Insulin-like Growth Factor 1 Regulates the Expression of ATP-Binding Cassette Transporter A1 in Pancreatic Beta Cells.

ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and cholesterol homeostasis. The present study investigates whether insulin-like growth factor 1 (IGF-1), which mediates stimulation of ABCA1 gene expression, could also interfere with the phosphatidylinositol 3-kinase (PI3-K) cascade.ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and a reporter gene assay in rat insulin-secreting INS-1 cells incubated with IGF-1. The binding of forkhead box O1 (FoxO1) protein to the ABCA1 promoter was assessed by a chromatin immunoprecipitation (ChIP) assay. ABCA1 protein levels increased in response to rising concentrations of IGF-1. Real-time PCR analysis showed a significant increase in ABCA1 mRNA expression. However, both effects were suppressed after silencing the IGF-1 receptor. In parallel with its effect on endogenous ABCA1 mRNA levels, IGF-1 induced the activity of a reporter construct containing the ABCA1 promoter, while it was abrogated by LY294002, a specific inhibitor of PI3-K. Constitutively active Akt stimulated activity of the ABCA1 promoter, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element in the ABCA1 promoter abolished the ability of IGF-1 to stimulate promoter activity. A ChIP assay showed that FoxO1 mediated its transcriptional activity by directly binding to the ABCA1 promoter region. The knockdown of FoxO1 disrupted the effect of IGF-1 on ABCA1 expression. Furthermore, IGF-1 promoted cholesterol efflux and reduced the pancreatic lipotoxicity. These results demonstrate that the PI3-K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF-1 stimulation.

Exendin-4 regulates the expression of the ATP-binding cassette transporter A1 via transcriptional factor PREB in the pancreatic ß cell line.

BACKGROUND: PRL regulatory element-binding (PREB) protein is a transcription factor that regulates insulin promoter activity in the rat anterior pituitary. The PREB protein is expressed not only in the anterior pituitary but also in pancreatic ß cells. Previously, we have reported that PREB plays an important role in glucose-mediated insulin gene expression in pancreatic ß cells. The ATP-binding cassette transporter A1 (ABCA1) in pancreatic ß cells influences insulin secretion and glucose homeostasis. Exendin-4 (Ex-4), a longacting agonist of the glucagon-like peptide 1, stimulates ABCA1 expression in pancreatic ß cells. AIMS: In this study, we examined the role played by PREB in Ex-4-induced ABCA1 expression in pancreatic ß cells. MATERIAL/SUBJECTS AND METHODS: PREB mRNA and protein expression were evaluated in pancreatic ß cell line (INS-1 cells) treated with Ex-4 (10 nM). RESULTS: Ex-4 stimulated PREB protein and mRNA expression in INS-1 cells. PREB stimulated the activity of the luciferase reporter protein that was under the control of the ABCA1 promoter. Chromatin immunoprecipitation assay showed that PREB mediates its transcriptional activity by directly binding to the ABCA1 promoter region. Finally, we used small interfering RNA to inhibit PREB expression in the cells and demonstrated that the knockdown of PREB expression attenuated the effects of Ex-4 on ABCA1 expression. CONCLUSION: PREB mediates Ex-4-stimulated transcription of the ABCA1 gene in pancreatic ß cells.

Suppression of prolactin expression by cabergoline requires prolactin regulatory element-binding protein (PREB) in GH3 cells.

