ORCID Profile
0000-0002-5772-2122
Current Organisation
Peter MacCallum Cancer Centre
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Publisher: American Association for the Advancement of Science (AAAS)
Date: 07-11-2003
Abstract: Apoptosis provoked by DNA damage requires the p53 tumor suppressor, but which of the many p53-regulated genes are required has remained unknown. Two genes induced by this transcription factor, noxa and puma ( bbc3 ), stand out, because they encode BH3-only proteins, proapoptotic members of the Bcl-2 family required to initiate apoptosis. In mice with either noxa or puma disrupted, we observed decreased DNA damage–induced apoptosis in fibroblasts, although only loss of Puma protected lymphocytes from cell death. Puma deficiency also protected cells against erse p53-independent cytotoxic insults, including cytokine deprivation and exposure to glucocorticoids, the kinase inhibitor staurosporine, or phorbol ester. Hence, Puma and Noxa are critical mediators of the apoptotic responses induced by p53 and other agents.
Publisher: Springer Science and Business Media LLC
Date: 16-01-2009
DOI: 10.1038/CDD.2008.195
Publisher: Elsevier BV
Date: 02-2013
Publisher: Cold Spring Harbor Laboratory
Date: 2005
Abstract: The Bcl-2 protein family, which largely determines commitment to apoptosis, has central roles in tumorigenesis and chemoresistance. Its three factions of interacting proteins include the BH3-only proteins (e.g., Bim, Puma, Bad, Noxa), which transduce erse cytotoxic signals to the mammalian pro-survival proteins (Bcl-2, Bcl-x(L), Bcl-w, Mcl-1, A-1), whereas Bax and Bak, when freed from pro-survival constraint, provoke the mitochondrial permeabilization that triggers apoptosis. We have discovered unexpected specificity in their interactions. Only Bim and Puma, which mediate multiple cytotoxic signals, engage all the pro-survival proteins. Noxa and Bad instead bind subsets and cooperate in killing, indicating that apoptosis requires neutralization of different pro-survival subsets. Furthermore, Mcl-1 and Bcl-x(L), but not Bcl-2, directly sequester Bak in healthy cells, and Bak is freed only when BH3-only proteins neutralize both its guards. BH3-only proteins such as Bim are tumor suppressors and mediate many of the cytotoxic signals from anticancer agents. Hence, compounds mimicking them may prove valuable for therapy. Indeed, the recently described ABT-737 is a promising "BH3 mimetic" of Bad. We find that, like Bad, ABT-737 kills cells efficiently only if Mcl-1 is absent or down-regulated. Thus, manipulation of apoptosis by targeting the Bcl-2 family has exciting potential for cancer treatment.
Publisher: Cold Spring Harbor Laboratory
Date: 08-2010
DOI: 10.1101/GAD.1940110
Abstract: Although tumor development requires impaired apoptosis, we describe a novel paradigm of apoptosis-dependent tumorigenesis. Because DNA damage triggers apoptosis through p53-mediated induction of BH3-only proteins Puma and Noxa, we explored their roles in γ-radiation-induced thymic lymphomagenesis. Surprisingly, whereas Noxa loss accelerated it, Puma loss ablated tumorigenesis. Tumor suppression by Puma deficiency reflected its protection of leukocytes from γ-irradiation-induced death, because their glucocorticoid-mediated decimation in Puma-deficient mice activated cycling of stem rogenitor cells and restored thymic lymphomagenesis. Our demonstration that cycles of cell attrition and repopulation by stem rogenitor cells can drive tumorigenesis has parallels in human cancers, such as therapy-induced malignancies.
