Telomerase plays a key role in the maintenance of chromosomal stability in tumors, but the mechanism regulating telomerase activity is still unclear. Recent studies have suggested that c-myc may be vital for regulation of hTERT mRNA expression and telomerase activity. In this study, we investigated the changes of telomerase activity and telomerase-related genes induced by herbimycin A in K562 human chronic myelogeous leukemic cells. Telomerase activity showed a biphasic pattern in herbimycin A-treated K562 cells. Initially, the telomerase activity decreased along with the decline of cells in S and G2/M phases, but it recovered slightly at the end of treatment. Expression of mRNA for the telomerase catalytic subunit (hTERT) was decreased before the decline of telomerase activity, and increased slightly before the reactivation of telomerase activity. During herbimycin A treatment, both c-myc and cyclin D1 mRNA showed transient downregulation before the increase of G1 cells. Herbimycin A treatment caused the downregulation of both telomerase activity and hTERT mRNA in cyclin D1-transfected K562 cells, while telomerase activity was partially restored in c-Myc-transfected cells. In contrast, hTERT-transfected K562 cells maintained a high level of telomerase activity during herbimycin A treatment. Neither the template RNA component of telomerase (hTERC) nor telomerase-associated protein (TEP-1) were altered in any of the transfected K562 cells. These results indicate that telomerase activity is mainly regulated by hTERT, and that c-Myc protein is one of the positive regulators of hTERT in leukemic cells but is not enough to counteract the downregulation of telomerase activity by herbimycin A completely.

In order to elucidate the relationship between the develop ment of Friend leukemia and erythroid differentiation in affected organs, C3H/He mice were given injections of high doses (about 2 x I o@spleen focus-forming units) of Friend leukemia virus, and changes in the heme synthesis rate, in responsive ness to erythropoietin in vitro, and in RNA-dependent DNA polymerase activity were investigated. By the 6th day following Friend leukemia virus inoculation, hyperbasophilic †̃ Friend cells―resembling proerythroblasts became dominant in the spleen. The heme synthesis rate in the infected spleen cells started to increase i 4 days after inoculation and reached a plateau at a level about five times higher than that of the uninfected control spleen cells after about 22 days. With respect to the response of the spleen cells to erythropoietin in vitro, however, while a normal response had been observed 4 hr after the inoculation, no response could be detected at 24 hr and thereafter. RNA-dependent DNA polymeraseactivityin the spleen cells began to increase on the 4th day, attaining a peak on the 8th and i 0th days and decreasing markedly thereafter. Enzyme activity again in creased on the 28th day. These results suggest that Friend leukemia virus affected the erythropoietin-responsive cells at a very early stage of infection, before morphological changes in the spleen cells or increases in RNA-dependent DNA polym erase activity had been observed.

Cytomegalovirus (CMV) infection is a major complication for patients who received allogeneic haematopoietic stem cell transplantation (HSCT). Accurate monitoring of CMV-specific T-cell reconstitution is required for appropriate decision on treatment, such as anti-viral drugs, which have adverse effects. Although human leucocyte antigen (HLA) tetramer and interferon-gamma-enzyme-linked immunospot (IFN-gamma-ELISPOT) assays have been used to measure CMV-specific T cells, detailed comparison of these assays and kinetics of anti-CMV T-cell reconstitution between reduced-intensity transplantation (RIST) and conventional HSCT has not yet been performed. In this study, we performed prospective comparative monitoring of CMV-specific T cells using HLA tetramer and IFN-gamma-ELISPOT assays with a single immunodominant CMV(495) peptide in 28 HLA-A*0201 and 9 HLA-A*0206 patients after various allogeneic HSCTs. The IFN-gamma-ELISPOT assay was more sensitive for evaluation of functional T cells than the HLA tetramer assay, and CMV-specific T cells were reconstituted earlier in patients who received RIST without anti-thymocyte globulin (ATG) than those receiving RIST with ATG or conventional HSCT. The threshold level for protection from CMV reactivation was estimated as over 1 x 10(6) cells/l peripheral blood with the IFN-gamma-ELISPOT assay. These results demonstrate that the IFN-gamma-ELISPOT assay with CMV(495) provides more accurate evaluation on CMV immunity in HLA-A*0201 and -A*0206 patients, and may be useful for determining timing of various treatments.

