Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice

The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2S394A mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2Y393F) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes.

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Improved Survival and Recovery of Hematopoiesis Following Lethal Radiation by Chloroquine-Mediated Activation of ATM

Blood ◽

10.1182/blood.v116.21.2228.2228 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2228-2228

Author(s):

Yiting Lim ◽

Mohammad Hedayati ◽

Akil Merchant ◽

Yonggang Zhang ◽

Theodore DeWeese ◽

...

Keyword(s):

Bone Marrow ◽

Dose Rate ◽

Cell Lines ◽

Research Funding ◽

Bone Marrow Cells ◽

Bone Marrow Failure ◽

Chloroquine Treatment ◽

Radiation Induced

Abstract Abstract 2228 Irreversible bone marrow damage and impaired blood formation is the primary cause of death following exposure to high doses of radiation. Moreover, the rate at which radiation is delivered may have a profound impact on cytotoxicity; prolonged exposure at a low dose-rate (LDR; 9.4 cGy/hr) has been found to induce greater cell death than the same total dose given at a high dose-rate (HDR; 4500 cGy/hr). Few non-toxic agents are presently available that can offer substantial protection against radiation induced bone marrow failure and death, especially during LDR exposure. We previously demonstrated that chloroquine, a commonly used agent in the treatment of malaria and rheumatologic diseases, can prevent LDR radiation induced cytotoxicity of cell lines in vitro and studied its effects on hematopoiesis in vivo. We initially quantified the effects of LDR radiation on C57/B6 mice and found that 9 Gy delivered at 9.4 cGy/hr for 95.7 hrs induced death in 13/19 (68%) of animals at 15–35 days after radiation. The administration of syngeneic bone marrow cells (1 × 106 cells) immediately after LDR radiation completely rescued animals (10/10) demonstrating that bone marrow failure was responsible for LDR radiation induced death similar to HDR radiation. Next we treated mice with chloroquine (0.0594 mg/17g body weight, i.p.) 24 hrs and 4 hrs prior to exposure to LDR radiation and found that it significantly improved survival (80%, p < 0.05) compared to untreated animals exposed to LDR radiation (32%). We examined hematopoietic recovery following LDR radiation and found that the peripheral WBC was significantly greater in mice treated receiving chloroquine (3.4 × 106/ml vs 1.1 × 106/ml at day 16, p<0.05). Similarly, we found that in vivo chloroquine treatment significantly increased the recovery of bone marrow myeloid CFC (p=0.02), suggesting that it impacted myeloid progenitors. To further validate this finding, we transplanted bone marrow from LDR irradiated mice into lethally irradiated CD45 congenic recipient mice, and found a significant improvement in early engraftment (4.2% vs. 0.4% engraftment at 6 weeks post-transplant, p=0.015). Chloroquine has been found to protect cancer cell lines from LDR radiation in vitro by activating ATM, an essential DNA damage sensor. We examined the effect of chloroquine on ATM and treated unradiated lin- bone marrow cells with chloroquine in vitro (35 ug/ml, 2 hr). Compared to control cells, chloroquine treated cells expressed 2.5-fold more phosphorylated ATM suggesting that the activation of ATM by chloroquine abrogated the lethal effects of LDR radiation in hematopoietic progenitors. We confirmed that ATM was required for chloroquine-mediated radioprotection by studying ATM null mice. In contrast to wild type mice, chloroquine treatment failed to protect ATM null mice from LDR radiation (9 Gy total) with 8/13 (62%) and 9/13 (69%) of animals surviving in treated or non treated mice, respectively (p=0.86). These data suggest that chloroquine exerts a radioprotective effect from LDR radiation by activating ATM in vivo, and may represent a novel means of limiting acute bone marrow failure in the event of widespread environmental LDR radiation exposure. Disclosures: Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding.

