Inhibitory effect of polysulfide, an endogenous sulfur compound, on oxidative stress-induced TRPA1 activation.

Polysulfide (PS), an endogenous sulfur compound, generated by oxidation of hydrogen sulfide, has a stimulatory action on the nociceptive TRPA1 channel. TRPA1 is also activated by reactive oxygen species such as hydrogen peroxide (H2O2) produced during inflammation. Here, we examined the effect of PS on H2O2-induced responses in native and heterologously expressed TRPA1 using a cell-based calcium assay. We also carried out behavioral experiments in vivo. In mouse sensory neurons, H2O2 elicited early TRPA1-dependent and late TRPA1-independent increases of [Ca2+]i. The former was suppressed by the pretreatment with PS. In cells heterologously expressed TRPA1, PS suppressed [Ca2+]i responses to H2O2. Simultaneous measurement of [Ca2+]i and the intracellular PS level revealed that scavenging effect of PS was not related to the inhibitory effect. Removal of extracellular Ca2+, a calmodulin inhibitor and dithiothreitol attenuated the inhibitory effect of PS. Pretreatment with PS diminished nociceptive behaviors induced by H2O2. The present data suggest that PS suppresses oxidative stress-induced TRPA1 activation due to cysteine modification and Ca2+/calmodulin signaling. Thus, endogenous sulfurs may have regulatory roles in nociception via functional changes in TRPA1 under inflammatory conditions.

Acute myelogenous leukemia with PIG-A gene mutation evolved from aplastic anemia-paroxysmal nocturnal hemoglobinuria syndrome.

We report a patient with aplastic anemia (AA)-paroxysmal nocturnal hemoglobinuria (PNH) syndrome who developed acute myelogenous leukemia (AML). Flow cytometric analysis showed that the leukemic cells in the bone marrow lacked CD59 antigen on their surface and were positive for P-glycoprotein. Heteroduplex and single-strand conformation polymorphism analysis followed by sequencing of the leukemic cells in the bone marrow disclosed 1 frameshift-type mutation in exon 2 of the phosphatidylinositol glycan-class A (PIG-A) gene, which deductively produces truncated PIG-A protein. These findings provide direct evidence that the leukemic cells evolved from the affected PNH clone. Cytogenetic analysis in the bone marrow in each stage of AA-PNH, AML, and at relapse of AML showed normal, -7, and -7 plus -20, respectively, showing evidence of a clonal evolution. Because complete remission of AML was not achieved by intensive chemotherapies, allogeneic peripheral blood stem cell transplantation (PBSCT) from the patient's HLA-matched sister was performed successfully with recovery of CD59 antigen on bone marrow hematopoietic cells; however, leukemia relapsed 4 months after PBSCT. Leukemia derived from PNH may be resistant to intensive chemotherapy, and a highly myeloablative regimen may be required for stem cell transplantation to eradicate the PNH-derived leukemia clone.

L-selectin expression in CD34 positive cells in chronic myeloid leukemia.

L-selectin is a cell adhesion molecule, expressed on leukocytes and involved in the regulation of leukocyte traffic. This adhesion receptor is implicated in hematopoiesis by the interaction of hematopoietic stem cells and progenitors to stroma in the bone marrow microenvironment. We found that L-selectin expression on CD34++ cells from patients with chronic myelogenous leukemia (CML) is decreased or deficient, reflecting one of the features of malignant CML progenitors. In this review, we briefly describe the structure and function of L-selectin, and its role in hematopoiesis and its expression in leukemia and lymphoma. Finally, we discuss the abnormal adhesiveness of CML progenitor cells, and the role of L-selectin in this defect.

XYY male and hematologic malignancy.

Two cases of XYY male with refractory anemia with excess of blasts are reported, and previous reported XYY males with hematologic malignancy are reviewed. Altogether 26 cases were collected for analysis: acute myeloid leukemia (10), acute lymphocytic leukemia (seven), acute leukemia (two), chronic myelocytic leukemia (three), myelodysplastic syndrome (three), and essential thrombocythemia (one). The age at the time of diagnosis ranged in age from 7.5 to 81 years. In three of six XYY/XY mosaicism cases, XYY clone was associated with malignancy. However, in two cases XYY clone was not involved. The evidence presented here suggests that the event of an XYY male with hematologic malignancy is incidental rather than a genetic etiology.