Publisher: Springer Science and Business Media LLC
Date: 18-02-2011
DOI: 10.1007/S10911-011-9199-Z
Abstract: Worldwide, more than one million women are diagnosed with breast cancer every year, making it the most common malignancy of females in the developed world. Germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 account for 4-6% of all breast cancer cases, and mutation carriers have a lifetime risk of 80% for developing breast cancer and 40% for developing ovarian cancer. Current treatment options are limited and often do not lead to cure. In the 17 years since the discovery of BRCA1, the generation of mouse models for BRCA1 deficiency has greatly aided our understanding of it's role in tumorigenesis. In contrast to human BRCA1 mutation carriers, mice carrying heterozygous mutations in Brca1 did not develop spontaneous tumors. This led to the generation of conditional mouse models in which tissue-specific Brca1 deletion induces formation of mammary tumors that closely resemble human BRCA1-mutated breast tumors. These models have proven useful for studying BRCA1-related tumor development, drug response and resistance. BRCA1-deficient cancer cells are defective in DNA repair mediated by homologous recombination (HR) and therefore highly sensitive to DNA-damaging agents such as platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors. However, BRCA1-mutated tumors can develop resistance to these drugs hence improved treatment strategies are critical. Existing mouse models have already proven useful for preclinical testing of (combinations of) therapeutic agents that may be beneficial for the treatment of patients with BRCA1-mutated tumors. In this review, we discuss the progress made towards modeling BRCA1-deficient breast cancer in mice and what we have learned from preclinical studies using these models.
Publisher: Informa UK Limited
Date: 10-2005
Publisher: Elsevier BV
Date: 05-2013
DOI: 10.1016/J.CELREP.2013.04.012
Abstract: Activation of apoptosis through transcriptional induction of Puma and Noxa has long been considered to constitute the critical (if not sole) process by which p53 suppresses tumor development, although G1/S boundary cell-cycle arrest via induction of the CDK inhibitor p21 has also been thought to contribute. Recent analyses of mice bearing mutations that impair p53-mediated induction of select target genes have indicated that activation of apoptosis and G1/S cell-cycle arrest may, in fact, be dispensable for p53-mediated tumor suppression. However, the expression of Puma, Noxa, and p21 was not abrogated in these mutants, only reduced therefore, the possibility that the reduced levels of these critical effectors of p53-mediated apoptosis and G1/S-cell-cycle arrest sufficed to prevent tumorigenesis could not be excluded. To resolve this important issue, we have generated mice deficient for p21, Puma, and Noxa (p21-/-puma-/-noxa-/- mice). Cells from these mice were deficient in their ability to undergo p53-mediated apoptosis, G1/S cell-cycle arrest, and senescence. Nonetheless, these animals remained tumor free until at least 500 days, in contrast to p53-deficient mice, which had all succumbed to lymphoma or sarcoma by 250 days. Interestingly, DNA lesions induced by γ-irradiation persisted longer in p53-deficient cells compared to wild-type or p21-/-puma-/-noxa-/- cells, and the former failed to transcriptionally activate several p53 target genes implicated in DNA repair. These results demonstrate beyond a doubt that the induction of apoptosis, cell-cycle arrest, and possibly senescence is dispensable for p53-mediated suppression of spontaneous tumor development and indicate that coordination of genomic stability and possibly other processes, such as metabolic adaptation, may instead be critical.
Publisher: American Society of Hematology
Date: 09-12-2010
DOI: 10.1182/BLOOD-2010-04-280818
Abstract: DNA-damaging chemotherapy is the backbone of cancer treatment, although it is not clear how such treatments kill tumor cells. In nontransformed lymphoid cells, the combined loss of 2 proapoptotic p53 target genes, Puma and Noxa, induces as much resistance to DNA damage as loss of p53 itself. In Eμ-Myc lymphomas, however, lack of both Puma and Noxa resulted in no greater drug resistance than lack of Puma alone. A third B-cell lymphoma-2 homology domain (BH)3-only gene, Bim, although not a direct p53 target, was up-regulated in Eμ-Myc lymphomas incurring DNA damage, and knockdown of Bim levels markedly increased the drug resistance of Eμ-Myc/Puma−/−Noxa−/− lymphomas both in vitro and in vivo. Remarkably, c-MYC–driven lymphoma cell lines from Noxa−/−Puma−/−Bim−/− mice were as resistant as those lacking p53. Thus, the combinatorial action of Puma, Noxa, and Bim is critical for optimal apoptotic responses of lymphoma cells to 2 commonly used DNA-damaging chemotherapeutic agents, identifying Bim as an additional biomarker for treatment outcome in the clinic.
