Improvement of criteria for refractory cytopenia with multilineage dysplasia according to the WHO classification based on prognostic significance of morphological features in patients with refractory anemia according to the FAB classification.

In the criteria of refractory cytopenia with multilineage dysplasia (RCMD) according to the WHO (World Health Organization) classification, the frequency threshold concerning dysplasia of each lineage was defined as 10%. To predict overall survival (OS) and leukemia-free survival (LFS) for patients with refractory anemia (RA) according to the French-American-British (FAB) classification, we investigated prognostic factors based on the morphological features of 100 Japanese and 87 German FAB-RA patients, excluding 5q-syndrome. In the univariate analysis of all patients, pseudo-Pelger-Huet anomalies >or=10% (Pelger+), micromegakaryocytes >or=10% (mMgk+), dysgranulopoiesis (dys G) >or=10% and dysmegakaryopoiesis (dys Mgk) >or=40% were unfavorable prognostic factors for OS and LFS (OS; P<0.001, LFS; P<0.001). The prognostic effects of the morphological features were similar in both Japanese and German patients. However, dys Mgk >or=10% was not correlated with OS and LFS. In the multivariate analysis, mMgk+ and dys Mgk>or=40% were adverse prognostic factors for OS for all patients, and dys G >or=10% and dys Mgk>or=40% were adverse prognostic factors for LFS for all patients. On the basis of the present analysis, we propose the following modified morphological criteria for RCMD. Modified RCMD should be defined as FAB-RA, excluding 5q-syndrome with dys G >or=10%, dys Mgk>or=40% or mMgk+.

A new method for measurement of bone deformation by echo tracking.

No method has been available to noninvasively detect bone deformation or strain under loading in vivo. We focused on ultrasonic measurement of the displacement at a certain point on a bone using the echo-tracking method (ET). To develop a method that can noninvasively detect bone deformation in vivo, a preliminary investigation was performed. We investigated the accuracy of measuring displacement with our echo tracking system by using a flat metal panel and found that the method could measure displacement with a precision of a few microns. A three-point bending test of a porcine tibia with both ends fully constrained was performed to measure bone surface displacement, and simultaneous measurement of the surface strain was done using two strain gauges. The correlation between the displacement measured by ET and the strain gauge readings was completely linear (r=0.999), showing that the method could precisely detect bone deformation. The loads versus displacement curves obtained with cyclic loading were typical hysteresis loops that showed viscoelastic properties of the measured bone. We also improved a multi-ET system capable of simultaneously tracking multiple points to detect deformation of the bone surface. Measurement by this echo tracking system was also compared with strain gauge readings during a three point bending test with both ends of the tibia supported. The linearity of both methods was very high (r=0.998). Our ET method might have considerable potential for noninvasive measurement of bone viscoelasticity and plasticity.

Biologic and biochemical analyses of p16(INK4a) mutations from primary tumors.

BACKGROUND: Point mutations in the tumor suppressor gene p16(INK4a) (also known as p16, CDKN2, MTS1, and INK4a) are found in many tumor types. Because the function of the products of these naturally occurring mutants has not been fully explored, we investigated the functional activities of a wide range of naturally occurring p16 mutant proteins. METHODS: Sixteen cancer-associated p16 mutant proteins, resulting from missense mutations, were characterized for their ability to bind and inhibit the cyclin-dependent kinases (CDK4 and CDK6) and to induce cell cycle arrest in G(1) phase. RESULTS/CONCLUSIONS: Among 16 mutants analyzed, nine had detectable functional defects. Three mutants (D84V, D84G, and R87P) had defects in CDK binding, kinase inhibition, and cell cycle arrest. The corresponding mutations are located in the third ankyrin repeat in a highly conserved region believed to form the CDK binding cleft. Three mutants (P48L, D74N, and R87L) had defects in kinase inhibition and cell cycle arrest. Among the 10 mutants with normal CDK binding and inhibitory activity, three mutants (N71S, R80L, and H83Y) had defects only in their ability to induce cell cycle arrest. Thus, p16 mutant proteins that retain CDK4 and CDK6 binding may have more subtle functional defects. All nine mutations leading to functional impairments mapped to the central portion of the p16 protein. Ankyrin repeats II and III appear more critical to p16 function, and mutations in ankyrin repeats I and IV are less likely to disrupt p16 function.