The prolactin regulatory element-binding protein (PREB) is a transcriptional factor that regulates prolactin (PRL) promoter activity in the anterior pituitary. Prolactinomas are the most common pituitary tumors. Administration of cabergoline, a selective dopamine D2-receptor agonist, has become the initial therapy of choice for most patients with prolactinomas. Although activation of the D2 receptor results in the inhibition of PRL synthesis, the details of the underlying mechanisms remain unknown. Samples of ten prolactinomas and ten nonfunctioning pituitary adenomas were analyzed by immunohistochemistry to detect the expression of PREB. The effect of cabergoline on PREB expression was assessed by western blotting and real-time polymerase chain reaction (PCR) analysis. Reporter gene analysis of PRL was employed to examine the role of PREB on cabergoline-induced suppression of PRL transcription. Immunohistochemical analysis revealed strong positive PREB expression in the prolactinoma tissue, but extremely weak or undetected expression in the nonfunctioning pituitary tumor tissue. Western blots probed with a PREB-specific antiserum revealed that the relative abundance of the PREB protein in the GH3 cells decreased in a dose-dependent manner in response to cabergoline treatment, as did the relative abundance of PREB mRNA. Although cabergoline inhibited the activity of the PRL promoter, mutation of PREB-binding site within the promoter abrogated the ability of cabergoline to inhibit the PRL promoter activity. We have demonstrated that PREB is expressed in prolactinomas and that the suppression of PRL expression by cabergoline requires the transcriptional factor PREB.

Hyperglycemia suppresses ABCA1 expression in vascular smooth muscle cells.

Hyperglycemia is a major risk factor for atherosclerotic disease. The ATP-binding cassette transporter A1 (ABCA1) functions as a pivotal regulator of lipid efflux from cells to apolipoproteins and is thus involved in lowering the risk of atherosclerosis. In this study, we have examined the glucose-mediated regulation of the ABCA1 gene expression in vascular smooth muscle cells. ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and reporter gene assay. The results showed that the expression of the ABCA1 mRNA and protein decreased after the cells were treated with 22.4 mM glucose for 48 h. The transcriptional activity of the ABCA1 promoter paralleled the endogenous expression of the ABCA1 gene. Next, we used inhibitors of certain signal transduction pathways to demonstrate that the glucose-induced ABCA1 suppression is sensitive to the p38-mitogen-activated protein kinase (MAPK) inhibitors. The expression of a constitutively active form of p38-MAPK in the cells inhibited the ABCA1 promoter activity, irrespective of the presence of glucose. A dominant-negative mutant of p38-MAPK abrogated the inhibitory effect of glucose on the ABCA1 promoter activity. These results indicate that the glucose-induced suppression of ABCA1 expression is partially mediated by the activation of the p38-MAPK pathway.

Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1.

Extracellular levels of the excitatory neurotransmitter glutamate in the nervous system are maintained by transporters that actively remove glutamate from the extracellular space. Homozygous mice deficient in GLT-1, a widely distributed astrocytic glutamate transporter, show lethal spontaneous seizures and increased susceptibility to acute cortical injury. These effects can be attributed to elevated levels of residual glutamate in the brains of these mice.

Telomerase activity in lung cancer cells obtained from bronchial washings.

BACKGROUND: Telomerase, a ribonucleoprotein enzyme that functions in the maintenance of telomeres (specialized structures at the ends of chromosomes), has been reported to be a novel diagnostic marker for malignant diseases. We sought to determine whether measurement of telomerase activity in bronchial washings is of value in the diagnosis of lung cancer. METHODS: Extracts of cells in bronchial washings were analyzed for telomerase activity by use of a telomeric repeat amplification protocol (TRAP) assay. Telomerase activity inside cells was evaluated by use of an in situ TRAP assay. The results of both TRAP assays were compared with those obtained from cytologic examination, which employed standard Papanicolaou staining. RESULTS: When results from the two TRAP assays were combined, telomerase activity was detected in bronchial washings from 18 (82%; 95% confidence interval [CI] = 60%-95%) of 22 patients with lung cancer. In contrast, cancer cells were detected by cytologic examination in the bronchial washings of nine (41%; 95% CI = 21%-64%) of the same 22 patients, a statistically significant difference (two-sided P = .0061). In patients with lung cancer, telomerase-positive cells could be detected in bronchial washings irrespective of tumor location--11 of 14 (79%; 95% CI = 49%-95%) peripheral cancerous lesions and seven of eight (88%; 95% CI = 47%-100%) central cancerous lesions were detected by use of TRAP assays (for comparison, two-sided P = .5349). CONCLUSIONS: A high percentage of patients with lung cancers had detectable telomerase activity in bronchial washings. Thus, the use of a cell extract-based or an in situ TRAP assay in addition to cytologic examination may make the diagnosis of lung cancer more reliable.