Decreased L-selectin expression in CD34-positive cells from patients with chronic myelocytic leukaemia.

Abnormal adhesive interaction between bone marrow stroma and progenitors, one of the causes of unregulated proliferation in chronic myelocytic leukaemia (CML), may be caused by some alterations in adhesion molecules on CML progenitors. We investigated the expression of adhesion molecules (CD44, VLA-5, VLA-4, LFA-1, ICAM-1, L-selectin and c-kit) on bone marrow CD34++ cells from 16 CML patients by three-colour flow cytometry. The mean percentage of cells expressing L-selectin in the CD34++CD38+(or)++ fraction from untreated CML patients was significantly lower, and that in the CD34++CD38- fraction tended to be lower than that from normal controls. Among 11 CML patients treated with interferon-alpha (IFN-alpha), the mean percentage of the cells expressing L-selectin in the CD34++CD38- fraction from three patients with a low percentage of Ph1(+) cells in bone marrow was significantly higher than that from five patients with a high percentage of Ph1(+) cells. In addition, L-selectin expression rate was inversely correlated to the percentage of Ph1(+) cells. There was no significant difference between the untreated patients and normal controls with regard to the expression rates of the other adhesion molecules in each CD34++ fraction except LFA-1. These data suggest that decreased L-selectin expression in CML CD34++ cells reflects one of the features of malignant CML progenitors.

Influence of M-BCR breakpoint sites on the duration of chronic phase in 100 patients with chronic myelocytic leukemia.

Rearrangements of the bcr (M-BCR) gene were studied in 100 patients with chronic myelocytic leukemia (CML). To determine the significance of a chimeric gene expression in the progression of CML, we analyzed 43 patients for bcr-ABL chimeric mRNA expression. Both DNA and RNA analyses revealed a possible influence of breakpoint sites in the bcr region on the duration of the chronic phase. Patients with the breakpoint located at about the 1-kb region between BamHI and HindIII in bcr exon 3 (region C2) had a significantly shorter chronic phase (31 months) (p = 0.028) than patients in whom the breakpoint was located in other regions. When the bcr locus was divided into 5' and 3' regions as for the BamHI cleavage site located near the 5' region of bcr exon 3, the chronic phase duration in patients with the 5' site (HindIII-BamHI) and 3' site (BamHI-EcoRI site) was 75 and 38 months, respectively. However, the difference was not statistically significant (p = 0.128). These results suggest that only the breakpoint site at C2 on the bcr locus, rather than breakpoint sites in other regions, has an important role in the progression of CML.

Amplification of c-MYC oncogene and point mutation of N-RAS oncogene point mutation in acute myelocytic leukemias with double minute chromosomes.

Two patients with acute myelocytic leukemia (AML) showing double minute (dmin) chromosomes were analysed to identify oncogene activation. Cytogenetic analysis showed 1-53 dmin chromosomes with the normal karyotype in the first patient and 1-84 dmin chromosomes with complex chromosome aberrations. Analysis of DNA from two patients revealed five- to tenfold amplification of c-MYC oncogene in the leukemic cells. The other sixteen oncogenes studied showed no increase in the gene content. Furthermore, a transforming gene, N-RAS was detected in the first patient by nude mouse tumorigenicity assay (in vivo selection assay). These results suggest that the amplification of c-MYC gene is common in dmin-positive AML patients and co-ordination of c-MYC and N-RAS oncogene might also play a significant role in the pathogenesis of some AML patients.

The prognostic value of cytogenetic analyses in patients with acute nonlymphocytic leukemia treated with the same intensive chemotherapy.