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TGF-β Pathway Inhibition Rescues the Function of Hematopoietic Stem and Progenitor Cells Derived from Patients with Fanconi Anemia

Blood ◽

10.1182/blood.v126.23.297.297 ◽

2015 ◽

Vol 126 (23) ◽

pp. 297-297

Author(s):

Haojian Zhang ◽

David Kozono ◽

Kevin O'Connor ◽

Alix Rousseau ◽

Lisa Moreau ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Failure ◽

Neutralizing Antibody ◽

Genotoxic Stress ◽

Cross Linking ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Pathway Inhibition

Abstract Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life, and frequently require an allogeneic or unrelated donor bone marrow transplant. FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutation in one of eighteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA is attributable to an impaired hematopoietic stem and progenitor cell (HSPC) pool. HSPCs in FA patients and FA mice exhibit reduced cell number and compromised stem cell function. Recent studies suggest that bone marrow failure in FA and impaired HSPC function result from the genotoxicity of endogenous cross-linking agents or from physiological stress. A greater understanding of the mechanisms of impairment of HSPC function could improve the therapeutic options for FA patients. Using a whole genome-wide shRNA screen, we have recently identified that the canonical transforming growth factor-β (TGF-β) pathway plays an important growth suppressive role in FA and targeting this pathway can reduce the genotoxic stress-induced growth inhibition of FA cells. Here, we investigated the possible suppressive function of the TGF-β pathway in HSPCs derived from patients with FA. Methods: We performed in vitro colony-forming assays using primary FA patient- derived bone marrow CD34+ cells which were either transduced with shRNA targeting SMAD3 or treated with the anti-human TGF-β neutralizing antibody GC1008. FA-like HSPCs were generated by stably knocking down FANCD2 with lentivirus encoded shRNA in primary human cord blood CD34+ cells. An in vivo engraftment assay was performed by transplanting the FA-like HSPCs into irradiated NSG mice. Results: The primary human FA bone marrow cells displayed elevated mRNA expression of multiple TGF-β pathway components. The TGF-β pathway inhibition, by knockdown of SMAD3 or anti-human TGF-β neutralizing antibody GC1008, rescued the in vitro clonogenic defects of primary CD34+ cells from bone marrow of five different FA patients. Similarly, the TGF-β pathway disruption by depletion of SMAD3 or GC1008 antibody in primary FA-like HSPCs, also rescued their clonogenic defect, and partially restored genotoxic stress-induced growth inhibition. Further, as the very low number of CD34+ cells in FA patients did not allow efficient xenograft assay to analyze in vivo clonogenicity, we performed a surrogate in vivo xenograft assay using FA-like primary CD34+ cells. Importantly, blockade of the TGF-β pathway by GC1008 antibody treatment enhanced the engraftment potential of primary FA-like CD34+ cells in vivo. Collectively, these results demonstrated that increased TGF-β pathway signaling impairs the hematopoietic function of primary human FA HSPCs. Conclusions: The TGF-β pathway signaling is increased in primary FA patient-derived hematopoietic cells and blockade of this pathway can restore the function of human FA-deficient primary HSPCs. The TGF-β signaling pathway-mediated growth suppression may account, at least in part, for bone marrow failure in FA. This work suggests that the TGF-β signaling pathway provides a novel therapeutic target for the treatment of bone marrow failure in FA. Disclosures No relevant conflicts of interest to declare.

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P53 Downregulation-Based Strategy to Protect Genomic Integrity from Radiation Therapy

Blood ◽

10.1182/blood-2020-139137 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 24-25

Author(s):

