Toshiko Motoji

Up-regulation of telomerase activity in human lymphocytes

We detected telomerase activity in human lymphocytes obtained from normal donors. Telomerase was up-regulated within 24 h when peripheral blood mononuclear cells were cultured in the presence of phytohemagglutinin. The activity increased gradually over 72 h, then remained stable for 96 h. During this period, cell number and the length of telomeric DNA remained constant. Anti-CD3 monoclonal antibody and Pansolbin also induced telomerase activity. These results demonstrate that telomerase is regulated during lymphocyte activation as cells progress from G0 to S phase. This system is useful for the study of telomerase during carcinogenesis, and in the testing of telomerase-inhibitory drugs.

Telomeric DNA in normal and leukemic blood cells.

We studied telomeric DNA in leukemic cells as well as in normal T cells, B cells, monocytes, polymorphonuclear leukocytes, and bone marrow hematopoietic progenitor cells. No marked differences were observed in the sizes of the telomeric repeats in the various populations of normal blood cells obtained from donors in their twenties to sixties, and the telomere length ranged between 8.5 and 9.0 kb. The leukemic cells of 12 patients with acute leukemia (seven with myeloid and five with lymphoid leukemia) showed a variable reduction in the length of telomeric DNA, ranging from 2.7 to 6.4 kb. The average telomere length was 4.8 and 4.7 kb in myeloid and lymphoid leukemia, respectively, while the telomere length in peripheral blood mononuclear cells obtained from the same patients during complete remission was 8.5 and 7.9 kb, respectively. When the same Southern blots were hybridized with Alu or alphoid sequences, no marked changes in the sizes of the repetitive DNA sequences were observed, indicating that the DNA abnormality in the leukemic cells was specific to the telomere region. Investigation of telomeric DNA changes may be helpful in determining the biological properties of leukemic cells.

Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype.

Point mutations of the transcription factor AML1 are associated with leukemogenesis in acute myeloblastic leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and mutations in the second tyrosine kinase domain of the Fms-like tyrosine kinase 3 (FLT3) gene represent the most frequent genetic alterations in AML. However, such mutations per se appear to be insufficient for leukemic transformation. To evaluate whether both AML1 and FLT3 mutations contribute to leukemogenesis, we analyzed mutations of these genes in AML M0 subtype in whom AML1 mutations were predominantly observed. Of 51 patients, eight showed a mutation in the Runt domain of the AML1 gene: one heterozygous missense mutation with normal function, five heterozygous frameshift mutations and two biallelic nonsense or frameshift mutations, resulting in haploinsufficiency or complete loss of the AML1 activities. On the other hand, a total of 10 of 49 patients examined had the FLT3 mutation. We detected the FLT3 mutation in five of eight (63%) patients with AML1 mutation, whereas five of 41 (12%) without AML1 mutation showed the FLT3 mutation (P=0.0055). These observations suggest that reduced AML1 activities predispose cells to the acquisition of the activating FLT3 mutation as a secondary event leading to full transformation in AML M0.

Prognostic significance of the null genotype of glutathione S-transferase-T1 in patients with acute myeloid leukemia: increased early death after chemotherapy

We investigated the prognostic significance of genetic polymorphism in glutathione-S transferase mu 1 (GSTM1), glutathione-S transferase theta 1 (GSTT1), NAD(P)H:quinone oxidoreductase (NQO1) and myeloperoxidase (MPO), the products of which are associated with drug metabolism as well as with detoxication, in 193 patients with de novo acute myeloid leukemia (AML) other than M3. Of the patients, 64.2% were either homozygous or heterozygous for GSTT1 (GSTT1+), while 35.8% showed homozygous deletions of GSTT1 (GSTT1−). The GSTT1− group had a worse prognosis than the GSTT1+ group (P = 0.04), whereas other genotypes did not affect the outcome. Multivariate analysis revealed that GSTT1− was an independent prognostic factor for overall survival (relative risk: 1.53; P = 0.026) but not for disease-free survival of 140 patients who achieved complete remission (CR). The rate of early death after the initiation of chemotherapy was higher in the GSTT1− group than the GSTT1+ group (within 45 days after initial chemotherapy, P = 0.073; within 120 days, P = 0.028), whereas CR rates and relapse frequencies were similar. The null genotype of GSTT1 might be associated with increased toxicity after chemotherapy.