Fusion of ETV6 to fibroblast growth factor receptor 3 in peripheral T-cell lymphoma with a t(4;12)(p16;p13) chromosomal translocation.

Fusions of the ETV6/TEL gene to receptor or protein tyrosine kinases (TKs), such as PDGFRbeta, JAK2, ABL, ABL2, TRKC, and Syk, have been reported in various hematological malignancies. Expression of the resultant chimeric proteins is believed to lead to constitutive TK activity through activation by the helix-loop-helix (HLH) domain of ETV6. We identified a novel ETV6 partner gene, fibroblast growth factor receptor 3 (FGFR3), in a patient with peripheral T-cell lymphoma (PTCL) with a t(4;12)(p16;p13) translocation. The ETV6-FGFR3 transcript showed a fusion of exon 5 of ETV6 to exon 10 of FGFR3, resulting in an open reading frame for a chimeric protein consisting of the HLH domain of ETV6 and the TK domains of FGFR3. This is the first report of ETV6 and FGFR3 involvement in PTCL.

BRCA1 protein level is not affected by peptide growth factors in MCF10A cell line.

The breast cancer susceptibility gene (BRCA1) has been identified as a putative tumor suppressor on chromosome 17. We raised antibody against Ring-finger domain of BRCA1. The antibody recognizes a specific BRCA1 protein doublet of about 220 kD. The majority of BRCA1 protein is localized to the nuclear fraction of untreated MCF10A cells. Though BRCA1 is thought to be a growth suppressor gene, no change in BRCA1 protein level was found when MCF10A cells were arrested by growth factor deprivation or stimulation of cell proliferation by re-addition of growth factors. Furthermore the subcellular localization of the BRCA1 protein does not change throughout the cell cycle. These results suggest that BRCA1 may not be directly involved in the regulation of the cell cycle of breast cancer cell line.

Long survival of leukemic mice by repeated combination treatment of cyclophosphamide and recombinant human granulocyte colony-stimulating factor.

The therapeutic and hematological effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in combination with cyclophosphamide (CY) were investigated in a murine myeloid leukemia model. Ten daily administrations of rhG-CSF following CY prolonged the survival time of leukemic mice more than either agent alone. Hematological examination indicated that this effect was attributable to suppression with rhG-CSF of the leukemic repopulation after CY injection. In addition, rhG-CSF accelerated recovery from CY-induced neutropenia. Based on these hematological changes, a treatment regimen was established consisting of a single injection of CY on day 1 and daily injections of rhG-CSF on days 2-6; this combination treatment was given to the leukemic mice for up to four cycles, with a pause of one day between each cycle. The leukemic mice completed each cycle of treatment with few failures, and it resulted in a long survival time for the leukemic mice. The mean survival time of the mice receiving four cycles of treatment was 47 days, 30 days longer than that of the untreated mice. Hematological examination performed at the end of each cycle showed that the leukemic cell population was controlled at a level equal to or below the pre-treatment level, and peripheral blood neutrophils were maintained at a level equal to or above the normal level. These results indicate the possible effectiveness of combining rhG-CSF with chemotherapeutic drugs in controlling leukemic cell growth, and the effectiveness of rhG-CSF in enhancing neutrophil recovery after chemotherapy. However, it was found that the leukemic cells became resistant to treatment with rhG-CSF after four cycles of combination treatment, suggesting that great care should be taken in the clinical application of rhG-CSF, even when the growth of acute myelogenous leukemia cells is not apparently stimulated by it.

Suppression of the development of murine myeloid leukemia with granulocyte colony-stimulating factor by inducing apoptosis of leukemic cells.