Hang Su ◽

Mei-Jun Long ◽

Joel E Michalek ◽

Michael Weil ◽

Chul S Ha

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Gene Deletion ◽

Bone Marrow Cells ◽

Genomic Integrity ◽

Normal Tissues ◽

Radiation Induced ◽

Marrow Cells

Background: Activation of p53 is one of major pathways by which DNA damaging agents (DDA) such as radiation and chemotherapy cause toxicity in normal tissues and it induces a cascade of events that eventually leads to cell senescence or cell death. We have reported that a brief pretreatment with low dose arsenic (LDA), by temporarily and reversibly downregulating p53 at the time of treatment with DDA, reduces the normal tissue toxicity without compromising tumor response to treatment. This protective effect is selective to normal tissues, as it requires functional p53. Though not every cancer cell has detectable p53 mutations, essentially every cancer cell has dysfunctional p53. Therefore most cancer cells will not be protected by this strategy. Genomic instability and inability to repair DNA damage from DDA in the hematopoietic stem cells have been attributed to the development of therapy-induced myelodysplastic syndrome (tMDS) and acute myeloid leukemia (AML). We have also been studying the effect of LDA on the genome in the setting of cancer therapy. We have reported that LDA pretreatment significantly reduces radiation-induced DNA double strand breaks (DSBs) and apoptosis in normal cells both in-vitro and in-vivo. Persistent DNA damage such as DSBs can trigger genomic instability and can be prevented by proper DNA repair. Our previous work using comet assay to quantify DNA damage after radiation has indicated that DNA repair capacity is enhanced by LDA pretreatment. A role for LDA in maintaining genomic integrity has been implicated in our in-vitro studies, where we found that LDA protected telomeres from enhanced erosion by DDA in Concanavalin A-activated normal human lymphocytes, and that LDA reduced spontaneous and radiation-induced mutations in mouse embryonic stem cells. Yet, whether this p53 downregulation-based strategy helps genome maintenance during cancer treatment using DDA has not been investigated in-vivo. CBA/Ca mice have 15-25% incidence of AML after 3 Gy of total body ionizing radiation (IR). About 95% of mice that develop radiation-induced AML (rAML) have a deletion on chromosome 2 encompassing the PU.1 gene. Since PU.1 deletion is a critical contributor to and a useful surrogate marker for leukemogenesis in the murine rAML model, we tested a hypothesis whether pretreatment with LDA before IR helps maintain genomic integrity by evaluating bone marrow cells for PU.1 gene deletion. Method: One hundred twenty mice were randomized into four groups: PBS+sham IR (control), LDA+sham IR, PBS+IR and LDA+IR. Prior to sham or 3 Gy of IR, CBA/Ca mice were injected with either PBS or LDA intraperitoneally at the dose of 0.4mg/kg for 3 days. At 7, 30 and 180 days after radiation, bone marrow cells were collected from femurs and fixed with Carnoy's Fixative. To assess the effect of LDA on PU.1 gene deletion, fluorescence in-situ hybridization (FISH) assay was performed. An ATTO550 labeled PU.1 probe was designed and used to detect deletions that occur in 2qE1 and involve the PU.1 gene locus, as well as two 6-FAM labeled probes for centromere and telomere respectively. Four to five hundred cells were analyzed for each mouse. Statistical significance was determined from a two-way analysis of variance in log units using SAS Version 9.4. Result: We successfully established the FISH assay that can specifically detect the PU.1 gene not only in metaphase cells but also in interphase cells. As shown in the figure, mice in the LDA+IR group have significantly fewer bone marrow cells exhibiting PU.1 gene deletion compared with PBS+IR group at all three time points examined (Day 7: 2±1.2% vs 3.7±2.6%, P=0.047; Day 30: 1.9±1.1% vs 3.2±1.9%, P=0.040; Day 180: 2.8±1.0% vs 5.6±3.5%, P=0.014). LDA treatment alone has a negligible effect on PU.1 loss as compared to the control group. Conclusion: Our result suggests that LDA pretreatment protects genomic integrity following IR treatment in-vivo. As the development of rAML is a multi-step process, the impact of LDA pretreatment on the actual incidence of secondary malignancy needs further validation in animal models. The genome-protective effect of LDA that we have revealed supports its potential use as a strategy to reduce the development of radiation-induced secondary malignances such as MDS and AML. Disclosures Ha: Protectum Oncology: Current Employment, Current equity holder in private company.

