Kazuhito Naka

Foxo3a Is Essential for Maintenance of the Hematopoietic Stem Cell Pool

Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone marrow niche. Here we show that Foxo3a, a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway, is critical for HSC self-renewal. We generated gene-targeted Foxo3a(-/-) mice and showed that, although the proliferation and differentiation of Foxo3a(-/-) hematopoietic progenitors were normal, the number of colony-forming cells present in long-term cocultures of Foxo3a(-/-) bone marrow cells and stromal cells was reduced. The ability of Foxo3a(-/-) HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired. Foxo3a(-/-) HSCs also showed increased phosphorylation of p38MAPK, an elevation of ROS, defective maintenance of quiescence, and heightened sensitivity to cell-cycle-specific myelotoxic injury. Finally, HSC frequencies were significantly decreased in aged Foxo3a(-/-) mice compared to the littermate controls. Our results demonstrate that Foxo3a plays a pivotal role in maintaining the HSC pool.

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.

TGF-β–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-β is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-β inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-β inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-β–FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo.

Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription

The mammalian Chk2 kinase is thought to mediate ATM‐dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2‐deficient mice. Although Chk2−/− mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR‐induced apoptosis. The IR‐induced G1/S cell cycle checkpoint, but not the G2/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2−/− mice. IR‐induced stabilization of p53 in Chk2−/− cells was 50–70% of that in wild‐type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR‐dependent but Chk2‐independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53‐dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2−/− cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.

Different anti‐HCV profiles of statins and their potential for combination therapy with interferon

We recently developed a genome‐length hepatitis C virus (HCV) RNA replication system (OR6) with luciferase as a reporter. The OR6 assay system has enabled prompt and precise quantification of HCV RNA replication. Pegylated interferon (IFN) and ribavirin combination therapy is the world standard for chronic hepatitis C, but its effectiveness is limited to about 55% of patients. Newer therapeutic approaches are needed. In the present study, we used the OR6 assay system to evaluate the anti‐HCV activity of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors, called statins, and their effects in combination with IFN‐α. Five types of statins (atorvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin) were examined for their anti‐HCV activities. Fluvastatin exhibited the strongest anti‐HCV activity (IC50: 0.9 μmol/L), whereas atorvastatin and simvastatin showed moderate inhibitory effects. However, lovastatin, reported recently as an inhibitor of HCV replication, was shown to exhibit the weakest anti‐HCV activity. The anti‐HCV activities of statins were reversed by the addition of mevalonate or geranylgeraniol. Surprisingly, however, pravastatin exhibited no anti‐HCV activity, although it worked as an inhibitor for HMG‐CoA reductase. The combination of IFN and the statins (except for pravastatin) exhibited strong inhibitory effects on HCV RNA replication. In combination with IFN, fluvastatin also exhibited a synergistic inhibitory effect. In conclusion, statins, especially fluvastatin, could be potentially useful as new anti‐HCV reagents in combination with IFN. (HEPATOLOGY 2006;44:117–125.)

Activated macrophages promote Wnt signalling through tumour necrosis factor-α in gastric tumour cells

The activation of Wnt/β‐catenin signalling has an important function in gastrointestinal tumorigenesis. It has been suggested that the promotion of Wnt/β‐catenin activity beyond the threshold is important for carcinogenesis. We herein investigated the role of macrophages in the promotion of Wnt/β‐catenin activity in gastric tumorigenesis. We found β‐catenin nuclear accumulation in macrophage‐infiltrated dysplastic mucosa of the K19‐Wnt1 mouse stomach. Moreover, macrophage depletion in ApcΔ716 mice resulted in the suppression of intestinal tumorigenesis. These results suggested the role of macrophages in the activation of Wnt/β‐catenin signalling, which thus leads to tumour development. Importantly, the conditioned medium of activated macrophages promoted Wnt/β‐catenin signalling in gastric cancer cells, which was suppressed by the inhibition of tumour necrosis factor (TNF)‐α. Furthermore, treatment with TNF‐α induced glycogen synthase kinase 3β (GSK3β) phosphorylation, which resulted in the stabilization of β‐catenin. We also found that Helicobacter infection in the K19‐Wnt1 mouse stomach caused mucosal macrophage infiltration and nuclear β‐catenin accumulation. These results suggest that macrophage‐derived TNF‐α promotes Wnt/β‐catenin signalling through inhibition of GSK3β, which may contribute to tumour development in the gastric mucosa.