Telomerase activity and cytogenetic changes in chronic myeloid leukemia with disease progression.

Progressive telomere shortening is thought to be important in the regulation of cellular senescence and that the upregulation or reactivation of telomerase activity may be a critical if not rate limiting step in the development of neoplastic cells. To obtain information about telomeres and telomerase activity in hematopoietic neoplasia at various disease stages, we evaluated 54 samples obtained from 41 patients with chronic myeloid leukemia (CML) using a combination of fluorescent-telomeric repeat amplification protocol and an internal telomerase assay standard. The terminal restriction fragment (TRF) lengths in the blast phase was reduced compared to that in the chronic phase (4.53 +/- 0.72 kb vs 6.13 +/- 1.68 kb; P = 0.0005). All samples obtained from CML in the chronic phase (n = 33) had detectable telomerase activity above background, regardless of age. In the blast phase (n = 21), a significant increase of telomerase activity was detected compared to that in the chronic phase (33.84 +/- 37.86% vs 6.08 +/- 3.21; P = 0.016). Among patients in the blastic phase, 50% of patients had moderate to high telomerase activity (>10 relative value), and the remaining patients had telomerase activity higher than that in the normal peripheral blood cells. No significant differences in hematologic findings, duration of chronic phase or blast phase, and telomere length in the blastic phase were noted between these two groups separated by telomerase activity. CML patients with moderate to high telomerase activity had a high frequency of additional cytogenetic changes (P = 0.01).

The Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes.

The Wilms' tumor gene, WT1, is a tumor marker for leukemic blast cells. The WT1 expression levels were examined for 57 patients with myelodysplastic syndromes (MDS) (refractory anemia (RA), 35; RA with excess of blasts (RAEB) 14; RAEB in transformation (RAEB-t), six; and MDS with fibrosis, two) and 12 patients with acute myeloid leukemia (AML) evolved from MDS. These levels significantly increased in proportion to the disease progression of MDS from RA to overt AML via RAEB and RAEB-t in both bone marrow (BM) and peripheral blood (PB). WT1 expression levels in PB significantly correlated with the evolution of RAEB or RAEB-t to overt AML within 6 months. Therefore, WT1 expression levels in PB were superior to those in BM for early prediction of the evolution to AML by means of quantitation of the WT1 expression levels. Furthermore, WT1 expression in PB of patients with overt AML evolved from MDS was significantly decreased by effective chemotherapy or allogeneic stem cell transplantation and became undetectable in long-term survivors. These results clearly showed that WT1 expression levels are a tumor marker for preleukemic or leukemic blast cells of MDS and thus reflect the disease progression of MDS. Therefore, monitoring of WT1 expression levels has made continuous assessment of the disease progression of MDS possible, as well as the prediction of the evolution of RAEB or RAEB-t to overt AML within 6 months. The results also showed that quantitation of WT1 expression levels is useful for diagnosis of minimal residual disease of MDS with high sensitivity, thus making it possible to evaluate the efficacy of treatment for MDS.

Clinical implications of telomerase activity levels in acute leukemia.