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New Bone Marrow Failure Genes: DNAJC21 and ERCC6L2

Blood ◽

10.1182/blood.v130.suppl_1.sci-22.sci-22 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. SCI-22-SCI-22

Author(s):

Inderjeet Dokal

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Nuclear Export ◽

Conflicts Of Interest ◽

Excision Repair ◽

Bone Marrow Failure ◽

Retinal Dystrophy ◽

Ribosomal Subunit ◽

Genotoxic Stress ◽

Biallelic Mutations

A significant number of cases with bone marrow failure present with one or more extra-hematopoietic abnormality. This suggests a constitutional or genetic basis, and yet many of them remain uncharacterized. Through exome sequencing, we have recently identified two sub groups of these cases, one defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21) and the other in ERCC6L2 (excision repair cross complementing 6 like-2). Patients with DNAJC21 mutations are characterized by global bone marrow failure in early childhood. They can also have a variable number of extra-hematopoietic abnormalities such as short stature and retinal dystrophy. The encoded protein associates with ribosomal RNA (rRNA) and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid patient cells exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced levels of rRNA. Characterisation of mutations has revealed impairment in interactions with cofactors (PA2G4, HSPA8 and ZNF622) involved in 60S maturation. DNAJC21 deficiency results in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles and increased cell death. Collectively these findings demonstrate that biallelic mutations in DNAJC21 cause disease due to defects in early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit. Patients harbouring biallelic ERCC6L 2 mutations are characterized by bone marrow failure (in childhood or early adulthood) and one or more extra-hematopoietic abnormality such as microcephaly. Knockdown of ERCC6L2 in human cells significantly reduces their viability upon exposure to the DNA damaging agent irofulven but not etoposide and camptothecin suggesting a role in nucleotide excision repair. ERCC6L2 knockdown cells and patient cells harbouring biallelic ERCC6L2 mutations also display H2AX phosphorylation that significantly increases upon genotoxic stress, suggesting an early DNA damage response. ERCC6L2 is seen to translocate to mitochondria as well as the nucleus in response to DNA damage and its knockdown induces intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger, N-acetyl-cysteine, attenuates the irofulven-induced cytotoxicity in ERCC6L2 knockdown cells and abolishes its traffic to mitochondria and nucleus in response to this DNA damaging agent. Collectively, these observations suggest that ERCC6L2has a pivotal rolein DNA repair and mitochondrial function. In conclusion, ERCC6L2 and DNAJC21 have an important role in maintaining genomic stability and ribosome biogenesis, respectively. They bring into focus new biological connections/pathways whose constitutional disruption is associated with defective hematopoiesis since patients harbouring germline biallelic mutations in these genes uniformly have bone marrow failure. Disclosures No relevant conflicts of interest to declare.

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Acceleration of hematopoietic reconstitution with a synthetic cytokine (SC-55494) after radiation-induced bone marrow aplasia

Blood ◽

10.1182/blood.v87.2.581.bloodjournal872581 ◽

1996 ◽

Vol 87 (2) ◽

pp. 581-591 ◽

Cited By ~ 38

Author(s):

AM Farese ◽

F Herodin ◽

JP McKearn ◽

C Baum ◽

E Burton ◽

...

Keyword(s):

Bone Marrow ◽

Pharmacokinetic Analysis ◽

Pharmacokinetic Parameters ◽

Platelet Recovery ◽

Hematopoietic Reconstitution ◽

Radiation Induced ◽

Complete Blood Counts ◽

60Co Gamma Radiation

The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3, while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1, cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] 500/microL) and thrombocytopenia (THROM; platelet count 20,000/microL) were assessed. Synthokine significantly (P .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered, the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine, SC55 94, administered therapeutically post-TBI, significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host.