BACKGROUND: Some specific chromosome abnormalities for the leukemias have been proven to be associated with the prognosis of acute nonlymphocytic leukemia (ANLL). However, most of these reports included patients treated with different protocols. Therefore, some bias has been involved in the evaluation of the prognostic factors in such reports. METHODS: The authors studied the morphologic, cytogenetic, and clinical features of 136 patients (86 males and 50 females) with de novo ANLL treated with the same protocol of intensive induction chemotherapy using multivariate analyses. RESULTS: Chromosome abnormalities were detected in 62.5% of the patients. The overall complete remission (CR) rate of disease was 85.5% in these patients. More than 90% of the patients with t(8;21) and pseudodiploid abnormalities achieved experienced CR. However, CR rates in the patients with abnormalities of chromosome 5 or 7 were 50%. With multivariate analyses by the type of karyotypic abnormality, CR duration and survival time of the patients with t(8;21) were longer than those of patients with normal karyotype and abnormalities of chromosome 5 or 7. Abnormalities of chromosome 5 or 7 and hyperdiploid were associated with poor prognosis. Older age and lower platelet counts also were factors contributing to shorter survival times. With the analysis with French-American-British (FAB) classification, only hypoplastic leukemia was a poor prognostic factor. CONCLUSIONS: These data suggest that cytogenetic analyses plays an important role in estimating the prognosis of patients treated with intensive induction chemotherapy.

Loss of heterozygosity at D3S2 locus of short arm of chromosome 3 in chronic myelogenous leukemia.

Loss of heterozygosity (LOH) on the short arm of chromosome 3 was studied in four patients with chronic myelogenous leukemia (CML). The bcr gene rearrangement-negative spleen cells and a B-cell line were used as normal tissue controls. Five probes showing restriction fragment length polymorphisms (RFLP) and a variable number of tandem repeats on chromosome 3 were used. DNA patterns in Southern blotting were compared between normal cells and leukemic cells. One of the four patients had LOH at the D3S2 locus mapped to 3p14.3-3p21.3. The LOH was detected in the blastic phase, but not in the chronic phase. This patient showed normal chromosomes 3 in the blastic phase. These data suggest the possibility of the existence of LOH in CML, occurring as a secondary event in the blastic phase, and which might have been induced by submicroscopic deletion or somatic recombination.

[Analysis of breakpoints on BCR gene in acute leukemia patients with Ph1 chromosome].

Eight cases with Ph1 positive acute leukemia (7 of acute lymphocytic leukemia: ALL, and one of acute myelocytic leukemia: AML) were studied molecular biologically to identify location of breakpoints on BCR gene in each patient. Six of the 8 patients (5 of ALL and 1 of AML) had rearrangements at bcr (M-BCR) region. Their locations of the breakpoint in M-BCR were similar to those of 59 chronic myelocytic leukemia patients. One of the remaining two patients had gene rearrangements at m-BCR-1 region in BCR intron 1, and the last patient did not have gene rearrangements at any site of m-BCR-1 and IgL C lambda region. Two cases had gene deletion at either 3' or 5' side of the bcr. A patient with bcr rearrangement was also analyzed by PCR method with reverse transcriptase (RT-PCR) and had simultaneous expressions of bcr3-abl and bcr2-abl chimeric mRNAs. These results indicate that Ph1 positive acute leukemia have heterogeneous characteristics in terms of the molecular biology. The molecular analysis will help for classifying the leukemic types and for elucidating the pathogenesis in Ph1 positive acute leukemia.

Cytogenetic and molecular changes in leukemia among atomic bomb survivors.

Seventy five radiation-related leukemia patients in Hiroshima including 16 patients exposed to more than one Gray were cytogenetically examined. Statistical analysis of data on the frequencies of chromosomal aberrations in the survivor groups according to bone marrow doses by DS86 estimation revealed that the heavily exposed group tended to have significantly higher aberration rates compared to the non-exposed group. Furthermore, the chromosomal aberrations in the survivors were observed to be of a more complex nature and had the characteristic findings of secondary leukemia. These observations therefore suggest that patients with a history of heavy exposure to atomic bomb radiation had leukemic cells originating from a stem cell which had been damaged by irradiation at the time of the bombing as well as cells involved in complex chromosome abnormalities. Molecular biologic studies on ras genes in acute and chronic leukemias and the bcr gene in chronic myelocytic leukemia were performed in exposed and non-exposed groups. So far, no distinctive differences have been observed in the frequency and sites of point mutations in N- and K-ras genes or in the rearrangement of the bcr gene. Further, retrospective analysis using DNA from leukemia patients who developed this disease in the early period from atomic bomb radiation exposure would be useful for the elucidation of the mechanisms of radiation-induced leukemia.