In the present study, we used the telomeric repeat amplification protocol assay, an internal telomerase assay standard, and an automatic DNA sequencer to detect and quantitate telomerase activity in blood samples obtained from normal and acute leukemia patients. Telomerase activity was analyzed in 78 acute leukemia patients and ranged from 0.65 to 147 relative to the internal standard. Compared to the age-matched normal levels of telomerase activity in the peripheral blood cells, we determined that 45 (81.8%) of 55 acute myeloid leukemia (AML) and 16 (69.6%) of 23 acute lymphoid leukemia patients had elevated telomerase activity. There was no relationship between peak telomere length and telomerase activity in both acute lymphoid leukemia and AML patients. In AML, the level of telomerase activity was associated with French-American-British subtypes and cytogenetics, and patients with elevated telomerase activity had high leukocyte counts and more frequent extramedullary involvement during the disease. Among 78 patients, 5 had high levels of telomerase activities similar to immortalized leukemia cell lines; these 5 patients had a very poor prognosis (P < 0.05). The levels of telomerase activity significantly decreased in patients in complete remission. Most of the patients in complete remission showed a normal level of telomerase activity; however, two of them had low to moderate telomerase activity, and they relapsed shortly after entering complete remission. In relapsed patients, there is a general trend for increased telomerase levels, and 2 of the 13 patients retained high telomerase activity, whereas the other 11 had normal to moderate telomerase activity. These results suggest that telomerase activity may be a useful additional method for monitoring the disease condition in acute leukemia patients.

Telomere stability is frequently impaired in high-risk groups of patients with myelodysplastic syndromes.

Genomic instability induces an accumulation of genetic changes and may play a role in the pathogenesis of myelodysplastic syndromes (MDS). To clarify the possible association between genomic instability and clinical outcome in MDS patients, we compared telomere dynamics to the recently established International Prognostic Scoring System (IPSS) risk groups for MDS. We measured the terminal restriction fragments (TRFs) of 93 patients with MDS at the time of diagnosis, and telomerase activity was analyzed in 62 patients with MDS using the PCR-based telomeric repeat amplification protocol (TRAP) assay. A total of 53 of 93 MDS patients had TRFs within the age-matched normal range, and the remaining patients showed shortened TRFs (35 patients) or elongated TRFs (5 patients). MDS patients with shortened TRFs had a significantly low hemoglobin concentration (P = 0.04), a high percentage of marrow blasts (P = 0.02), and a high incidence of cytogenetic abnormalities (P < 0.05). The incidence of leukemic transformation was significantly high in patients with shortened TRF length (P < 0.05). In addition, patients with shortened TRF length were frequently seen in the IPSS high-risk group (P < 0.01). Most of the MDS patients had normal-to-low levels of telomerase activity, suggesting that changes in TRF length rather than telomerase activity may more accurately reflect the pathophysiology of MDS. MDS patients with shortened TRF length had a very poor prognosis (P < 0.01), suggesting that telomere dynamics may be linked to clinical outcome in MDS patients. Thus, an abnormal mechanism of telomere maintenance in subgroups of MDS patients may be an early indication of genomic instability. This study demonstrates that telomere stability is frequently impaired in a high-risk group of MDS patients and suggests that, in combination with the IPSS classification system, measurement of TRFs may be useful in the future to stratify MDS patients according to risk and manage the care of MDS patients.

Replication errors in hematological neoplasias: genomic instability in progression of disease is different among different types of leukemia.

Genetic alteration, including genomic instability, is an ultimate step toward the malignant process. One approach to delineating replication errors in cancer cells is to determine the alterations of microsatellites, which are short, repeated nucleotide sequences existing throughout the genomes. We used a fluorescent system to assess microsatellite changes in seven loci (D2S123, D3S643, D5S107, LPL, D17S261, TP53, and D18S34) of 73 consecutive patients with various hematological neoplasias. De novo acute leukemia patients had a low frequency (<1%) of microsatellite alterations at each locus, and none of them demonstrated multiple microsatellite changes. In chronic myeloid leukemia patients, no microsatellite instability was detected in the chronic phase, whereas a relatively high frequency (25%) of multiple microsatellite changes was evident in the blastic phase, and half of these patients had multiple microsatellite changes. About 50% of the patients with myelodysplastic syndrome (MDS) and post-MDS acute myeloid leukemia (post-MDS AML) had microsatellite alterations. We next compared microsatellite alterations in two different hematological phases (MDS and post-MDS AML phases); 5 of 11 patients with post-MDS AML had de novo appearance of microsatellite instability during disease progression. This indicates that genomic instability at multiple microsatellite loci could occur either before or after leukemic transformation in MDS patients. We concluded that genomic instability in chronic myeloid leukemia might be linked to blastic transformation in combination with cytogenetic changes. In contrast, MDS patients had replication errors as a relatively early genetic event as well as a late genetic event. These results suggest that the involvement of genomic instability in the progression of disease is different among various types of leukemia.