We studied the antileukemic effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by using a radiation-induced murine myeloid leukemia cell line C2M-A5. Intravenous inoculation of C2M-A5 cells into C3H/He mice resulted in the development of myeloid leukemia. However, the leukemic death of the mice was completely suppressed by the subcutaneous injections of rhG-CSF. In order to clarify the mechanism of the suppression, effects of rhG-CSF on C2M-A5 cells were studied in vitro. While C2M-A5 cells grew exponentially in the absence of rhG-CSF, the viability, the growth, and the self-renewal capacity of C2M-A5 cells were all suppressed in cultures in the presence of rhG-CSF. Preincubation with rhG-CSF for 48 h deprived C2M-A5 cells of the ability to induce leukemia in syngeneic mice. Morphological examination revealed the appearance of apoptotic changes of C2M-A5 cells in cultures containing rhG-CSF over the 2-day incubation period. In gel electrophoresis, the DNA from C2M-A5 cells incubated with rhG-CSF for 48 h showed a ladder of degradated DNA bands compatible with apoptosis. From these results, we concluded that the apoptosis of C2M-A5 cells played a key role in the antileukemic effect of rhG-CSF.

Effects of recombinant human granulocyte colony stimulating factor (rG-CSF) on murine myeloid leukemia: stimulation of proliferation of leukemic cells in vitro and inhibition of development of leukemia in vivo.

We have established an experimental murine myeloid leukemia model and investigated the effects of recombinant granulocyte colony stimulating factor (rG-CSF) on myeloid leukemia in vitro and in vivo. rG-CSF stimulated colony formation by the leukemic cells in semisolid agar medium, and exponential growth of the clonogenic cells in suspension medium. Thus, rG-CSF was able to stimulate both the differentiation and self-renewal processes of the leukemic stem cells in vitro. However, 14 consecutive daily injections of rG-CSF prolonged the mean survival time of the mice implanted with the leukemic cells. This effect of rG-CSF was accompanied by a delay in the emergence of the blast cells in peripheral blood and by a decreased blast population in the spleen, suggesting that development of leukemia was suppressed in the rG-CSF-treated-mice. The prolongation of the survival time by rG-CSF was more evident when rG-CSF was administered in therapeutic combination with cyclophosphamide. These results indicate that the effect of rG-CSF on the development of leukemia is not exactly predicted from in vitro experiments.

A clonal study of hematopoiesis using the M27 beta probe: aberrant band patterns caused by incomplete digestion of a methyl-sensitive enzyme in the the inactive X-chromosome.

The M27 beta probe has been used to determine the clonality of human tumors, based upon X-chromosome inactivation. However, it occasionally gives rise to aberrant results. In this study, the M27 beta probe was used for clonal analysis in Japanese women with clonal stem cell disorders and in those with normal hematopoiesis. Restriction digestion with PstI indicated heterozygosity for the DXS255 locus in 41 out of 50 individuals (82%). Further digestion with HpaII in heterozygous women led to four distinct band patterns: I, both fragments were partially digested; II, either one of the two fragments was completely digested; III, a three-band pattern; and IV, neither fragment was digested. Of 21 hematologically normal females, 17 (81%) and four (19%) had patterns I and III, respectively. In some subjects with pattern I, imbalanced HpaII digestion in the two alleles was seen. Fifteen (65%) of the 23 patients with clonal stem cell disorders had pattern II, while the remainder (35%) had pattern IV. The normal tissues of three acute myeloid leukemia patients with pattern IV all revealed pattern I. It is possible that the aberrant band patterns could be caused by incomplete HpaII digestion in inactive X-chromosomes. In this study, we propose a hypothesis whereby, in normal tissues, aberrant cells, the DXS255 locus of which is not digested with HpaII despite their inactive status, would be mixed with cells demonstrating the usual methylation pattern. In normal tissues, complex of proportion of aberrant cells and skewed Lyonization could produce a variety of band patterns. If a cell with the usual methylation pattern proliferated monoclonally, pattern II would be seen: whereas if an aberrant cell proliferated, pattern IV would be demonstrated.

Granulocyte-colony stimulating factor induced apoptosis in radiation-induced murine leukemia cell line.