Regulation of Reactive Oxygen Species and Genomic Stability in Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are defined by their ability both to self-renew and to give rise to fresh blood cells throughout the lifetime of an animal. The failure of HSCs to self-renew during aging is believed to depend on several intrinsic (cell-autonomous) and extrinsic (non-cell-autonomous) factors. In this review, we focus on how dysregulation of reactive oxygen species (ROS) and disruptions of genomic stability can impair HSC functions. Recently, it was shown that long-term self-renewing HSCs normally possess low levels of intracellular ROS. However, when intracellular ROS levels become excessive, they cause senescence or apoptosis, resulting in a failure of HSC self-renewal. Repression of intracellular ROS levels in HSCs by treatment with an antioxidant that scavenges ROS can rescue HSC functions, indicating that excess ROS levels are at the root of HSC failure. Products of numerous genes that are involved in either DNA-damage responses or longevity-related signaling contribute to the maintenance of the HSC self-renewal capacity. Further investigations on the molecular mechanisms of ROS regulation and on the manipulation of excess ROS levels could lead to the development of novel therapeutics for hematopoietic diseases, regenerative medicine, and the prevention of leukemia.

Effect of trichostatin A on cell growth and expression of cell cycle- and apoptosis-related molecules in human gastric and oral carcinoma cell lines

The effect of trichostatin A (TSA), histone deacetylase inhibitor, on cell growth and the mechanism of growth modulation was examined in 8 gastric and 3 oral carcinoma cell lines which included 9‐cis‐retinoic acid resistant (MKN‐7 and Ho‐1‐N‐1) and IFN‐β resistant cell lines (MKN‐7, ‐28 and ‐45). TSA inhibited growth in all cell lines examined. Apoptotic cell death was confirmed by apoptotic ladder formation and induction of a cleaved form (85 kDa) of poly (ADP‐ribose) polymerase (PARP) induction. TSA enhanced the protein expression of p21WAF1, CREB‐binding protein, cyclinE, cyclin A, Bak and Bax, while it reduced the expression of E2F‐1, E2F‐4, HDAC1, p53 and hyperphosphorylated form of Rb. Furthermore, TSA induced morphological changes, such as elongation of cytoplasm and cell‐to‐cell detachment, in gastric and oral carcinoma cell lines. These results suggest that TSA may inhibit cell growth and induce apoptosis of gastric and oral carcinoma cells through modulation of the expression of cell cycle regulators and apoptosis‐regulating proteins. Int. J. Cancer 88:992–997, 2000.

Regulation of Reactive Oxygen Species by Atm Is Essential for Proper Response to DNA Double-Strand Breaks in Lymphocytes

The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm−/− mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm−/− mice. Reduction of ROS by the antioxidant N-acetyl-l-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm−/− mouse embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm−/− mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm−/− mice. Impairment of Ig class switch recombination seen in Atm−/− mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm−/− mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis.

Expression of POT1 is Associated with Tumor Stage and Telomere Length in Gastric Carcinoma

Pot1, a telomere end-binding protein in fission yeast and human, is proposed not only to cap telomeres but also to recruit telomerase to the ends of chromosomes. No study has been performed regarding Pot1 expression status in human cancers. Thus, we examined POT1 mRNA expression in 51 gastric cancer (GC) tissues and evaluated telomere length and 3′ telomeric overhang signals in 20 of the 51 GC tissues. Quantitative reverse transcription-PCR analysis showed that POT1 expression levels in the tumor relative to those in nonneoplastic mucosa (T/N ratio) were significantly higher in stage III/IV tumors than in stage I/II tumors (P = 0.005). Down-regulation of POT1 (T/n < 0.5) was observed more frequently in stage I/II GC (52.4%, 11 of 21) than in stage III/IV GC (23.3%, 7 of 30; P = 0.033), whereas up-regulation of POT1 (T/n > 2.0) was observed more frequently in stage III/IV GC (33.3%, 10 of 30) than in stage I/II GC (9.5%, 2 of 21; P = 0.048). POT1 expression levels showed decreased in accordance with telomere shortening (r = 0.713, P = 0.002). In-gel hybridization analysis showed that 3′ telomeric overhang signals decreased in accordance with decreases in POT1 expression levels (r = 0.696, P = 0.002) and telomere shortening (r = 0.570, P = 0.013). Reduced POT1 expression was observed in GC cell lines with telomeres shortened by treatment with azidothymidine. In addition, inhibition of Pot1 by antisense oligonucleotides led to telomere shortening as well as inhibition of telomerase activity in GC cells. Moreover, inhibition of Pot1 decreased 3′ overhang signals and increased the frequency of anaphase bridge (P = 0.0005). These data suggest that Pot1 may play an important role in regulation of telomere length and that inhibition of Pot1 may induce telomere dysfunction. Moreover, changes in POT1 expression levels may be associated with stomach carcinogenesis and GC progression.