Sphingosine kinase 1 expression is downregulated during differentiation of Friend cells due to decreased c-MYB.

Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between -53bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.

A prospective dose-finding trial using a modified continual reassessment method for optimization of fludarabine plus melphalan conditioning for marrow transplantation from unrelated donors in patients with hematopoietic malignancies.

BACKGROUND: Because of the less graft-facilitating effect by bone marrow (BM), we need to assess a dosage of conditioning more accurately particularly in combination with reduced-intensity conditioning. Thus we examined that modified continual reassessment method (mCRM) is applicable for deciding appropriate conditioning of allogeneic BM transplantation. PATIENTS AND METHODS: The conditioning regimen consisted of i.v. fludarabine (125 mg/m2) plus an examination dose of i.v. melphalan. The primary endpoint was a donor-type T-cell chimerism at day 28 with successful engraftment defined as >90% donor cells. Five patients per dose level were planned to be accrued and chimerism data were used to determine the next dose. RESULTS: Seventeen patients were enrolled at doses between 130 and 160 mg/m2. The dose was changed from 160 to 130 mg/m(2) (second level) after five full-donor chimerisms. With one patient of 0% chimera in the second level, the dose was increased to 135 mg/m2 (third level). Following five full-donor chimerisms in the third level, the study was complete as projected. CONCLUSIONS: mCRM was shown to be a relevant method for dose-finding of conditioning regimen. The melphalan dose of 135 mg/m2 was determined as the recommended phase II dose to induce initial full-donor chimerism.

p27 deregulation by Skp2 overexpression induced by the JAK2V617 mutation.

Janus kinase 2 (JAK2) V617F mutation has been regarded as the major cause of myeloproliferative disorders (MPD). However, the mechanisms of abnormal cell growth by JAK2V617F have not been elucidated. In this study, cell cycle regulatory protein expression was analyzed using JAK2V617F-Ba/F3 and mock-Ba/F3. JAK2V617F-Ba/F3, but not mock-Ba/F3, showed IL-3 independent cell growth and constitutive STATs activation. Deregulation of p27(Kip1), the cell cycle regulator at the G1 to S transition, was observed in JAK2V617F-Ba/F3 but not in mock-control. p27(Kip1) deregulation was not due to p27(Kip1) mRNA level but due to high Skp2 expression, a subunit of ubiquitin E3 ligase, through the STAT binding in the Skp2 promoter. Like JAK2V617F overexpression, constitutively active STAT5 or STAT3 induced aberrant p27(Kip1) expression of Ba/F3 cells. Similar findings were observed in BCR/ABL-transfected Ba/F3. Our results elucidate the regulatory mechanism by which JAK2V617F modulates Skp2 gene expression through the STAT transcription factors.

HDAC2 Regulates Site-Specific Acetylation of MDM2 and Its Ubiquitination Signaling in Tumor Suppression.

Histone deacetylases (HDACs) are promising targets for cancer therapy, although their individual actions remain incompletely understood. Here, we identify a role for HDAC2 in the regulation of MDM2 acetylation at previously uncharacterized lysines. Upon inactivation of HDAC2, this acetylation creates a structural signal in the lysine-rich domain of MDM2 to prevent the recognition and degradation of its downstream substrate, MCL-1 ubiquitin ligase E3 (MULE). This mechanism further reveals a therapeutic connection between the MULE ubiquitin ligase function and tumor suppression. Specifically, we show that HDAC inhibitor treatment promotes the accumulation of MULE, which diminishes the t(X; 18) translocation-associated synovial sarcomagenesis by directly targeting the fusion product SS18-SSX for degradation. These results uncover a new HDAC2-dependent pathway that integrates reversible acetylation signaling to the anticancer ubiquitin response.

Clinical characteristics of chronic graft-versus-host disease following umbilical cord blood transplantation for adults.