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Increases in circulating megakaryocyte growth-promoting activity in the plasma of rats following whole body irradiation

Blood ◽

10.1182/blood.v63.5.1060.1060 ◽

1984 ◽

Vol 63 (5) ◽

pp. 1060-1066 ◽

Cited By ~ 15

Author(s):

M Miura ◽

CW Jackson ◽

SA Lyles

Keyword(s):

Bone Marrow ◽

Plasma Concentration ◽

Bone Marrow Cells ◽

Single Cells ◽

Rat Plasma ◽

Whole Body ◽

Growth Promoting ◽

Marrow Cells

Abstract To gain insight into the regulation of megakaryocyte precursors in vivo, we assayed (in vitro) megakaryocyte growth-promoting activity (Meg-GPA) in plasma of rats in which both marrow hypoplasia and thrombocytopenia had been induced by irradiation. Rats received whole body irradiation of 834 rad from a 137Cs source. Plasma was collected at intervals of hours to days, up through day 21 postirradiation, and was tested, at a concentration of 30%, for Meg-GPA on bone marrow cells cultured in 1.1% methylcellulose with 5 X 10(-5) M 2-mercaptoethanol. With normal rat plasma, no megakaryocyte colonies (defined as greater than or equal to 4 megakaryocytes) were seen and only a few single megakaryocytes and clusters (defined as 2 or 3 megakaryocytes) were formed. Two peaks of plasma Meg-GPA were observed after irradiation. The first appeared at 12 hr, before any decrease in marrow megakaryocyte concentration or platelet count. The second occurred on days 10–14 after irradiation, after the nadir in megakaryocyte concentration and while platelet counts were at their lowest levels. A dose-response study of plasma concentration and megakaryocyte growth, using plasma collected 11 days postirradiation, demonstrated that patterns of megakaryocyte growth were related to plasma concentration; formation of single megakaryocytes was optimal over a range of 20%-30% plasma concentration, while cluster and colony formation were optimal at a plasma concentration of 30%. All forms of megakaryocyte growth were decreased with 40% plasma. There was a linear relationship between the number of bone marrow cells plated and growth of single cells, clusters, and colonies using a concentration of 30% plasma collected 11 days after irradiation. We conclude that irradiation causes time- related increases in circulating megakaryocyte growth-promoting activity. We suggest that the irradiated rat is a good model for studying the relationships between Meg-GPA and megakaryocyte and platelet concentration in vivo.

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Direct Kinase-to-Kinase Signaling Mediated by the FHA Phosphoprotein Recognition Domain of the Dun1 DNA Damage Checkpoint Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.23.4.1441-1452.2003 ◽

2003 ◽

Vol 23 (4) ◽

pp. 1441-1452 ◽

Cited By ~ 62

Author(s):

Vladimir I. Bashkirov ◽

Elena V. Bashkirova ◽

Edwin Haghnazari ◽

Wolf-Dietrich Heyer

Keyword(s):

Dna Damage ◽

Target Genes ◽

Genotoxic Stress ◽

Dna Damage Checkpoint ◽

Kinase Signaling ◽

M Cell ◽

Fha Domain ◽

Checkpoint Pathway

ABSTRACT The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. It belongs to the Chk2 family of checkpoint kinases, which includes S. cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2. Dun1 is required for DNA damage-induced transcription of certain target genes, transient G2/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55. Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G2/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.

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Xeroderma Pigmentosum as a Model for Treatment of Bone Marrow Failure in Fanconi Anemia and Aplastic Anemia.