[Parallel loss of c-FMS and GM-CSF genes in myeloid leukemias with 5q-chromosome].

DNA contents of c-FMS and GM-CSF genes were analyzed by densitometer in nine patients with myelodysplastic syndrome or acute myeloid leukemia associated with abnormality of chromosome 5. Five patients with deletion in the long arm of chromosome 5 had loss of both c-FMS and GM-CSF genes. These findings suggest that c-FMS oncogene and GM-CSF gene locating in the critical region on chromosome 5 seem to have an important role in the process of leukemogenesis.

Blastic transformation in essential thrombocythemia. In vitro differentiation of blast cells into granulocytic, erythroid, and megakaryocytic lineages.

A 57-year-old man with essential thrombocythemia (ET) developed myelofibrosis, that progressed to a blastic transformation state. The characteristics of the blastic cells were serially studied both morphologically and phenotypically as well as in cell culture. The blastic cells that were first detected in peripheral blood had features of myeloid stem cells with slight differentiation toward megakaryocytic lineage. However, later in the course, most of the blastic cells were immature. During culture in the presence of human plasma-derived serum (PDS), some blastic cells obtained at the initial stage differentiated, mainly to both granulocytes and macrophages morphologically, but later tended to differentiate into both megakaryocytes and macrophages. Finally the blasts appeared to have lost their ability to differentiate morphologically. However, the blasts formed mixed colonies consisting of erythroblasts, granulocytes, macrophages, and immature blasts when cultured in methylcellulose with PHA-leukocyte conditioned medium. In addition, the blastic cells in suspension culture strongly expressed phenotypic features which are characteristic of erythroblasts, in the presence of both PDS and 12-0-tetradecanoylphorbol 13-acetate (TPA), whereas they expressed features of megakaryoblasts in the presence of PDS alone. These results suggest that essential thrombocythemia is of myeloid stem cell origin. This is the first case in the literature in which a clonal evolution in ET has been followed closely, essential events were identified serially, and the blastic cells, which appeared as a result of the progression of ET, were found to have the capability to differentiate toward the three myeloid lineages.

9;22 translocation and bcr rearrangements in chronic myelocytic leukemia patients among atomic bomb survivors.

To elucidate the mechanism of leukemia induced by radiation, we studied both chromosome abnormalities and bcr rearrangements of seven CML patients with a history of atomic bomb exposure and 14 CML patients without the exposure. All patients, irrespective of radiation exposure, had 9;22 translocation and rearrangement of the bcr gene in the leukemic cells. Further analysis of breakpoints within the bcr gene demonstrated no distinct difference between the exposed and the non-exposed groups. The present study suggests that formation of the chimeric bcr-abl gene and its genetic products may play an important role in the development of leukemia in either radiation-induced or de novo CML.

[Late appearance of Philadelphia chromosome with bcr gene rearrangement in an acute myelocytic leukemia patient].

A case of acute myelocytic leukemia (AML-M2) with a late appearance of Philadelphia chromosome (Ph1) is presented. Chromosome analysis revealed a normal karyotype at the time of diagnosis and for 23 months, when hematological relapse occurred, accompanied by abnormal clones, 46, XX, t(9;22) (q34;q11) (78%) and 45,XX, -16, t(9;22) (q34;q11), del (5) (q13q31) (22%). The patient died of GVHD after bone marrow transplantation. Molecular analysis confirmed bcr gene rearrangement in the cells with Ph1 chromosome. Acquisition of Ph1 chromosome during the course of hematological malignancies other than CML is extremely rare. This case is undoubtedly important for the understanding of leukemogenesis and the evolution of leukemia clones. The authors discussed possible mechanisms of Ph1 acquisition in the late stages of AML.