Rishu Takimoto

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.

Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site.

KILLER/DR5, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor gene, has been shown to be induced by DNA damaging agents and radiation in a p53-dependent manner. Although TRAIL is a potential therapeutic agent for cancer, the induction mechanism of its receptors is poorly understood. Here we show the identification of three p53 DNA-binding sites in the KILLER/DR5 genomic locus located upstream (BS1; −0.82 Kb) of the ATG site, within Intron 1 (BS2; +0.25 Kb downstream of the ATG) and within Intron 2 (BS3; +1.25 Kb downstream of the ATG). A modified p53-binding and immunoselection protocol using a wild-type p53-expressing adenovirus vector (Ad-p53) was used to identify the binding sites and to show that each binding site can bind specifically to wild-type p53 protein (wt-p53). A reporter assay revealed that only BS2 could enhance luciferase expression driven by a basal promoter. We constructed a reporter plasmid carrying the genomic regulatory region of KILLER/DR5 including the three p53 DNA-binding sites but no additional basal promoter. The genomic fragment showed basal transcriptional activity which was induced by wt-p53 but not by mutant p53, and human papilloma virus E6 inhibited the p53-dependent activation. Mutation of BS2 abrogated not only the binding activity of wt-p53 but also the induction of the KILLER/DR5 genomic promoter-reporter gene, indicating that BS2 is responsible for the p53-dependent transactivation of KILLER/DR5. In p53-wild-type but not -mutant or -null cell lines, doxorubicin treatment stabilized p53 protein, and increased specific binding to BS2 as revealed by EMSA, and upregulated the KILLER/DR5 promoter-luciferase reporter gene. These results suggest that the transactivation of KILLER/DR5 is directly regulated by exogenous or endogenous wt-p53 and establishes KILLER/DR5 as a p53 target gene that can signal apoptotic death.

The Bile Acid Glycochenodeoxycholate Induces TRAIL-Receptor 2/DR5 Expression and Apoptosis

Toxic bile salts induce hepatocyte apoptosis by both Fas-dependent and -independent mechanisms. In this study, we examined the cellular mechanisms responsible for Fas-independent, bile acid-mediated apoptosis. HuH-7 cells, which are known to be Fas deficient, were stably transfected with the sodium-dependent bile acid transporting polypeptide. The toxic bile acid glycochenodeoxycholate (GCDC)-induced apoptosis in these cells in a time- and concentration-dependent manner. Apoptosis and mitochondrial cytochrome c release were inhibited by transfection with dominant negative FADD, CrmA transfection, or treatment with the selective caspase 8 inhibitor IETD-CHO. These observations suggested the Fas-independent apoptosis was also death receptor mediated. Reverse transcriptase-polymerase chain reaction demonstrated tumor necrosis factor-R1, tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-R1/DR4, -R2/DR5, and TRAIL, but not tumor necrosis fator-α expression by these cells. GCDC treatment increased expression of TRAIL-R2/DR5 mRNA and protein 10-fold while expression of TRAIL-R1 was unchanged. Furthermore, aggregation of TRAIL-R2/DR5, but not TRAIL-R1/DR4 was observed following GCDC treatment of the cells. Induction of TRAIL-R2/DR5 expression and apoptosis by bile acids provides new insights into the mechanisms of hepatocyte apoptosis and the regulation of TRAIL-R2/DR5 expression.

BRCA1 Directs a Selective p53-Dependent Transcriptional Response towards Growth Arrest and DNA Repair Targets