Epstein-Barr virus detection in kidney biopsy specimens correlates with glomerular mesangial injury.

To determine the relationship between the detection of Epstein-Barr virus (EBV)-specific DNA and glomerular injury, 33 renal needle-biopsy specimens that had been formalin-fixed and paraffin-embedded were analyzed using polymerase chain reaction (PCR) with subsequent nonradioactive Southern blot technique. Light microscopic examination and immunofluorescence were also performed. In 30 of 33 renal biopsy specimens, the beta globin gene could be successfully amplified as integrity controls. These 30 patients consisted of 12 patients with immunoglobulin A nephropathy (IgAN), 10 patients with minor glomerular abnormalities, 6 patients with membranous nephropathy, and 2 patients with focal/segmental lesions. EBV was detected in 7 of 12 patients with IgAN (58%), 3 of 6 patients with membranous nephropathy (50%), 0 of 10 patients with minor glomerular abnormalities (0%), and 2 of 2 patients with focal/segmental lesions. EBV detection was not disease specific. The EBV detection ratio of the group with glomerular mesangial lesions (64%; 9 of 14 patients) was significantly greater than those without (19%; 3 of 16 patients; P < 0.012, chi-square test). The EBV detection ratio of the group with glomerular lesions (60%; 12 of 20 patients) was significantly greater than those without (0%; 0 of 10 patients; P < 0.0016, Fisher's exact test), and the EBV detection ratio of the group with fibrinogen deposits observed in immunofluorescence (73%; 11 of 15 patients) was significantly greater than those without (7%; 1 of 15 patients; P < 0.0002, chi-square test). The EBV detection ratio of the group with immunoglobulin deposits (57%; 12 of 21 patients) was also significantly greater than those without (0%; 0 of 9 patients; P < 0.0040, Fisher's exact test). These data suggest that EBV can damage the glomerular mesangium beyond disease units and be mediated by immunoglobulin in patients with various chronic glomerulonephritides.

Refractory anemia with ringed sideroblasts with a low IPSS score progressed rapidly with de novo appearance of multiple karyotypic abnormalities and into acute erythroleukemia (AML-M6A).

We report here a case of refractory anemia with ringed sideroblasts (RARS) with a low risk group by the International Prognostic Scoring System (IPSS) at the time of diagnosis but had a rapid disease progression. Although the patient showed a normal male karyotype at the time of RARS diagnosis, his marrow cells had del(5)(q14) and add(17)(p12) abnormalities 2 months after the diagnosis, and later the marrow cells had multiple abnormalities and the patient expired 6 months after the initial diagnosis of RARS. The patient was diagnosed as having RARS with a low risk group by the IPSS classification, however, one should keep in mind that some patients with myelodysplastic syndromes with low risks by either the French-American-British (FAB) classification or the IPSS classification may have progressive disease and subsequential cytogenetic analysis could predict the disease progression.

Impaired telomere regulation mechanism by TRF1 (telomere-binding protein), but not TRF2 expression, in acute leukemia cells.