Cytokines regulate proliferation and differentiation of hematopoietic progenitor cells. Recently it has been clarified that physiological cell death, apoptosis plays important role of hematopoiesis. So we evaluated the effects of granulocyte-colony stimulating factor (G-CSF) on leukemic cells, especially focused on apoptosis. Intravenous inoculation of radiation-induced murine leukemia cell line, C2M-A5 into the parent C3H mice resulted in the development of myeloid leukemia. However, the leukemic death of the mice was completely suppressed by the daily subcutaneous injection of recombinant human (rh)G-CSF from the next day (Bessho M. et al., Leuk Res 1989:13:1001-1007). In the in vitro study using C2M-A5 cells, we found that apoptosis appears on the cells at 48 hours after addition of G-CSF in culture. The cells in this stage lost the leukemogenicity to C3H mice (Bessho M. et al., Leukemia 8:1185-1190:1994). To clarify the mechanism of the induction of apoptosis by G-CSF we studied cell cycle and molecular changes in C2M-A5 cells cultured in medium with or without rhG-CSF by means of using the flowcytometry and Northern and Western blot analyses. After addition of rh G-CSF to culture, C2M-A5 cells removed to S phase, next arrested at G0/G1 phase on and after 24 hours, and 48 hours later, apoptosis was observed. Overexpression of mRNAs for c-myc (3-24 hours later) and for p53 (6-24 hours later), were observed in the cell cultured in rhG-CSF administered medium with a concomitant down-expression of bcl-2 mRNA (from 6 hours later). Tyrosine-phosphorylated protein (17 kd) appeared at 48 hours after administration of rhG-CSF to cell culture. This protein was suggested for specific apoptosis induction by rhG-CSF. These results are summarized as follows. (1) rhG-CSF induced apoptosis to C2M-A5 and deprived its leukemogenicity to mice. (2) Induction of apoptosis was associated with cell cycle and correlated to the changes of the expression rates of c-myc,p53, and bcl-2. (3) Tyrosine kinase may play an important role in apoptosis induction to C2M-A5 by rhG-CSF.

Incidence and characteristics of clonal hematopoiesis in remission of acute myeloid leukemia in relation to morphological dysplasia.

We studied 34 patients in remission of acute myeloid leukemia (AML) by performing clonal analysis of peripheral blood polymorphonuclear (PMN) cells and mononuclear (MN) cells, using X-linked DNA polymorphisms, in conjunction with the assessment of morphological myelodysplastic changes, performed by a scoring method. Nine patients demonstrated a non-random or skewed X-chromosome inactivation pattern in PMN cells. Three of these nine patients had an apparently random pattern in MN cells (group A), whereas the remaining six patients demonstrated no difference between the inactivation patterns of PMN and MN cells (group B). The PMN cells of the other 25 patients showed a random X-chromosome inactivation pattern, and the patterns of the PMN cells did not differ from those of the MN cells (group C). The scores for myelodysplasia were high (> or = 4) in all three patients in group A, intermediate (2-3) in two patients and low (score < 2) in four patients in group B, and intermediate in five patients and low in 20 patients in group C. The duration of remission in patients with a myelodysplasia score of > or = 2 was significantly shorter than that of patients with a score of < 2 (P < 0.01). We conclude that clonal remission actually occurs with myelodysplastic features in some patients with AML (around 10%, group A). It is possible that this clonal analysis may not be sensitive enough to detect the preleukemic clone with myelodysplastic features when this clone constitutes only a minor population of remission hematopoiesis. To further elucidate the biology of such preleukemic clones it is essential to develop more sensitive molecular methods for the detection of genetic abnormalities specific to preleukemic hematopoiesis.

Refractory anemia with severe dysplasia: clinical significance of morphological features in refractory anemia.