Blood ◽

10.1182/blood.v104.11.4235.4235 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4235-4235

Author(s):

W. Clark Lambert ◽

Santiago A. Centurion

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Fanconi Anemia ◽

Xeroderma Pigmentosum ◽

Bone Marrow Failure ◽

S Phase ◽

White Female ◽

Cross Linking

Abstract We have previously shown that the primary cell cycle defect in the inherited, cancer-prone, bone marrow failure associated disease, Fanconi anemia (FA), is not in the G2 phase of the cell cycle, as had been thought for many years, but rather in the S phase. FA cells challenged with the DNA cross-linking agent, psoralen coupled with long wavelength, ultraviolet (UVA) radiation (PUVA), fail to slow their progression through the S phase of the subsequent cell cycle, as do normal cells. FA cells are extremely sensitive to the cytotoxic and clastogenic effects of DNA cross-linkers, such as PUVA, so much so that the diagnosis of FA is based on an assay, the “DEB test”, in which cells are examined for clastogenic and cytotoxic effects of diepoxybutane (DEB), a DNA cross-linking agent. More recently, we have shown that artificially slowing the cell cycle of FA cells exposed to PUVA by subsequent treatment with agents which slow their progression through S phase leads to markedly increased viability and reduced chromosome breakage in vitro. We now show that similar results can be obtained in vivo in patients with another DNA repair deficiency disease, xeroderma pigmentosum (XP), a recessively inherited disorder associated with defective repair of sunlight induced adducts in the DNA of sun-exposed tissues followed by development of numerous mutations causing large numbers of cancers in these same tissues. We treated two patients with XP, a light complected black male and a white female, both 14 years of age, in sun-exposed areas with 5-fluorouracil, an inhibitor of DNA synthesis, daily for three months. In contrast to normal patients, who only show clinical results if an inflammatory response is invoked, marked improvement in the clinical appearance of the skin was seen with no inflammation observed. This effect was confirmed histologically by examining epidermis adjacent to excised lesions in sun-exposed areas and further verified by computerized image analysis. Treatment with agents that slow progression through S phase, such as hydroxyurea, may similarly improve clinical outcomes in patients with FA or others who are developing bone marrow failure.

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Hsp90 regulates the Fanconi anemia DNA damage response pathway

Blood ◽

10.1182/blood-2006-08-038638 ◽

2007 ◽

Vol 109 (11) ◽

pp. 5016-5026 ◽

Cited By ~ 46

Author(s):

Tsukasa Oda ◽

Toshiya Hayano ◽

Hidenobu Miyaso ◽

Nobuhiro Takahashi ◽

Takayuki Yamashita

Keyword(s):

Dna Damage ◽

Fanconi Anemia ◽

Heat Shock Protein 90 ◽

Genotoxic Stress ◽

Cross Linker ◽

Cellular Tolerance ◽

Underlying Mechanisms ◽

Dna Damage Response Pathway

Abstract Heat shock protein 90 (Hsp90) regulates diverse signaling pathways. Emerging evidence suggests that Hsp90 inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), enhance DNA damage-induced cell death, suggesting that Hsp90 may regulate cellular responses to genotoxic stress. However, the underlying mechanisms are poorly understood. Here, we show that the Fanconi anemia (FA) pathway is involved in the Hsp90-mediated regulation of genotoxic stress response. In the FA pathway, assembly of 8 FA proteins including FANCA into a nuclear multiprotein complex, and the complex-dependent activation of FANCD2 are critical events for cellular tolerance against DNA cross-linkers. Hsp90 associates with FANCA, in vivo and in vitro, in a 17-AAG–sensitive manner. Disruption of the FANCA/Hsp90 association by cellular treatment with 17-AAG induces rapid proteasomal degradation and cytoplasmic relocalization of FANCA, leading to impaired activation of FANCD2. Furthermore, 17-AAG promotes DNA cross-linker–induced cytotoxicity, but this effect is much less pronounced in FA pathway-defective cells. Notably, 17-AAG enhances DNA cross-linker–induced chromosome aberrations. In conclusion, our results identify FANCA as a novel client of Hsp90, suggesting that Hsp90 promotes activation of the FA pathway through regulation of intracellular turnover and trafficking of FANCA, which is critical for cellular tolerance against genotoxic stress.

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