ABSTRACT The pathway leading to BRCA1-dependent tumor suppression is not yet clear but appears to involve activities in DNA repair as well as gene transcription. Moreover, it has been shown that BRCA1 can regulate p53-dependent transcription. Because BRCA1 overexpression stabilizes wild-type p53 but does not lead to apoptosis of most cell lines, we investigated the selectivity of BRCA1 for p53-dependent target gene activation. We find that BRCA1-stabilized p53 regulates transcription of DNA repair and growth arrest genes while p53 stabilized by DNA-damaging agents induces a wide array of genes, including those involved in apoptosis. This differential expression profile was reflected in the treatment outcome—apoptosis following DNA damage and growth arrest after expression of BRCA1. Depletion of BRCA1 in wild-type-p53-expressing cells abolished the induction of such repair genes as p53R2, while the expression of PIG3, an apoptosis-inducing gene, was still induced. BRCA1 also conferred diminished cell death in a p53-dependent manner in response to adriamycin compared to that conferred by controls. These results suggest that BRCA1 selectively coactivates the p53 transcription factor towards genes that direct DNA repair and cell cycle arrest but not towards those that direct apoptosis.

Stabilization of p53 by CP-31398 inhibits ubiquitination without altering phosphorylation at serine 15 or 20 or MDM2 binding.

ABSTRACT CP-31398, a styrylquinazoline, emerged from a high throughput screen for therapeutic agents that restore a wild-type-associated epitope (monoclonal antibody 1620) on the DNA-binding domain of the p53 protein. We found that CP-31398 can not only restore p53 function in mutant p53-expressing cells but also significantly increase the protein level and promote the activity of wild-type p53 in multiple human cell lines, including ATM-null cells. Cells treated with CP-31398 undergo either cell cycle arrest or apoptosis. Further investigation showed that CP-31398 blocks the ubiquitination and degradation of p53 but not in human papillomavirus E6-expressing cells. Of note, CP-31398 does not block the physical association between p53 and MDM2 in vivo. Moreover, unlike the DNA-damaging agent adriamycin, which induces strong phosphorylation of p53 on serines 15 and 20, CP-31398 exposure leads to no measurable phosphorylation on these sites. We found that CP-31398 could also stabilize exogenous p53 in p53 mutant, wild-type, and p53-null human cells, even in MDM2-null p53−/− mouse embryonic fibroblasts. Our results suggest a model wherein CP-31398-mediated stabilization of p53 may result from reduced ubiquitination, leading to high levels of transcriptionally active p53. Further understanding of this mechanism may lead to novel strategies for p53 stabilization and tumor suppression in cancers, even those with absent ARF or high MDM2 expression.

The mutant p53-conformation modifying drug, CP-31398, can induce apoptosis of human cancer cells and can stabilize wild-type p53 protein.

CP-31398, a styrylquinazoline, emerged from a screen for therapeutic agents that restore a wild-type DNA-binding conformation of mutant p53 to suppress tumors in-vivo (Science 286, 2507, 1999). We investigated the growth inhibitory mechanism of CP-31398 using nine human cancer cell lines containing wild-type, mutant or no p53 expression. Six of nine cell lines underwent apoptosis after exposure to CP-31398, while two cell lines, DLD1 colon cancer and H460 lung cancer, underwent exclusively cell cycle arrest. Cell cycle arrest preceded the apoptosis in some cases. CP-31398 did not inhibit growth of the p53 non-expressing ovarian cancer cell line SKOV3. Interestingly, we found that wild-type p53 protein is stabilized upon CP-31398 exposure. p53 target genes such as p21WAF1/Cip1, and KILLER/DR5 were upregulated by CP-31398, but their expression did not correlate with arrest or apoptosis induction. Combination of CP-31398 and TRAIL or chemotherapeutic agents enhanced cancer cell killing effect possibly through upregulation of p53-regulated genes such as KILLER/DR5. Bax-/-, wild-type p53-expressing cells displayed reduced susceptibility to killing by CP-31398. An Affymetrix GeneChip Array screen revealed that CP-31398 alters expression of non-p53 target genes in addition to p53-responsive genes. Although our preliminary data suggest that CP-31398 does not alter wild-type p53:MDM2 interaction, further efforts are required to elucidate the mechanism of wild-type p53 stabilization by CP-31398. The results increase our understanding of CP-31398 action, and suggest strategies for improving its specificity, possibly through use of microarrays to screen related compounds with higher mutant p53-specificity.

Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells.