Refractory anemia (RA) in myelodysplastic syndromes (MDS) are very heterogeneous diseases regarding their morphology, clinical features and survival. We proposed the new designations 'RA with severe dysplasia (RASD)' and 'RA with minimal dysplasia (RAminiD)'. In our criteria, RASD is considered present if a bone marrow (BM) examination shows Pseudo-Pelger-Huet anomalies of mature neutrophils > or =3% and/or micromegakaryocytes (mMgk) of megakaryocytes > or =10% in RA patients. RAminiD is defined as RA cases other than RASD. After the reclassification of 58 primary RA patients, the group was composed of 45 RAminiD and 13 RASD patients. The blast percentage in the BM and the frequency of cytogenetic abnormalities observed in the RASD patients were intermediate between those in the RAminiD and RAEB patients. The analysis of survival curves revealed differences among the three groups; the RASD patients had lower survival probabilities than those of the RAminiD group, and significantly higher probabilities than those of the RAEB group. (RAminiD vs RASD, P=0.06; RASD vs RAEB, P=0.004.) Our data indicate that in RA patients, RASD is a distinct subset of RA with an unfavorable clinical outcome.

New system for assessing the prognosis of refractory anemia patients.

Refractory anemia (RA) is a very heterogeneous disease regarding biological and clinical features. The International Prognostic Scoring System (IPSS) was useful for assessing the prognosis in the whole group of 219 myelodysplastic syndrome (MDS) patients. However, the IPSS was not sufficient in 132 RA patients. To predict survival and freedom from acute myeloid leukemia (AML) evolution, we investigated individual prognostic factors based on the clinical parameters (age, gender, morphologic features, cytopenias and cytogenetics) of 132 RA patients using univariate and multivariate analyses. Based on the results, we devised a new system for assessing the prognosis of RA patients. In our system, RA patients with pseudo-Pelger-Huët anomalies >/=3% were classified as high risk (12 patients); of patients without pseudo-Pelger-Huët anomalies >/=3%, those with intermediate/poor karyotype according to IPSS, Hb /=10% were classified as intermediate risk (57 patients); and those without high or intermediate risk were classified as low risk (67 patients). In our system, the analyses of both survival times and leukemia-free survival times revealed significant differences among the three groups (P < 0.0001).

I kappa B-mediated apoptotic gene therapy against acute myelogenous leukemia using replication-defective HSV-1 vector expressing TK and mutant I kappa B alpha.

Overexpression of NF-kappa B reportedly plays anti-apoptotic roles in the growth of AML cells. Control of AML cell growth was attempted using a replication-defective herpes simplex virus-1 vector, T0I kappa B alpha, overexpressing mutant I kappa B alpha to inhibit NF-kappa B in vitro. T0I kappa B alpha displays defective ICP4/ICP22/ICP27, isogenic thymidine kinase, and mutant I kappa B alpha. T0Z.1 expressing lacZ instead of I kappa B was used for controls. Infection of T0I kappa B alpha at 15 multiplicity of infection (MOI) with cells of AML lines, HL60, K562, and NB4 displaying >90% infection efficiency and tumor killing in vitro. Use of 10 microM of Ara-C alone was clinically equivalent to high-dose Ara-C, displaying 11% tumor killing. Neither ganciclovir (GCV) nor Ara-C enhanced T0I kappa B- alpha mediated tumor killing. Attenuation of NF-kappa B by T0I kappa B alpha was confirmed by EMSA. T0I kappa B alpha induced caspase-3 activity, with subsequent apoptosis confirmed by colorimetric and TUNEL assays. Fresh AML cells from 8 patients were infected with T0I kappa B alpha at 3 MOI, with or without GCV or 10 microM of Ara-C in vitro. Infection efficiency was 10%. T0I kappa B alpha displayed 8-15% tumor killing, superior to Ara-C in 6 of the 8 patients. Administration of Ara-C enhanced tumor killing in 5 of these 6 cases. Our results suggest that T0I kappa B alpha-mediated gene therapy induces apoptosis of AML cells in vitro.

Effects of chloramphenicol on hematopoietic inductive microenvironment.