This study detected clonal T cells in patients with acquired pure red-cell aplasia (PRCA) by Southern blotting and polymerase chain reaction (PCR). Twenty-nine adult patients with acquired PRCA were enrolled in this study. Seventeen patients had primary acquired PRCA, while 12 patients had the secondary form. Twenty-two of 29 (76%) patients demonstrated TCR rearrangement by at least one method. We divided the patients into three groups depending on T-cell clonality. The CD4/8 ratio of patients who were positive on Southern blotting was significantly lower than that of other groups. Except for the CD4/8 ratio, other laboratory findings did not significantly differ among the three groups. The CD4/8 ratio should be a useful surrogate marker to detect T-cell clonality.

CMV infection is a major complication after allogeneic HSCT. Although peptide/HLA tetramer and IFN-g ELISPOT assays have been used to measure CMV-specific T cells to monitor the reactivation of CMV, detailed evaluations of these assays and reconstitution kinetics of anti-CMV T cells in RIST and conventional HSCT have not yet been made. In this study, we prospectively monitored CMV-specific T cells after allogeneic peripheral blood HSCT using HLA-tetramer and IFN-g ELISPOT assays specific for previously identified HLA-A*0201 binding pp65 495–503 (NLVPMVATV) peptide in 28 HLA-A*0201- and 9 HLA-A*0206-positive patients. This peptide was able to induce CTL from PBMC in 7 of 8 and 6 of 6 HLA-A*0201- and HLA-A*0206-positive healhy individuals, respectively, indicating that this is an immunodominant CMV peptide for individuals with these HLA-A2 subtypes. The mean of the first day of detectable CMV-specific T cells in 23 patients after allogeneic HSCT was 52.9 days by the HLA-tetramer assay and 29.5 days by the IFN-g ELISPOT assay, indicating that the IFN-g ELISPOT assay is more sensitive than the HLA-tetramer assay, and reconstitution of CMV-specific T cells was detected significantly earlier by the IFN-g ELISPOT assay (p<0.001). The absolute numbers of CMV-specific T cells calculated based on the HLA-tetramer assay were always higher than those with the IFN-g ELISPOT assay, suggesting that the IFN-g ELISPOT assay was able to measure functional T cells. We then compared the recovery of functional CMV-specific T cells using the IFN-g ELISPOT assay among patients who received various HSCT protocols, including RIST without anti-thymocyte globulin (ATG), RIST with ATG, and conventional HSCT. The mean of the first day of detectable functional CMV-specific T cells was 23 days, 39 days, and 79 days, respectively. Thus, the recovery of functional CMV-specific T cells in patients with conventional HSCT was profoundly delayed compared to those in patients who received RIST without ATG (p=0.0001) and RIST with ATG (p=0.02). The recovery of functional CMV-specific T cells in patients who received RIST with ATG was significantly delayed compared to that in patients who received RIST without ATG (p=0.006). We found that patients who had more than 1x10[sup6] functional CMV-specific T cells/L peripheral blood did not develop CMV antigenemia, indicating that this level of recovery was clinically sufficient for protection against CMV infection. The number of functional CMV-specific T cells in patients with RIST without ATG reached 1x10[sup6] cells/L significantly earlier than that in patients who received RIST with ATG (P=0.014). Therefore, RIST without ATG offers the advantage of early reconstitution of anti-CMV immunity. These results indicate that the measurement of functional T cells specific for the single immunodominant CMV peptide using the ELISPOT assay may be useful for evaluating immune recovery for CMV.

We examined the efficacy of cyclosporin A (CsA) in 50 patients with myelodysplastic syndrome (MDS) consisting from 47 of RA, 1 of RARS, and 2 of RAEB. These patients showed various marrow cell types including hypo-, normo-, and hypercellularity. Patients belonged to the following International Prognostic Scoring System (IPSS) risk groups: 4 of low, 41 of intermediate-1, and 5 of intermediate-2. The median CsA dose was 4.58 mg/kg, and treatment responses were classified according to the International Working Group (IWG) criteria. Hematological improvement (HI) was observed in 30 (60%) patients, and all of them were belonged to RA. In the patients with RARS or RAEB, no efficacy was observed. Four (8%) of the responders achieved partial remission (PR) with granulocytes ≥1500 μl−1, Hb >11 g/dl and platelets ≥100,000 μl−1. Higher response rate (53%) was shown in erythroid lineage (HI-E) compared to platelet (HI-P, 36%) or neutrophil lineage (HI-N, 35%). When we analyzed the correlation between the response to CsA therapy and the karyotype or HLA type, there were significantly more responders with good karyotype or DRB1∗1501 than with intermediate/poor karyotypes or with other HLA types. These results indicate the usefulness of CsA therapy for MDS patients with any marrow cellularity, especially for erythroid lineage and patients with good karyotype or DRB1∗1501.