Telomere regulation is suggested to be an important mechanism in cellular proliferation and cellular senescence not only in normal diploid cells but also in neoplastic cells, including human leukemia cells. We studied the possible correlation among telomere length, telomerase (a ribonuclear protein that synthesizes the telemeres de novo) activity, hTERT (a catalytic subunit of telomerase) expression, and TRF1 and TRF2 (telomere DNA binding proteins) expression in human acute leukemia cells. The hTERT expression level was strongly associated with telomerase activity (P=0.0001), indicating that the expression level of the catalytic subunit (hTERT) regulates telomerase activity in human acute leukemia cells. TRF1 expression, which is believed to control telomere length, was significantly elevated in patients with acute lymphoblastic leukemia (ALL) (P=0.0232) compared to those in acute myeloid leukemia (AML); TRF1 expression tended to be higher in patients without telomere shortening (P=0.077) and in those with hTERT expression (P=0.055). This indicates that TRF1 may act to monitor telomere length under the condition of up-regulated telomerase activity in some neoplastic cells. In contrast, TRF2 expression in acute leukemia did not show any correlation with telomere parameters in this study. Although the precise regulation mechanism of telomere length is still uncertain, these results may suggest that regulation of telomere length is partially associated with TRF1 expression, whereas dysfunction of TRF1 expression may be speculated in a subset of acute leukemia.

Telomerase reactivation in leukemia cells.

We utilized fluorescent-labeled primers and modified the telomeric repeal amplification protocol (TRAP) to assess telomerase activity during the process of in vitro establishment in four different leukemia cell lines. We demonstrate that three of four leukemia cell lines had elevated telomerase activity, shortened telomeres, and the appearance of either dicentric chromosomes or acentric fragments. By contrast, one leukemia cell line (HAL-01) had high levels of telomerase activity, stable telomeres, but no obvious additional cytogenetic rearrangements. These experiments indicate that there may be several pathways involving telomerase activity in the establishment of leukemia cell lines.

Serum soluble CD44 levels for monitoring disease states in acute leukemia and myelodysplastic syndromes.

To determine the clinical implications of soluble CD44 (sCD44) levels in hematologic neoplasias, we developed an enzyme-linked immunosorbent assay for sCD44 using two monoclonal antibodies to the standard 90 kDa form, and assessed the serum concentration of sCD44 in normal healthy volunteers, patients with acute leukemia, myelodysplastic syndromes (MDS), and those with chronic myeloid leukemia (CML). Compared to that in normal individuals (n=51; 145. 1 24.6 ng/ml), the serum sCD44 level was significantly elevated in patients with acute myeloid leukemia (AML; n=18; 331.9 99.0 ng/ml, P=0.0001), acute lymphoid leukemia (ALL; n=16; 551.3 427.8 ng/ml, P=0.0001) and CML (n=18; 262.0 97.5 ng/ml, P=0.0001). The sCD44 level was slightly elevated in patients with MDS (n=43; 173.8 54.9 ng/ml, P=0.0071). In patients with acute leukemia, serum sCD44 concentrations decreased significantly in response to treatment and reached nearly normal levels after complete remission (P=0.0005 in AML and P=0.0032 in ALL). The sCD44 levels in patients with MDS increased after they developed acute leukemia, whereas no significant difference in sCD44 levels was observed between the chronic and the blastic phases in patients with CML. Our results indicate that serum sCD44 levels may be a useful marker for monitoring response to treatment and disease progression, especially in acute leukemia.

Time course of activated coagulation time at various sites during continuous haemodiafiltration using nafamostat mesilate.