Chronic GVHD is a significant complication following allogeneic hematopoietic stem cell transplantation; however, the clinical characteristics of chronic GVHD following cord blood transplantation (CBT) in adults have not been well described. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers of the Nagoya Blood and Marrow Transplantation Group. Of 77 patients, 29 survived without graft failure or progression of underlying diseases for at least 100 days after transplantation. The median age of the 29 patients was 42 years (range, 18-67 years). Seven patients developed chronic GVHD (extensive, n=4; limited, n=3) disease. The cumulative incidence of chronic GVHD 1 year after day 100 was 24% (95% confidence interval (CI), 11-41%), and the organs involved were the skin (n=6), oral cavity (n=4), liver (n=1) and gastrointestinal tract (n=1). In three patients, chronic GVHD was resolved with supportive care. The remaining four were successfully treated with additional immunosuppressive therapy. Event-free survival rates of the 29 patients with and without chronic GVHD 3 years after day 100 were 83 (95% CI, 27-97%) and 36% (95% CI, 17-56%), respectively (P=0.047). These results suggest that chronic GVHD following CBT is mild and has a graft-versus-malignancy effect.

L1503R is a member of group I mutation and has dominant-negative effect on secretion of full-length VWF multimers: an analysis of two patients with type 2A von Willebrand disease.

Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF); this in turn is associated with an absence of high-molecular-weight multimers. Sequence analysis of the VWF gene from two unrelated type 2A VWD patients showed an identical, novel, heterozygous T-->G transversion at nucleotide 4508, resulting in the substitution of L1503R in the VWF A2 domain. This substitution, which was not found in 60 unrelated normal individuals, was introduced into a full-length VWF cDNA and subsequently expressed in 293T cells. Only trace amount of the mutant VWF protein was secreted but most of the same was retained in 293T cells. Co-transfection experiment of both wild-type and mutant plasmids indicated the dominant-negative mechanism of disease development; as more of mutant DNA was transfected, VWF secretion was impaired in the media, whereas more of VWF was stored in the cell lysates. Molecular dynamic simulations of structural changes induced by L1503R indicated that the mean value of all-atom root-mean-squared-deviation was shifted from those with wild type or another mutation L1503Q that has been reported to be a group II mutation, which is susceptible to ADAMTS13 proteolysis. Protein instability of L1503R may be responsible for its intracellular retention and perhaps the larger VWF multimers, containing more mutant VWF subunits, are likely to be mal-processed and retained within the cell.

Skewed X chromosome inactivation in fraternal female twins results in moderately severe and mild haemophilia B.

Female carriers of haemophilia B are usually asymptomatic; however, the disease resulting from different pathophysiological mechanisms has rarely been documented in females. In this study, we investigated the mechanisms responsible for haemophilia B in fraternal female twins. We sequenced the factor IX gene (F9) of the propositus, her father, a severe haemophilia B patient and the other family members. X chromosome inactivation was assessed by the methylation-sensitive HpaII-PCR assay using X-linked polymorphisms in human phosphoglycerate kinase 1 gene (PGK1) and glutamate receptor ionotropic AMPA 3 gene (GRIA3). The twins were found to be heterozygotes with a nonsense mutation (p.Arg384X) inherited from their father. The propositus, more severely affected twin, exhibited a significantly higher percentage of inactivation in the maternally derived X chromosome carrying a normal F9. The other twin also showed a skewed maternal X inactivation, resulting in a patient with mild haemophilia B. Thus, the degree of skewing of maternal X inactivation is closely correlated with the coagulation parameters and the clinical phenotypes of the twins. Furthermore, we identified a crossing-over in the Xq25-26 region of the maternal X chromosome of the more severely affected twin. This crossing-over was absent in the other twin, consistent with their fraternal state. Differently skewed X inactivation in the fraternal female twins might cause moderately severe and mild haemophilia B phenotypes, respectively.

Lyn is an important component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.