Prognostic value of p53 gene mutations and the product expression in de novo acute myeloid leukemia

Abstract: In acute myeloid leukemia (AML), p53 mutations are reportedly infrequent but associated with a poor prognosis. The majority of mutations are missense mutations, which generally lead to accumulation of nuclear p53 protein. However, the prognostic significance of the accumulation remains unknown in AML. In this study, we compared the prognostic value of p53 mutations versus accumulation of the product. p53 mutations were found in 9 (4.5%) of 200 patients with de novo AML. The p53 mutation detectable (mutation+) group had a worse prognosis (p=0.0009) than the mutation not detectable (mutation−) group. Multivariate analysis showed that the p53 mutation was an independent factor (p=0.005) for short overall survival as well as 60 yr or older (p=0.001) and unfavorable karyotypes (p=0.001). In 79 of the 200 patients, the expression of p53 was studied by immunocytochemistry (ICC) using anti‐p53 monoclonal antibody (DO‐7). All samples carrying missense mutations (N=6) were positive for ICC in over 15% of nuclei of each sample, chosen as the optimized cutoff value of p53 accumulation. Accumulation was thus found in 14 of the 79 patients. However, there was no prognostic difference according to the accumulation, because the mutation−/accumulation+ group (N=8) tended to have a good prognosis. These findings indicate that molecular detection of p53 mutations yields better prognostic information than ICC. In a subset of AML, p53 protein might be accumulated without mutation presumably due to upstream signals of p53.

Granulocyte colony-stimulating factor(G-CSF) receptors on acute myeloblastic leukaemia cells and their relationship with the proliferative response to G-CSF in clonogenic assay

Summary. The number and the affinity of granulocyte colony‐stimulating factor (G‐CSF) receptors expressed by blast cells in acute myeloblastic leukaemia (AML) were determined using radiolabeled recombinant human G‐CSF (rhG‐CSF). Eighteen of 20 patients demonstrated specific binding, and Scatchard analysis revealed a single class of high affinity (Kd 15‐130 pM) G‐CSF receptors on the AML blasts. The number of G‐CSF receptors varied from 55 to 1200 per cell (mean 278). In the remaining two patients, specific binding was not observed. The number of G‐CSF receptors did not differ significantly between various AML subtypes, but the mean receptor number was the highest on type M2 blasts. A chemical cross‐linking study revealed that the G‐CSF receptor has an approximate molecular weight of 140 000. Autoradiography showed heterogeneity of the distribution of G‐CSF receptors on the AML blasts obtained from a single patient. The number of colonies stimulated by the addition of rhG‐CSF varied from 0 to 566 per dish, and blast colony formation was observed in eight of 20 patients. The population mean of G‐CSF receptor number expressed by blasts that formed colonies on stimulation with rhG‐CSF was significantly higher than that on blasts which did not form colonies. These results suggest that a proliferative response of AML blasts to G‐CSF may be predicted when the blasts express a large number of G‐CSF receptors. Accordingly, it may be safer to restrict the clinical use of G‐CSF to AML patients who have blasts with a low G‐CSF receptor expression and no response to G‐CSF in blast colony assay.

Circulating megakaryocyte progenitors in myeloproliferative disorders are hypersensitive to interleukin-3

Summary. Previous studies have reported that megakaryocyte progenitors in myeloproliferative disorders (MPD) formed spontaneous megakaryocyte colonies without the addition of megakaryocyte colony‐stimulating factor (Meg‐CSF). To determine whether this spontaneous colony formation is due to autocrine proliferation of MPD megakaryocyte progenitors or to hypersensitivity to Meg‐CSF that might exist in the culture system, we investigated colony‐forming unit‐megakaryocytes (CFU‐Meg) in the peripheral blood of 11 MPD patients, using serum‐free cultures. Spontaneous megakaryocyte colonies were observed in serum‐free cultures of nonadherent mononuclear cells (NAdMNC) obtained from MPD patients with thrombocytosis, whereas the NAdMNC of MPD patients without thrombocytosis, that of patients with reactive thrombocytosis and normal subjects never formed spontaneous colonies. However, the spontaneous colonies from MPD patients with thrombocytosis disappeared in cultures using highly purified CD34‐positive cells as target cells.