OBJECTIVE: To determine the adequate site of activated coagulation time (ACT) measurement during continuous haemodiafiltration (CHDF) using nafamostat mesilate. DESIGN: Prospective, consecutive, clinical study. SETTING: An intensive care unit of a general hospital. PATIENTS: Ten patients with acute organ failure including kidney, lung and liver, caused by sepsis after major surgery. INTERVENTIONS: A CHDF circuit with a haemofilter made of polymethylmethacrylate membrane was primed with 50 mg nafamostat in 500 ml saline, and was started at a circuit flow rate of 100 ml/min. Continuous injection of 0.5 mg/kg per h nafamostat, 700 ml/h of dialysis fluid and 1000 ml/h of filtrate fluid was set. MEASUREMENTS AND RESULTS: The circuit pressure at the inlet and outlet of the circuit were monitored, and ACT was measured every 2 h at the site of nafamostat injection, outlet, patient's artery and inlet until 24 h. A value of standard deviation of ACT at each site was regarded as the variation value of ACT. The circuit pressure did not change significantly. The ACT did not change significantly at any measurement site. The variation value of ACT at the inlet of the circuit was significantly lower than that at the site of nafamostat injection. CONCLUSIONS: The ACT value at the inlet of the circuit may be adequate to monitor anticoagulation during CHDF using nafamostat.

Near-hexaploid Ph-positive acute myeloid leukemia with major-BCR/ABL transcript.

We describe the first case of acute myeloid leukemia (AML) with a Philadelphia (Ph) translocation and a near-hexaploid range chromosome number, whose leukemic cells had the major-BCR/ABL transcript. The genesis of near-hexaploid leukemic cells might be due to endoreduplication of triploid leukemic cells with the Ph, since the relapsed leukemic cells had triploid range chromosomes with double Ph chromosomes.

Chronic lymphocytic leukemia complicated by plasmacytoma originating from different clones.

In a woman with chronic lymphocytic leukemia (CLL), a plasmacytoma developed on the back region after four years. CLL cases complicated with plasmacytoma are rare. In the present case, the plasmacytoma showed kappa cytoplasmic immunoglobulin (Ig), and the CLL showed gamma lambda surface Ig. To reveal the clonal origin of CLL and plasmacytoma, we analyzed Ig gene rearrangements in the patient's peripheral blood and plasmacytoma. Ig gene DNA analysis confirmed the presence of different rearrangements in the heavy and light chain genes of CLL and plasmacytoma. These findings suggest that in this patient, the two B cell malignancies arose from expansion of two phenotypically and genotypically distinct clones.

Telomere dynamics in myelodysplastic syndromes and acute leukemic transformation.

Myelodysplastic syndromes (MDS) are characterized by cytopenias in the blood and dysplastic features in the hematopoietic cells. Although the impact of cytogenetic abnormalities is considerable for prognosis, the exact genetic mechanism of MDS remains undetermined. In this study we assessed cytogenetic changes, microsatellite alterations, and telomere dynamics in order to obtain further insight into the pathogenesis of MDS. Thirty-three percentage of MDS patients and 60% of post-MDS acute leukemia (post-MDS AML) had de novo microsatellite changes. In the MDS phase, however, > 60% of patients showed reduction of telomere lengths without microsatellite changes, indicating that telomere reduction in most MDS patients does not seem to be directly linked to genome instability, or that reduction of telomere length does not induce microsatellite changes in the MDS phase. Some MDS patients had microsatellite changes without telomerase elevation, indicating that genome instability might accumulate during the disease progression in some MDS patients, and this condition (cellular senescence) may be related to ineffective hemopoiesis in MDS patients. In contrast, 40% of post-MDS AML patients had elevated telomerase activity with microsatellite changes, indicating that approximately 40% of patients with post-MDS AML patients had accumulation of genome instability resulting in elevated telomerase activity in an attempt to obtain genetic stability. However, the remaining MDS patients had microsatellite changes without telomerase up-regulation, suggesting that some MDS had genome instability even after leukemic transformation. Most MDS patients with elevated telomerase activity in the AML phase had elevated telomerase activity even in the MDS phase without apparent change in telomere length before and after leukemic transformation. These findings indicate that telomerase activity in the MDS phase may be independent of telomere length, although telomere shortening seems to be related to genomic instability, and this process may be linked to apoptosis of MDS cells.