Fms-like tyrosine kinase 3 (FLT3) is expressed in hematopoietic progenitor cells. An internal tandem duplication (ITD) of FLT3 (FLT3/ITD) is the most frequent mutation in human adult acute myeloid leukemia (AML). FLT3/ITD contributes to the constitutive activation of FLT3 itself and its downstream signal components, mitogen-activated protein kinase and signal transducers and activators of transcription 5 (STAT5), and enables interleukin (IL)-3-dependent cell lines to grow autonomously. In the present study, we showed the specific association of FLT3/ITD with Lyn, which led to the phosphorylation of Lyn in vivo. We also demonstrated that FLT3/ITD receptors displayed a higher affinity to bind to Lyn than wild-type FLT3 receptors in vitro and that this affinity was relative to the intensity of tyrosil phosphorylation of the receptor. Both treatment with small interfering RNA (siRNA) targeting Lyn and the Src family kinase inhibitor PP2 suppressed the IL-3-independent growth of FLT3/ITD-expressing 32D cells (FLT3/ITD-32D), reducing the constitutive phosphorylation of Lyn and STAT5. PP2 treatment of mice transplanted with FLT3/ITD-32D cells blocked the onset of tumors and decreased the size of established tumors. These results demonstrate that Lyn is an important component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.

Homing, proliferation and survival sites of human leukemia cells in vivo in immunodeficient mice.

The cellular components of the hematopoietic stem cell niche have been gradually identified. However, the niche for malignant hematopoiesis remains to be elucidated. Here, using human leukemia cells, which could be transplanted to immunodeficient mice, we studied the in vivo homing, proliferation and survival sites by immunohistopathology, compared with the corresponding sites for cord blood CD34(+) (CBCD34(+)) cells. The human leukemia cells initially localized on the surface of osteoblasts in the epiphysial region, and expanded to the inner vascular and diaphysial regions within 4 weeks. The percentage of CD34(+) leukemia cells in the bone marrow was transiently increased up to 50%. In vivo 5-bromo-2'-deoxyuridine labeling revealed that the epiphysis was the most active site for leukemia cell proliferation. CBCD34(+) cells showed the similar pattern of homing and proliferation to leukemia cells. After high-dose administration of cytosine-1-beta-D-arabinofuranoside, residual leukemia cells were localized in the perivascular endothelium as well as in contact with the trabecular endosteum. These findings suggest that xenotransplantation into immunodeficient mice provides a useful model to study the leukemia niche.

Skp2-dependent reactivation of AKT drives resistance to PI3K inhibitors.

The PI3K-AKT kinase signaling pathway is frequently deregulated in human cancers, particularly breast cancer, where amplification and somatic mutations of PIK3CA occur with high frequency in patients. Numerous small-molecule inhibitors targeting both PI3K and AKT are under clinical evaluation, but dose-limiting toxicities and the emergence of resistance limit therapeutic efficacy. Various resistance mechanisms to PI3K inhibitors have been identified, including de novo mutations, feedback activation of AKT, or cross-talk pathways. We found a previously unknown resistance mechanism to PI3K pathway inhibition that results in AKT rebound activation. In a subset of triple-negative breast cancer cell lines, treatment with a PI3K inhibitor or depletion of PIK3CA expression ultimately promoted AKT reactivation in a manner dependent on the E3 ubiquitin ligase Skp2, the kinases IGF-1R (insulin-like growth factor 1 receptor) and PDK-1 (phosphoinositide-dependent kinase-1), and the cell growth and metabolism-regulating complex mTORC2 (mechanistic target of rapamycin complex 2), but was independent of PI3K activity or PIP3 production. Resistance to PI3K inhibitors correlated with the increased abundance of Skp2, ubiquitylation of AKT, cell proliferation in culture, and xenograft tumor growth in mice. These findings reveal a ubiquitin signaling feedback mechanism by which PI3K inhibitor resistance may emerge in aggressive breast cancer cells.

High-dose methotrexate therapy significantly improved survival of adult acute lymphoblastic leukemia: a phase III study by JALSG.