Publisher: American Society of Hematology
Date: 03-10-2013
DOI: 10.1182/BLOOD-2013-01-478651
Abstract: Caloric restriction reduces Mcl-1 expression and sensitizes lymphoma cells to ABT-737 in vivo. Caloric restriction mimetics can sensitize lymphomas to ABT-737–induced death independently of p53 and of the main BH3-only proteins.
Publisher: American Society of Hematology
Date: 15-12-2005
DOI: 10.1182/BLOOD-2005-04-1595
Abstract: 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 γ-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 erse types of lymphocytes from the effects of γ-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 γ-radiation– and glucocorticoid-induced apoptosis in lymphoid cells in vivo.
Publisher: Wiley
Date: 12-10-2012
DOI: 10.1002/PATH.4096
Abstract: The leucine-rich repeat-containing heterotrimeric guanine nucleotide-binding protein-coupled receptor 5 (LGR5) has been identified as a marker of cycling stem cells in several epithelial tissues, including small intestine, colon, stomach and hair follicle. To investigate whether LGR5 also marks mammary epithelial stem cells, we performed in situ lineage-tracing studies and mammary gland reconstitutions with LGR5-expressing mammary epithelial cells. Interestingly, the LGR5 progeny population in mammary epithelium switches from the luminal to the myoepithelial compartment during the first 12 days of postnatal development, likely reflecting local changes in Wnt signalling. Together, our findings point to a stage-specific contribution of LGR5-expressing cells to luminal and basal epithelial lineages during postnatal mammary gland development.
Publisher: Springer Science and Business Media LLC
Date: 08-07-2007
DOI: 10.1038/NI1487
Publisher: Oxford University Press (OUP)
Date: 09-2013
DOI: 10.1002/STEM.1437
Abstract: Specification of the cellular hierarchy in the mammary gland involves complex signaling that remains poorly defined. Polycomb group proteins are known to contribute to the maintenance of stem cell identity through epigenetic modifications, leading to stable alterations in gene expression. The polycomb protein family member EZH2 is known to be important for stem cell maintenance in multiple tissues, but its role in mammary gland development and differentiation remains unknown. Our analyses show that EZH2 is predominantly expressed in luminal cells of the mouse mammary epithelium. As mammary gland development occurs mostly after birth, the analysis of EZH2 gene function in postnatal development is precluded by embryonic lethality of conventional EZH2 knockout mice. To investigate the role of EZH2 in normal mammary gland epithelium, we have generated novel transgenic mice that express doxycycline-regulatable short hairpin (sh) RNAs directed against Ezh2. Knockdown of EZH2 results in delayed outgrowth of the mammary epithelium during puberty, due to impaired terminal end bud formation and ductal elongation. Furthermore, our results demonstrate that EZH2 is required to maintain the luminal cell pool and may limit differentiation of luminal progenitors into CD61+ differentiated luminal cells, suggesting a role for EZH2 in mammary luminal cell fate determination. Consistent with this, EZH2 knockdown reduced lobuloalveolar expansion during pregnancy, suggesting EZH2 is required for the differentiation of luminal progenitors to alveolar cells.Stem Cells 2013 :1910-1920
Publisher: Springer Science and Business Media LLC
Date: 08-02-2008
DOI: 10.1038/CDD.2008.16
Publisher: Society for Neuroscience
Date: 05-07-2006
DOI: 10.1523/JNEUROSCI.0196-06.2006
Abstract: Neural precursor cells (NPCs) are highly sensitive to genotoxic injury, which triggers activation of the intrinsic mitochondria-dependent apoptotic pathway. This pathway is typically initiated by members of the BH3 (Bcl-2 homology 3)-only subgroup of the Bcl-2 (B-cell CLL/lymphoma 2) protein family, which are positioned upstream in the apoptotic pathway to respond to specific death stimuli. We have shown previously that NPCs deficient in the tumor suppressor protein p53 show significantly less death after exposure to genotoxic injury or to staurosporine (STS), a broad kinase inhibitor and potent apoptosis inducer. p53 has been shown to regulate the expression of both Noxa and Puma, two BH3-only proteins, although their involvement in p53-dependent cell death appears to be cell-type and stimulus specific. A systematic comparison of the relative contributions of Noxa and Puma to NPC apoptosis has not yet been performed. We hypothesized that p53-dependent transcription of Noxa and Puma leads to death in telencephalic NPCs exposed to genotoxic stress. We found that genotoxic injury induces a rapid p53-dependent increase in expression of Noxa and Puma mRNA in telencephalic NPCs. Furthermore, deficiency of either Noxa or Puma inhibited DNA damage-induced caspase-3 activation and cell death in telencephalic NPCs in vitro . However, only Puma deficiency protected telencephalic ventricular zone NPCs from death in vivo . In contrast to genotoxic injury, STS produced a p53-independent increase in Noxa and Puma expression, but neither Noxa nor Puma was required for STS-induced NPC death. Together, these experiments identify Noxa and Puma as important regulators of genotoxin-induced telencephalic NPC death.