OBJECTIVE To compare the hematopoietic support provided by telomerized human mesenchymal stem cells (MSCs) and telomerized MSC-derived stromal cells. METHODS We transfected the human telomerase catalytic subunit (hTERT) gene into primary MSCs to establish hTERT-transduced MSCs (hTERT-MSCs). Stromal induction of hTERT-MSCs was performed by replacing the culture medium with Dexter-type culture medium. Hematopoietic support was examined by coculture with cord blood CD34(+) cells. RESULTS The hTERT-MSCs were morphologically identical with the primary MSCs and expressed surface antigens including CD105, CD73, and CD166. hTERT-MSCs showed a similar doubling time as primary MSCs and continued to proliferate to over 80 population doublings (PD), although the primary MSCs underwent crisis in vitro at 16 PD. The osteogenic, chondrogenic, adipogenic, neurogenic, and stromal differentiation potential of hTERT-MSCs were maintained up to at least 40 PD. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-C) upon 12-day coculture with the hTERT-MSC-derived stromal cells were nearly the same as those upon 12-day coculture with hTERT-MSCs (CD34, 33.0-fold+/-2.8-fold vs 36.1-fold+/-1.7-fold of the initial cell number; CFUs, 344.4-fold+/-62.5-fold vs 239.3-fold+/-87.0-fold; CFU-mix, 368.4-fold+/-113.7-fold vs 341.3-fold+/-234.3-fold). However, on day 18 of coculture, the number of cobblestone areas (CA) observed beneath the stromal cells was 15 times higher than that beneath hTERT-MSCs (CA, 146.9+/-54.6 vs 9.4+/-8.1, p<0.01). CONCLUSION Stromal induction of hTERT-MSCs exclusively enhanced the support of CA formation provided by hTERT-MSCs. Our human hTERT-MSCs will be useful for elucidating the mechanism of the formation of CAs.

hADA3 is required for p53 activity

The tumor suppressor protein p53 is a transcription factor that is frequently mutated in human cancers. In response to DNA damage, p53 protein is stabilized and activated by post‐translational modifications that enable it to induce either apoptosis or cell cycle arrest. Using a novel yeast p53 dissociator assay, we identify hADA3, a part of histone acetyltransferase complexes, as an important cofactor for p53 activity. p53 and hADA3 physically interact in human cells. This interaction is enhanced dramatically after DNA damage due to phosphorylation event(s) in the p53 N‐terminus. Proper hADA3 function is essential for full transcriptional activity of p53 and p53‐mediated apoptosis.

Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells

Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-l-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3.

BRCA1 Transcriptionally Regulates Damaged DNA Binding Protein (DDB2) In the DNA Repair Response Following UV-Irradiation

The p53 and BRCA1 tumor suppressors are involved in repair processes and may cooperate to transactivate certain genes, including p21WAF1/CIP1 and GADD45. We find that the Xeroderma Pigmentosum Complementation group E (XPE) mutated Damaged-DNA binding protein p48 (DDB2) is upregulated by BRCA1 in a p53-dependent manner following UVC, Adriamycin, or Cisplatin exposure. BRCA1 enhances p53 binding to the DDB2 promoter in vivo as well as p53-dependent transactivation of DDB2 promoter-reporter constructs through a classical p53 DNA responsive element. Antisense abrogation of BRCA1 expression abrogates upregulation of DDB2 after UVC or cisplatin exposure. Using a host cell reactivation assay, DNA repair activity is more significantly restored by introduction of BRCA1 into wt as compared to DDB2-deficient cells. Furthermore disappearance of the photoproducts cyclobutane pyrimidine dimer (CPD) and 6-4 photoproduct (6-4PP) was delayed by antisense abrogation of BRCA1 expression in UV-exposed human cells. Thus the DNA repair function of BRCA1 may be attributed in part to p53-dependent transcriptional induction of DDB2. Loss of BRCA1-dependent DDB2 repair function may contribute to cancer susceptibility and cellular sensitivity to DNA damage.