High-dose methotrexate (Hd-MTX) therapy has recently been applied to the treatment of adult acute lymphoblastic leukemia (ALL) based on pediatric protocols; however, its effectiveness for adult ALL has not yet been confirmed in a rigorous manner. We herein conducted a randomized phase III trial comparing Hd-MTX therapy with intermediate-dose (Id)-MTX therapy. This study was registered at UMIN-CTR (ID: C000000063). Philadelphia chromosome (Ph)-negative ALL patients aged between 25 and 64 years of age were enrolled. Patients who achieved complete remission (CR) were randomly assigned to receive therapy containing Hd-MTX (3 g/m2) or Id-MTX (0.5 g/m2). A total of 360 patients were enrolled. The CR rate was 86%. A total of 115 and 114 patients were assigned to the Hd-MTX and Id-MTX groups, respectively. The estimated 5-year disease-free survival rate of the Hd-MTX group was 58%, which was significantly better than that of the Id-MTX group at 32% (P=0.0218). The frequencies of severe adverse events were not significantly different. We herein demonstrated the effectiveness and safety of Hd-MTX therapy for adult Ph-negative ALL. Our results provide a strong rationale for protocols containing Hd-MTX therapy being applied to the treatment of adult ALL.

Characterization of the immunoglobulin heavy chain complementarity determining region (CDR)-III sequences from human B cell precursor acute lymphoblastic leukemia cells.

Sequence analysis of the immunoglobulin heavy chain complementarity determining region (CDR)-III of B-lineage cells at various stages has provided important insights concerning B cell maturation and selection. Knowledge of human CDR-III sequences has been relatively limited compared with that of the murine system. We analyzed the CDR-III sequences of B cell precursor acute lymphoblastic leukemia (pre-B ALL) cells in 23 newly diagnosed and 10 relapsed patients, in order to elucidate the organization of CDR-III in B cell precursors. We found a very low frequency of somatic mutations in D and JH regions, preferential use of DLR, DXP, DHQ52, and DN elements, and of 3' side JH segments, and no predominant usage of D coding frames. Unusual joinings such as VH-D-D-JH and VH-JH were observed in three, and one sequences, respectively. We compared the CDR-III sequences derived from 10 patients between diagnosis and relapse. Two of them had three spots of mutated nucleotides at relapse, all of which were found in the N region near the D segments. Our data showed the possibility of somatic mutation at relapse, in addition to developmentally regulated rearrangement of the immunoglobulin gene at the stage of B cell precursors.

High incidence of secondary failure of platelet recovery after autologous and syngeneic peripheral blood stem cell transplantation in acute promyelocytic leukemia.

Secondary failure of platelet recovery (SFPR), which is a delayed decline in platelet count after primary recovery following myeloablative hematopoietic SCT, is a significant problem in allogeneic SCT. However, its clinical characteristics have not been well described in autologous SCT for acute myeloid leukemia. We reviewed 11 consecutive patients who had received autologous or syngeneic SCT for acute promyelocytic leukemia. Seven of 11 patients (64%) had SFPR, which is defined as a decline in the platelet count to less than 30,000/microl for more than 7 days. The median onset of SFPR was day 36 (range, 25-51 days) and the median duration of thrombocytopenia was 13 days (range, 4-25 days). Of nine patients who received busulfan-containing preparative regimens, seven (78%) had SFPR and one had delayed primary platelet count recovery. Neither patient who received cyclophosphamide and total body irradiation as preparative regimens had SFPR. The clinical courses of SFPR were transient and self-limited. SFPR was not associated with relapse of underlying diseases, graft failure or other fatal morbidities. The unexpectedly high prevalence and the characteristics of SFPR may provide additional information on management following autologous SCT for acute myeloid leukemia.

Significance of additional high-dose cytarabine in combination with cyclophosphamide plus total body irradiation regimen for allogeneic stem cell transplantation.