Publisher: Proceedings of the National Academy of Sciences
Date: 22-06-2015
Abstract: Poly(ADP-ribose) polymerase (PARP) inhibitors hold promise for patients with breast cancer 1 ( BRCA1 )-associatated cancers but are anticipated to give rise to resistance. We show that mouse mammary tumors resembling BRCA1-associated metaplastic breast carcinoma display intrinsic resistance to the PARP inhibitor olaparib as a result of increased P-glycoprotein drug efflux transporter expression. These findings may have implications for ongoing clinical trials.
Publisher: Rockefeller University Press
Date: 21-01-2008
Abstract: Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2–like proteins, Mcl-1 stability is acutely regulated. For ex le, the Bcl-2 homology 3 (BH3)–only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, BimS2A, which is highly selective for Mcl-1. Unlike Noxa, BimS2A is unable to trigger Mcl-1 degradation, yet, like Noxa, BimS2A promotes cell killing only when Bcl-xL is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.
Publisher: Elsevier BV
Date: 06-2005
DOI: 10.1016/J.BBRC.2005.03.183
Abstract: p53 is a tumour suppressor that is found mutated or functionally inactivated in more than half of all human cancers. p53 function is activated by DNA damage, hypoxia, expression of certain oncogenes, and many cytotoxic stimuli. p53 is a transcription factor that regulates expression of target genes which promote apoptotic cell death, cell cycle arrest, cellular senescence, and some other processes. In this review we summarise current knowledge of p53 target genes implicated in apoptosis signalling.
Publisher: EMBO
Date: 15-01-2014
Publisher: Springer Science and Business Media LLC
Date: 03-07-2019
DOI: 10.1038/S41580-019-0143-1
Abstract: Chromatin is a macromolecular complex predominantly comprising DNA, histone proteins and RNA. The methylation of chromatin components is highly conserved as it helps coordinate the regulation of gene expression, DNA repair and DNA replication. Dynamic changes in chromatin methylation are essential for cell-fate determination and development. Consequently, inherited or acquired mutations in the major factors that regulate the methylation of DNA, RNA and/or histones are commonly observed in developmental disorders, ageing and cancer. This has provided the impetus for the clinical development of epigenetic therapies aimed at resetting the methylation imbalance observed in these disorders. In this Review, we discuss the cellular functions of chromatin methylation and focus on how this fundamental biological process is corrupted in cancer. We discuss methylation-based cancer therapies and provide a perspective on the emerging data from early-phase clinical trial therapies that target regulators of DNA and histone methylation. We also highlight promising therapeutic strategies, including monitoring chromatin methylation for diagnostic purposes and combination epigenetic therapy strategies that may improve immune surveillance in cancer and increase the efficacy of conventional and targeted anticancer drugs.
Publisher: Wiley
Date: 06-02-2006
Publisher: Elsevier BV
Date: 06-2007
DOI: 10.1016/J.CELL.2007.04.027
Abstract: Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a erse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPalpha-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.