FLT3 is fused to ETV6 in a myeloproliferative disorder with hypereosinophilia and a t(12;13)(p13;q12) translocation.

The FMS-like tyrosine kinase 3 (FLT3) gene, belonging to the receptor tyrosine kinase (TK) subclass III family, plays an important role in normal hematopoiesis and is one of the most frequently mutated genes in hematologic malignancies as well as an attractive target for directed inhibition. Activating mutations of this gene, including internal tandem duplication in the juxtamembrane (JM) domain and point mutations in the TK domain, are found in approximately one-third of patients with acute myeloid leukemia and in a smaller subset of patients with acute lymphoblastic leukemia. We report here that FLT3 may contribute to leukemogenesis in a patient with myeloproliferative disorder and a t(12;13)(p13;q12) translocation through generating a fusion gene with the ETS variant gene 6 (ETV6) gene. ETV6 has been reported to fuse to various partner genes, including TK and transcription factors. Both ETV6/FLT3 and reciprocal FLT3/ETV6 transcripts were detected in the patient mRNA by reverse transcriptase-polymerase chain reaction. At the protein level, however, only ETV6/FLT3 products were expressed. Among them, one retains the helix–loop–helix (HLH) oligomerization domain of ETV6 and the JM as well as TK domain of FLT3. FLT3 receptor in leukemic cells might be inappropriately activated through dimerization by HLH domain of ETV6, which consequently interfered with proliferation and differentiation of hematopoietic cells.

Mutations of the p53 tumour suppressor gene in haematologic neoplasms

Mutations of the p53 tumour suppressor gene have frequently been observed in several types of solid tumours and are believed to be implicated in the development of these tumours. To determine the relevance of p53 mutations in haematologic neoplasms, we performed polymerase chain reaction‐single strand conformation polymorphism analysis on the p53 gene in 45 patients with various types of haematologic neoplasms. In exons 5–8 containing highly conserved regions, mobility shifts indicating sequence alterations were detected in four of the 45 patients, and subsequent sequencing was performed. A point mutation resulting in a novel stop codon was detected at codon 213 in one of 23 cases of chronic myelogenous leukaemia (one of five cases of blast crisis). Point mutations causing amino acid substitutions were detected in one of four cases of myelodysplastic syndrome at codon 195, one of three cases of adult T‐cell leukaemia at codon 281, and one of eight cases of acute lymphoblastic leukaemia at codon 281, and these missense mutations were accompanied by loss of the wild type allele. Patients harbouring these nonsense and missense mutations were in advanced disease stages. These findings suggest that mutational inactivation of the p53 gene is infrequent but is involved in the tumorigenesis of several types of haematologic neoplasms at least in some cases.

Prognostic significance of the cell cycle inhibitor p27Kip1 in acute myeloid leukemia

There are few molecular biologic determinants that are prognostic for patients with acute myeloid leukemia (AML). Hence, we examined whether cellular levels of the cyclin-dependent kinase inhibitor p27Kip1 in acute myeloid leukemia could be used to predict clinical outcome in AML. Using immunoblot analysis, levels of p27 were assessed in blast cells from 72 AML patients who were registered and treated by the identical chemotherapy protocol. AML cases were classified into three groups on the basis of the percentage of the expression level of p27 compared to a control cell line. AML cases exhibiting p27 expression at low, moderate, and high levels were 43, 9, and 20 cases, respectively. No significant differences in the rates of complete remission (CR) were observed among the three groups. Although the level of p27 expression was not correlated with any other possible prognostic markers, such as age, white blood cell count, chromosome abnormalities, and FAB subclasses, patients with high p27 expression had a significantly increased disease-free survival (DFS) (78% vs 19%, P = 0.004). We further examined the expression of cyclin E at the protein level in all 72 AML cases. We observed a statistically significant correlation between a high cyclin E level and a high p27 level (P < 0.005). However, we failed to find any correlation between the rates of cr or dfs and cyclin e expression. The present study reveals that levels of p27 expression can be one of the useful prognostic molecular markers for aml.