The combination of cyclophosphamide (CY) and total body irradiation (TBI) has been used as a standard conditioning regimen for allogeneic transplantation. Several studies showed an advantage of adding high-dose cytarabine (HDCA) to this regimen. To clarify the significance of additional HDCA, we conducted a retrospective multicenter study and compared the clinical results of these two regimens. From June 1985 to March 2003, 219 patients with hematological malignancies underwent allogeneic transplantation after conditioning with CY+TBI 12Gy (n=73) or CA+CY+TBI 12Gy (n=146). Engraftment, overall survival, transplant-related mortality (TRM), relapse rate and incidence of graft-versus-host disease (GVHD) were compared according to risks and donors. Addition of HDCA had no impact on the relapse rate in all subgroups, and it was associated with lower TRM among standard-risk patients after related transplantation, and with higher TRM and worse survival among standard-risk patients after unrelated transplantation. The incidence of acute GVHD was not significantly different between the two regimens, and HDCA resulted in a higher incidence of chronic GVHD among standard-risk patients after related transplantation. In summary, addition of HDCA is not beneficial for high-risk patients, and is not recommended for standard-risk patients receiving unrelated transplantation.

Diminished DYRK2 sensitizes hormone receptor-positive breast cancer to everolimus by the escape from degrading mTOR.

Mammalian target of rapamycin (mTOR) inhibitor, everolimus, provides benefit for metastatic hormone receptor positive breast cancer after failure of the endocrine therapy. The present report highlights Dual Specificity Tyrosine Phosphorylation Regulated Kinase 2 (DYRK2) as a predictive marker for everolimus sensitivity. The key node and KEGG pathway analyses revealed that mTORC1 pathway is activated in DYRK2-depleted cells. Everolimus was more effective in DYRK2-depleted cells compared with control cells. In xenograft model, everolimus treatment significantly inhibited tumor growth compared with vehicle or eribulin treatment. In clinical analysis, patients with low DYRK2 expression acquired longer treatment period and had higher clinical benefit rate than those with high DYRK2 expression (171 vs 82 days; P < 0.05 and 50% vs 12.5%, respectively). We further investigated the underlying mechanism by which DYRK2 regulates mTORC1 pathway. The ectopic expression of DYRK2 promoted phosphorylation of Thr631 for the ubiquitination and degradation of mTOR. DYRK2 expression levels may thus predict clinical responses to everolimus.

Acetylation-dependent regulation of MDM2 E3 ligase activity dictates its oncogenic function.

Abnormal activation of the oncogenic E3 ubiquitin ligase murine double minute 2 (MDM2) is frequently observed in human cancers. By ubiquitinating the tumor suppressor p53 protein, which leads to its proteasome-mediated destruction, MDM2 limits the tumor-suppressing activity of p53. On the other hand, by ubiquitinating itself, MDM2 targets itself for destruction and promotes the p53 tumor suppressor pathway, a process that can be antagonized by the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP). We investigated the regulation of MDM2 substrate specificity and found that acetyltransferase p300-mediated acetylation and stabilization of MDM2 are molecular switches that block self-ubiquitination, thereby shifting its E3 ligase activity toward p53. In vitro and in cancer cell lines, p300-mediated acetylation of MDM2 on Lys182 and Lys185 enabled HAUSP to bind, presumably deubiquitinate, and stabilize MDM2. This acetylation within the nuclear localization signal domain decreased its interaction with the acidic domain, subsequently increased the interaction between the acidic domain and RING domain in MDM2, enabled the binding of HAUSP to the acidic domain in MDM2, and shifted MDM2 activity from autoubiquitination to p53 ubiquitination. However, upon genotoxic stress through exposure to etoposide, the deacetylase sirtuin 1 (SIRT1) deacetylated MDM2 at Lys182 and Lys185, thereby promoting self-ubiquitination and less ubiquitination and subsequent degradation of p53, thus increasing p53-dependent apoptosis. Therefore, this study indicates that dynamic acetylation is a molecular switch in the regulation of MDM2 substrate specificity, revealing further insight into the posttranslational regulation of the MDM2/p53 cell survival axis.

Frequent somatic mutations in epigenetic regulators in newly diagnosed chronic myeloid leukemia.