STI571, an abl tyrosine kinase inhibitor, is less effective in chronic myelogenous leukemia (CML) patients in the accelerated phase and in blastic crisis. We addressed whether STI571 is effective for the CML blastic crisis cell line K562 and the P-glycoprotein (P-gp) positive, multidrug resistance cell line K562/ADM. The present results demonstrate that P-gp positive K562/ADM cells were more resistant than K562 cells to the anti-proliferative and apoptotic effect of STI571, but the co-addition of a P-gp modulator augmented the sensitivity of K562/ADM cells to STI571. For patients in CML blastic crisis, simultaneous use of a P-gp modulator may increase the efficacy of STI571.

Summary. Myelodysplastic syndrome (MDS) is a common neoplasm of haematopoietic pluripotent stem cells. Although one third of MDS patients evolve to acute myeloid leukaemia (AML), little is understood about the mechanisms responsible for this progression. We have previously detected the frequent loss of heterozygosity (LOH) on the short arm of chromosome 1 in blast crisis of chronic myelocytic leukaemia. In this study, we examined the chromosomal arm 1p for allelic loss in the progression of MDS to AML, using 17 microsatellite markers spanning chromosome 1 in 20 patients who progressed from MDS to AML. DNA was extracted from slides of bone marrow smears. In each patient, DNA from MDS was analysed alongside DNA from AML. Allelic loss on 1p was observed in six of the 20 individuals (30%). Serial cytogenetic information was available in five of the six patients with LOH on 1p; no deletions in this region were detected. Three samples showed LOH at all informative loci on 1p. The other three samples showed LOH on at least one but not all loci on 1p with consensus regions of LOH located distal to D1S253 (1p36.3) and probably proximal to D1S496 (1p32−). Our results suggest that tumour suppressor genes that play an important role in the progression of MDS to AML may reside in at least two different regions on 1p.

To understand molecular pathways underlying 9p21 deletions, which lead to inactivation of the p16/CDKN2A, p14/ARF, and/or p15/CDKN2B genes, in lymphoid leukemia, 30 breakpoints were cloned from 15 lymphoid leukemia cell lines. Seventeen (57%) breakpoints were mapped at five breakpoint cluster sites, BCS-LL1 to LL5, each of <15 bp. Two breakpoint cluster sites were located within the ARF and CDKN2Bloci, respectively, whereas the remaining three were located >100 kb distal to the CDKN2A, ARF, andCDKN2B loci. The sequences of breakpoint junctions indicated that deletions in the 11 (73%) cell lines were mediated by illegitimate V(D)J recombination targeted at the five BCS-LL and six other sites, which contain sequences similar to recombination signal sequences for V(D)J recombination. An extrachromosomal V(D)J recombination assay indicated that BCS-LL3, at which the largest number of breakpoints (i.e. five breakpoints) was clustered, has a V(D)J recombination potential 150-fold less than the consensus recombination signal sequence. Three other BCS-LLs tested also showed V(D)J recombination potential, although it was lower than that of BCS-LL3. These results indicated that illegitimate V(D)J recombination, which was targeted at several ectopic recombination signal sequences widely distributed in 9p21, caused a large fraction of 9p21 deletions in lymphoid leukemia.

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusions. The maintenance of telomeres is essential for chromosomal stability. Telomerase is regulated by human telomerase reverse transcriptase (hTERT). c-Myc oncoprotein is also implicated in the positive regulation of hTERT expression. We show here that two clones of hTERT-transfected K562 erythroleukemia cells have elongated telomeres (22.5 and 24.0 kb), whereas telomere length of both c-Myc-transfected K562 cells and parental K562 cells is 6.5 kb. Telomerase activity and hTERT mRNA expression increased in hTERT-transfected K562 cells, while the expression levels of telomerase activity and hTERT in c-Myc-transfected K562 cells were similar to that in parental K562 cells, despite an overexpression of c-Myc. Importantly, we found that hTERT-transfected K562 cells are protected against apoptosis induced by serum deprivation and double-stranded DNA break inducing agents (ionizing irradiation, and etoposide (VP-16)), but not against DNA synthesis inhibitors (1-beta-D-arabinofuranosylcytosine and hydroxyurea). These findings suggest that overexpression of telomerase by transfecting hTERT confers telomere-elongation and resistance to double-stranded DNA break inducing agents.