Publisher: Rockefeller University Press
Date: 05-02-2007
Abstract: To identify the mechanisms of ultraviolet radiation (UVR)–induced cell death, for which the tumor suppressor p53 is essential, we have analyzed mouse embryonic fibroblasts (MEFs) and keratinocytes in mouse skin that have specific apoptotic pathways blocked genetically. Blocking the death receptor pathway provided no protection to MEFs, whereas UVR-induced apoptosis was potently inhibited by Bcl-2 overexpression, implicating the mitochondrial pathway. Indeed, Bcl-2 overexpression boosted cell survival more than p53 loss, revealing a p53-independent pathway controlled by the Bcl-2 family. Analysis of primary MEFs lacking in idual members of its BH3-only subfamily identified major initiating roles for the p53 targets Noxa and Puma. In the transformed derivatives, where Puma, unexpectedly, was not induced by UVR, Noxa had the dominant role and Bim a minor role. Furthermore, loss of Noxa suppressed the formation of apoptotic keratinocytes in the skin of UV-irradiated mice. Collectively, these results demonstrate that UVR activates the Bcl-2–regulated apoptotic pathway predominantly through activation of Noxa and, depending on cellular context, Puma.
Publisher: Wiley
Date: 23-09-2016
Publisher: Public Library of Science (PLoS)
Date: 06-08-2018
Publisher: Springer Science and Business Media LLC
Date: 15-10-2015
Abstract: Preclinical in vivo validation of target genes for therapeutic intervention requires careful selection and characterization of the most suitable animal model in order to assess the role of these genes in a particular process or disease. To this end, genetically engineered mouse models (GEMMs) are typically used. However, the appropriate engineering of these models is often cumbersome and time consuming. Recently, we and others described a modular approach for fast-track modification of existing GEMMs by re-derivation of embryonic stem cells (ESCs) that can be modified by recombinase-mediated transgene insertion and subsequently used for the production of chimeric mice. This 'GEMM-ESC strategy' allows for rapid in vivo analysis of gene function in the chimeras and their offspring. Moreover, this strategy is compatible with CRISPR/Cas9-mediated genome editing. This protocol describes when and how to use the GEMM-ESC strategy effectively, and it provides a detailed procedure for re-deriving and manipulating GEMM-ESCs under feeder- and serum-free conditions. This strategy produces transgenic mice with the desired complex genotype faster than traditional methods: generation of validated GEMM-ESC clones for controlled transgene integration takes 9-12 months, and recombinase-mediated transgene integration and chimeric cohort production takes 2-3 months. The protocol requires skills in embryology, stem cell biology and molecular biology, and it is ideally performed within, or in close collaboration with, a transgenic facility.
Publisher: Rockefeller University Press
Date: 18-12-2006
DOI: 10.1084/JEM.20061552
Abstract: The physiological role of B cell lymphoma 2 (Bcl-2) homology 3–only proteins has been investigated in mice lacking the in idual genes identifying rate-limiting roles for Bim (Bcl-2–interacting mediator of cell death) and Puma (p53–up-regulated modulator of apoptosis) in apoptosis induction. The loss of Bim protects lymphocytes from apoptosis induced by cytokine deprivation and deregulated Ca++ flux and interferes with the deletion of autoreactive lymphocytes and the shutdown of immune responses. In contrast, Puma is considered the key mediator of p53-induced apoptosis. To investigate the hypothesis that Bim and Puma have overlapping functions, we generated mice lacking both genes and found that bim−/− uma−/− animals develop multiple postnatal defects that are not observed in the single knockout mice. Most strikingly, hyperplasia of lymphatic organs is comparable with that observed in mice overexpressing Bcl-2 in all hemopoietic cells exceeding the hyperplasia observed in bim−/− mice. Bim and Puma also have clearly overlapping functions in p53-dependent and -independent apoptosis. Their combined loss promotes spontaneous tumorigenesis, causing the malignancies observed in Bcl-2 transgenic mice, but does not exacerbate the autoimmunity observed in the absence of Bim.
No related grants have been discovered for Ewa Michalak.