence indicates that the cell cycle inhibitor p21, a downstream effector of p53, might be involved in DD processing in SCs. In the absence of p21, SCs exhaust prematurely (16) and after a low radiation dose display reduced reconstitution capacity (17). Here we report our studies on the role of p53 and p21 in DD processing of highly purified hematopoietic SCs (HSCs) and mammary SCs (MaSCs).     

p53 status dictates responses of B lymphomas to monotherapy with proteasome inhibitors

The proapoptotic function of p53 is thought to underlie most anticancer modalities and is also activated in response to oncogenic insults, such as overexpression of the Myc oncoprotein. Here we generated tractable B lymphomas using retroviral transduction of the MYC oncogene into hematopoietic cells with 2 knock-in alleles encoding a fusion between p53 and 4-hydroxytamoxifen (4OHT) receptor (p53ER(TAM)). In these polyclonal tumors, Myc is the only oncogenic lesion, and p53ER(TAM) status can be rapidly toggled between "off" and "on" with 4OHT, provided that the Trp53 promoter has been independently activated. Although 4OHT can trigger widespread apoptosis and overt tumor regression even in the absence of DNA-damaging agents, in tumors with high levels of Mdm2 these responses are blunted. However, cotreatment with proteasome inhibitors fully restores therapeutic effects in vivo. Similarly, human Burkitt lymphomas with wild-type p53 and overexpression of Hdm2 are highly sensitive to proteasome inhibitors, unless p53 levels are reduced using the HPV-E6 ubiquitin ligase. Therefore, proteasome inhibitors could be highly effective as a monotherapy against Myc-induced lymphomas, with no need for adjuvant chemotherapy or radiation therapy. On the other hand, their efficacy is crucially dependent on the wild-type p53 status of the tumor, placing important restrictions on patient selection.     

Differential requirement for p19ARF in the p53-dependent arrest induced by DNA damage, microtubule disruption, and ribonucleotide depletion

p19ARF has been implicated as a key regulator of p53 stability and activation. While numerous stresses activate the p53 growth arrest pathway, those requiring p19ARF remain to be elucidated. We used p19ARF knockout mouse embryo fibroblasts to show that DNA damage and microtubule disruption require p19ARF to induce p53 responses, whereas ribonucleotide depletion and inhibition of RNA synthesis by low doses of actinomycin D do not. The data provide evidence that the arrest pathway activated by ribonucleotide depletion involves some different signal transducers than those activated by DNA damage or microtubule disruption. We also present biochemical analyses that provide insights into the mechanism by which p53 and p19ARF cooperate in normal cells to induce cell cycle arrest.     

BCR-ABL-mediated inhibition of apoptosis with delay of G2/M transition after DNA damage: a mechanism of resistance to multiple anticancer agents

A critical determinant of the efficacy of antineoplastic therapy is the response of malignant cells to DNA damage induced by anticancer agents. The p53 tumor-suppressor gene is a critical component of two distinct cellular responses to DNA damage, the induction of a reversible arrest at the G1/S cell cycle checkpoint, and the activation of apoptosis, a genetic program of autonomous cell death. Expression of the BCR-ABL chimeric gene produced by a balanced translocation in chronic myeloid leukemia, confers resistance to multiple genotoxic anticancer agents. BCR-ABL expression inhibits the apoptotic response to DNA damage without altering either the p53-dependent WAF1/CIP1-mediated G1 arrest or DNA repair. BCR-ABL-mediated inhibition of DNA damage-induced apoptosis is associated with a prolongation of cell cycle arrest at the G2/M restriction point; the delay of G2/M transition may allow time to repair and complete DNA replication and chromosomal segregation, thereby preventing a mitotic catastrophe. The inherent resistance of human cancers to genotoxic agents may result not only by the loss or inactivation of the wild-type p53 gene, but also by genetic alterations such as BCR-ABL that can delay G2/M transition after DNA damage.