Allelic status of the BAT26 and BAT25 loci was examined in 117 leukemia/lymphoma cell lines consisting of 44 B-lymphoid lineage cell lines, 30 T-lymphoid cell lines and 43 myeloid cell lines to define the lineage specificity of microsatellite instability (MSI) in hematological malignancies. Seventeen (15%) cell lines were defined as having MSI. The incidence of MSI was significantly (P<0.01) higher in cell lines of lymphoid lineage (15/74; 20%) than in those of myeloid lineage (2/43; 5%). In the cell lines of lymphoid lineage, the incidence of MSI in T cell acute lymphoblastic leukemia (T-ALL) (11/30; 37%) was significantly (P<0.01) higher than those in B-lineage malignancies (4/44; 9%). The 17 cell lines with MSI were subjected to the mutation analysis of the coding microsatellites in 13 candidate genes. Frameshift mutations were most frequently detected in the BAX gene (14/17, 82%), while the hMSH3, hMSH6, TGFβRII, DRP and IGFIIR genes were less frequently mutated (24–47%). The present result indicates that MSI is involved in the development and/or progression of lymphoid malignancies, especially of T-ALL, through the inactivation of BAX and several other genes.

Myelodysplastic syndrome (MDS) with erythroid hypoplasia, a rare form of MDS, has not yet been clearly defined. We report four patients with MDS with erythroid hypoplasia who received immunosuppressive therapy. All were elderly, had severe transfusion‐dependent anaemia, morphological evidence of myelodysplasia and a low percentage (3·2–13·6%) of erythroid precursors. Administration of cyclosporin A (CsA) improved their anaemia; all transfusion‐dependent patients achieved transfusion‐independence. An inverted CD4/8 ratio was seen in three patients who also demonstrated T‐cell receptor (TCR)‐β and ‐γ gene rearrangements by Southern blotting and clonality by polymerase chain reaction. Treatment with CsA can be an attractive alternative treatment for patients with MDS with erythroid hypoplasia, which may be associated with a clonal abnormality in T cells.

Refractory anemia has a relatively low incidence of the subsequent development of acute leukemia and a relatively long survival among the myelodysplastic syndromes (MDS). We observed hematological improvement due to high-dose methylprednisolone in 9 of 18 patients with refractory anemia. The patients' age range was from 28 to 78 years old (mean age: 54), including 14 male and 4 females. A complete response was obtained in 5 patients, minimal response in 4 patients, and no response in 9 patients. Laboratory data of peripheral blood counts and differential counts of bone marrow aspirates were not different, except that fewer chromosomal abnormalities (P = 0.086) were observed in the responder group. Side effects were seen in two patients but were controllable. Overall survival was significantly longer in the responder group (Log-rank P = 0.040, Wilcoxon P = 0.045). The overall survival of responders did not reach the median and 85% of the patients were alive after 180 months, while the median overall survival of the nonresponders was 61.8 months. Disease progression was more frequently seen in the non-responder group (P = 0.045). Furthermore, we investigated retrospectively immunohistochemical bone marrow staining, and a significantly higher percentage of CD68-positive (22.6% +/- 7.1%) and CD45RA-positive cells was observed in the responder group compared to the non-responder group (6.5% +/- 1.3%). Our present results indicate that high-dose methylprednisolone is valuable as a primary treatment before other immunosuppressive treatments, because of its ease of use. High efficacy with high-dose methylprednisolone is expected, especially in patients in which increments in CD68-positive cells in bone marrow are observed.

A 77-year-old man with relapsed non-Hodgkin's lymphoma, diffuse large B-cell type, was treated with naproxen, a nonsteroidal anti-inflammatory drug (NSAID), for paraneoplastic fever. A dramatic disappearance of not only the fever but also generalized lymphadenopathy was observed. Naproxen was continued, and he maintained remission for 10 months. When relapse of lymphoma occurred in spite of continuous naproxen administration, indomethacin, another type of NSAID, was tried. Surprisingly, rapid regression of lymphoma occurred again and was maintained for almost 1 year. These results indicate that NSAIDs are effective in some patients with non-Hodgkin's lymphoma.