Although tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML), the ability of TKIs to eradicate CML remains uncertain and patients must continue TKI therapy for indefinite periods. In this study, we performed whole-exome sequencing to identify somatic mutations in 24 patients with newly diagnosed chronic phase CML who were registered in the JALSG CML212 study. We identified 191 somatic mutations other than the BCR-ABL1 fusion gene (median 8, range 1-17). Age, hemoglobin concentration and white blood cell counts were correlated with the number of mutations. Patients with mutations ⩾6 showed higher rate of achieving major molecular response than those<6 (P=0.0381). Mutations in epigenetic regulator, ASXL1, TET2, TET3, KDM1A and MSH6 were found in 25% of patients. TET2 or TET3, AKT1 and RUNX1 were mutated in one patient each. ASXL1 was mutated within exon 12 in three cases. Mutated genes were significantly enriched with cell signaling and cell division pathways. Furthermore, DNA copy number analysis showed that 2 of 24 patients had uniparental disomy of chromosome 1p or 3q, which disappeared major molecular response was achieved. These mutations may play significant roles in CML pathogenesis in addition to the strong driver mutation BCR-ABL1.

Fatal thrombosis of antithrombin-deficient mice is rescued differently in the heart and liver by intercrossing with low tissue factor mice.

BACKGROUND: We previously reported that the targeted disruption of murine antithrombin (AT) gene resulted in embryonic lethality before 16.5 gestational days (gd) because of severe cardiac and hepatic thrombosis. OBJECTIVE AND METHODS: To investigate the influences of lowered tissue factor (TF) activity upon hypercoagulation of AT-/- embryos, we crossed AT+/- with low TF (mTF-/- hTF+) mice to yield homozygous AT-deficient mice with the extremely low TF activity, that is expressed from the inserted human TF mini gene. RESULTS: AT-/- embryos either with 50% TF (AT-/- mTF+/- hTF+) or with low (approximately 1% TF, AT-/- mTF-/- hTF+) were not born, although the survival was prolonged until 18.5 gd. In both genotypes, histological examination showed disseminated thrombosis in hepatic sinusoidal space or in the portal veins, suggesting that the thrombogenesis caused loss of hepatic blood flow. As in original AT-/-, AT-/- mTF+/- hTF+ showed subcutaneous (s.c.) bleeding and also suffered from the myocardial degeneration apparently because of coronary thrombus formation. However, AT-/- mTF-/- hTF+ had no skin hemorrhage and the thrombosis and degeneration were completely abolished in the heart. Myocardium of adult low TF mice had exhibited fibrosis secondary to hemorrhage; however, it was significantly decreased in low TF mice with AT+/-. CONCLUSIONS: Our current model suggests that, in the heart, TF plays an important role in the thrombogenesis and it counterbalances AT-dependent anticoagulation. AT may be a potent anticoagulant during mice development and the activation and subsequent regulation of TF-procoagulant activity take place differently between the liver and the heart. These differences appear to point to local regulatory mechanisms in murine hemostasis.

In vitro characterization of missense mutations associated with quantitative protein S deficiency.

OBJECTIVE: To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. PATIENTS: Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. RESULTS: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. CONCLUSION: These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.

Increased risk for treatment-related mortality after bone marrow transplantation in GSTM1-positive recipients.

Treatment-related mortality (TRM) is a major obstacle to successful allogeneic hematopoietic stem cell transplantation (HSCT). A variety of drugs are used in allogeneic HSCT, and a genetic polymorphism in metabolic enzymes could affect the metabolism of drugs and potentially influence TRM. Here, we focused attention on GSTM1 and GSTT1 enzymes, which metabolize chemotherapeutic agents, chemical carcinogens and by-products of oxidative stress and are absent from more than 50% of some populations. To assess the significance of homozygous GSTM1 and GSTT1 gene deletion in HSCT, we analyzed DNA from 373 patients with hematological disease and their HLA-identical unrelated bone marrow donors using PCR. Homozygous GSTM1 and GSTT1 gene deletions were observed in 56 and 45% of patients, respectively, and 57 and 46% of donors, respectively. There was no significant association between GSTT1 polymorphism and any outcome. However, a GSTM1-positive genotype in recipients was significantly associated with higher TRM and lower survival. These results suggest that a GSTM1-null genotype in recipients protects against TRM after allogeneic HSCT. Further studies are needed to elucidate a mechanism of increased risk for TRM in GSTM1-positive recipients.