HMGA2 as a potential molecular target in KMT2A‐AFF1‐positive infant acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) in infants is an intractable cancer in childhood. Although recent intensive chemotherapy progress has considerably improved ALL treatment outcome, disease cure is often accompanied by undesirable long‐term side effects, and efficient, less toxic molecular targeting therapies have been anticipated. In infant ALL cells with KMT2A (MLL) fusion, the microRNA let‐7b (MIRLET7B) is significantly downregulated by DNA hypermethylation of its promoter region. We show here that the expression of HMGA2, one of the oncogenes repressed by MIRLET7B, is reversely upregulated in infant ALL leukaemic cells, particularly in KMT2A‐AFF1 (MLL‐AF4) positive ALL. In addition to the suppression of MIRLET7B, KMT2A fusion proteins positively regulate the expression of HMGA2. HMGA2 is one of the negative regulators of CDKN2A gene, which encodes the cyclin‐dependent kinase inhibitor p16^INK4A. The HMGA2 inhibitor netropsin, when combined with demethylating agent 5‐azacytidine, upregulated and sustained the expression of CDKN2A, which resulted in growth suppression of KMT2A‐AFF1‐expressing cell lines. This effect was more apparent compared to treatment with 5‐azacytidine alone. These results indicate that the MIRLET7B‐HMGA2‐CDKN2A axis plays an important role in cell proliferation of leukaemic cells and could be a possible molecular target for the therapy of infant ALL with KMT2A‐AFF1.     

Chronic obstructive pulmonary disease (COPD) burden in Japan--confronting COPD Japan survey

To grasp the burden and management status of COPD in Japan, a large telephone survey was conducted. In initial screening 400 individuals > or =45 years were identified as either having been given a diagnosis of COPD or fulfilling criteria for their respiratory-related symptoms and smoking history (baseline population) and they were recruited for a detailed investigation (interview sample). They were asked about demographic information, exacerbation, impact of COPD on daily life, and management and treatment. Of the 400 interview samples, 209 subjects (52%) had a diagnosis of COPD, and the remaining 191 ones (48%) were not, retrospectively. It was confirmed that proportions of a current smoker in the COPD (35.4%) and non-COPD (35.6%) groups were almost at the same level. The use of inhaled bronchodilators, recommended by guidelines in 157 treated subjects, was 16% or less, whereas respiratory conditions affected daily activities in 70% of all the subjects. In conclusion, COPD in Japanese subjects significantly affects daily life yet is undiagnosed; there is a need to improve COPD diagnosis and management by general practitioners through disseminating guidelines for diagnosis, management, and prevention of COPD in Japan.     

Efficacy of the polo-like kinase inhibitor rigosertib,alone or in combination with Abelson tyrosine kinase inhibitors,against break point cluster region-c-Abelson-positive leukemia cells

The potency of Abelson (ABL) tyrosine kinase inhibitors (TKIs) against chronic myeloid leukemia (CML) has been demonstrated. However, ABL TKI resistance can develop. In this study, we investigated the efficacy of a combination therapy including rigosertib (ON 01910.Na), a polo-like kinase (PLK) and phosphoinositide 3-kinase (PI3K) inhibitor, and ABL TKIs. A 72-h rigosertib treatment was found to inhibit cell growth, induce apoptosis, reduce phosphorylation of the breakpoint cluster region-c (BCR)-ABL and its substrate Crk-L, and increase the activities of caspase 3 and poly (ADP-ribose) polymerase (PARP). This combination therapy also exerted a synergistic inhibitory effect on Philadelphia chromosome (Ph)-positive cell proliferation and reduced the phosphorylation of BCR-ABL and Crk-L while increasing that of cleaved PARP and the H2A.X histone. Rigosertib also potently inhibited the growth of ABL TKI-resistant cells, and cotreatment with ABL TKIs and rigosertib induced higher cytotoxicity. These results indicate that rigosertib treatment may be a powerful strategy against ABL TKI-resistant cells and could enhance the cytotoxic effects of ABL TKIs.     

Effects of depletion of dihydropyrimidine dehydrogenase on focus formation and RPA phosphorylation

Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase (DPYD), partially inhibits homologous recombination (HR) repair and has a radiosensitizing effect as well as enhanced sensitivity to Camptothecin (CPT). DPYD is the target protein for radiosensitization by Gimeracil. We investigated the mechanisms of sensitization of radiation and CPT by DPYD inhibition using DLD-1 cells treated with siRNA for DPYD. We investigated the focus formation of various kinds of proteins involved in HR and examined the phosphorylation of RPA by irradiation using Western blot analysis. DPYD depletion by siRNA significantly restrained the formation of radiation-induced foci of Rad51 and RPA, whereas it increased the number of foci of NBS1. The numbers of colocalization of NBS1 and RPA foci in DPYD-depleted cells after radiation were significantly smaller than in the control cells. These results suggest that DPYD depletion is attributable to decreased single-stranded DNA generated by the Mre11/Rad50/NBS1 complex-dependent resection of DNA double-strand break ends. The phosphorylation of RPA by irradiation was partially suppressed in DPYD-depleted cells, suggesting that DPYD depletion may partially inhibit DNA repair with HR by suppressing phosphorylation of RPA. DPYD depletion showed a radiosensitizing effect as well as enhanced sensitivity to CPT. The radiosensitizing effect of DPYD depletion plus CPT was the additive effect of DPYD depletion and CPT. DPYD depletion did not have a cell-killing effect, suggesting that DPYD depletion may not be so toxic. Considering these results, the combination of CPT and drugs that inhibit DPYD may prove useful for radiotherapy as a method of radiosensitization.     

Current and future perspectives on the TARGET system: the registration system for Glivec® established by the JSH

Since hematology is a highly specialized field, an individual physician may not have much direct treatment experience with a given disease. Therefore, the Japanese Society of Hematology (JSH) has discussed establishing a registry of hematologic disorders, in order to contribute to improving the quality of treatments and clinical outcomes in this field. As a first step, the Timely and Appropriate Registration System for GLIVEC Therapy (TARGET), a registration system for chronic myeloid leukemia (CML) patients treated with imatinib, was established in October 2003. We present the preliminary results of the first 4 years’ experience with this system. CML patients treated with imatinib in Japan were registered through the website and the patient’s clinical course, including parameters and events like imatinib dose, blood counts, adverse events, and efficacy were recorded in months 1, 3, 6, 9, and 12 and then every 6 months thereafter. By September 2007, 862 patients from 176 hospitals were registered. Follow-up period was 0–54 months, and 127 patients were followed-up for more than 36 months. Based on these cumulative data, present imatinib treatment trends were analyzed and safety and efficacy were compared with international trial data. The part of content in this article was presented in the Joint Annual Conference of the 68th Japanese Society of Hematology (JSH) and the 48th Japanese Society of Clinical Hematology (JSCH) in Fukuoka (Japan) at 7 October 2006. Other participants in the TARGET system are listed in the Appendix.     

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth- factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G- protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.     

Morphological age changes in the diaphragm of rats under different protein diets

Male Donryu rats were housed in a specific pathogen free room and fed with 60% of the mean daily quantity of diet consumed by rats fed ad libitum The animals were divided into three groups of 50 and each group fed different amounts and types of dietary protein. The group P40 was fed with a diet containing 40% of total volume as vegetable protein; the agroup P10 with 10% of total volume as vegetable protein; and the group CE7 with 18% of total volume as mixed vegetable and animal proteins. Subsets of the animals were sacrificed at the ages of 6, 12, 18, 24 and 30 months. The P40 group gained body weight faster than other two groups during the first months. However, after 12 months, the body weight began to decrease, reaching the lowest mean body weight among the three groups by the end of the study. The three groups did not show any significative difference in the muscle fiber number index and in the number of capillaries per muscle fiber. The calculated diaphragm volume index increased up to a peak at 12 months in all three groups decreasing gradually thereafter, but did not show any significant difference due to dietary protein intake. The data suggest that the morphological age changes occurring in the diaphragm muscle are likely unrelated to dietary conditions and are more likely due to other factors such as changes in the neural activity during aging.     

Japanese individuals do not harbor the T594M mutation but do have the P592S mutation in the C-terminus of the beta-subunit of the epithelial sodium channel: the Ohasama study

OBJECTIVE: To assess the implications of polymorphisms of the amiloride-sensitive epithelial sodium channel in essential hypertension in the Japanese population by determining the incidence of the T594M mutation in the , subunit of the epithelial sodium channel, and by screening the C-terminus of the epithelial sodium channel. METHODS: Single-strand confirmational polymorphism (SSCP) analysis using two sets of primers which cover the last two-thirds of the last exon coding the B epithelial sodium channel and modification of a specific enzyme restriction site (NlaIII) for the T594M mutation were performed on 803 Japanese subjects. They were randomly selected from the study participants representative of a general population of Ohasama, Japan, who measured their home blood pressure. Polymerase chain reaction (PCR) products presenting a shift in SSCP gel, as well as controls, were directly sequenced by autoanalyser to identify the mutation. RESULTS: SSCP analysis identified altered migration in five subjects. Four SSCP variants found by sequencing were heterogeneous for the P592S (CCT to TCT) mutation conserving the PY motif, although it was not significantly associated with either home or casual blood pressure values. The resting polymorphism was at codon Thr 594, leading to no change in the amino acid sequence (ACG to ACA). None of the PCR products were modified by NlaIII, indicating the absence of the T594M mutation. CONCLUSIONS: The epithelial sodium channel variants at the C-terminus are not involved in the common form of essential hypertension in Japanese.