Effects of chloramphenicol (CP) on hematopoietic inductive microenvironment (HIM) were studied using in vitro and in vivo assay systems. HIM was represented as fibroblast colonies (CFUF) in in vitro culture. CP suppressed the growth of not only granuloid committed progenitor cells (CFUC), but also CFUF in in vitro culture at the concentration of 10, 50 and 100 micrograms/ml. To analyze the function of HIM in vivo, the subcutaneous bone implantation method was used. The recovery of hematopoietic stem cells in subcutaneously implanted femora of mice, which were treated with a 500 mg/kg dose of CP daily for 6 days, was significantly decreased compared to the sham treated group. Suppressive effect of CP on HIM was shown. The important role of the derangement of HIM on the pathogenesis of CP-induced aplastic anemia was discussed.

Differential effects of TGF-beta 1 on normal and leukemic human hematopoietic cell proliferation.

We evaluated the effects of transforming growth factor-beta 1 (TGF-beta 1) on the growth of hematopoietic progenitors in normal donors and in patients with hematologic malignancies now designed as clonal disorders of multipotential stem cells. TGF-beta 1 at 80 pM exhibited differential effects on the normal hematopoietic progenitors when cells were stimulated with different growth factors, such as G-CSF, GM-CSF, interleukin-3 (IL-3), or stem cell factor (SCF). The suppressive effect by TGF-beta 1 was increased for growth with GM-CSF, IL-3, and SCF, and growth with G-CSF was unaffected in hematologic malignancies, TGF-beta 1 suppression for growth with G-CSF was increased for essential thrombocythemia (ET) and polycythemia vera; chronic myelogenous leukemia (CML) in chronic phase; CML in accelerated phase; CML in myeloid crisis; myelodysplastic syndrome (MDS) in refractory anemia; MDS in refractory anemia with an excess of blasts; and acute myeloblastic leukemia (AML). In CML-myeloid crisis and AML, TGF-beta 1 almost completely abolished the growth, with some patient-to-patient variation. The mean ED50s for the growth of leukemic blast progenitors were 1.6, 1.2, 0.7, and 0.2 pM in the presence of G-CSF, GM-CSF, IL-3, and SCF, respectively, c-myc and c-myb antisense oligonucleotides significantly suppressed the growth of leukemic blast progenitors, but not that of clonogenic cells from normal donors and patients with ET. We also demonstrated that TGF-beta 1 inhibits mRNA expression by AML blasts for c-myc and/or c-myb. When the data are taken together, growth suppression by TGF-beta 1 appears to increase with the progression of clonal evolution in hematologic malignancies.

Differential regulation by hematopoietic growth factors of apoptosis and mitosis in acute myeloblastic leukemia cells.

We evaluated the effects of various hematopoietic growth factors (HGFs) on the prevention of apoptosis in blasts from 19 patients with acute myeloblastic leukemia (AML) by assessing DNA ladder formation. After incubation without HGF, apoptosis was noted in all but two patients. HGFs prevented, did not affect, or enhanced apoptosis in 39 (60%), 14 (22%), or 12 (18%) of 65 suspension cultures, respectively. HGFs that prevented apoptosis also stimulated and/or synergized blast colony formation in 35 of 39 corresponding methylcellulose cultures. HGFs that alone stimulated colony formation also prevented apoptosis in all but two of 28 corresponding suspension cultures. In contrast, HGFs that did not prevent apoptosis also failed to stimulate growth in 17 of 26 corresponding methylcellulose cultures. HGFs that enhanced apoptosis alone never stimulated colony formation. After incubation, we noted enhanced c-fos and cjun genes as well as induction of p21 protein. An appropriate dose of HGF elevated c-fos, reduced c-jun and p21, induced G1/S transition, and inhibited apoptosis. In two patients, apoptosis was not induced after incubation. Cells not treated with HGF expressed no c-fos, c-jun, or c-myc, and remained in G0/G1. Taken together, our results support the conclusion that not only c-fos, cjun, and c-myc, but also p53 and p21 are required for blast apoptosis. HGF differentially prevents apoptosis and induces mitosis, and both events seem to be integral to the self-renewal of AML clonogenic cells.

Poor response of Wv/Wx mice to a grafted neutrophilia-inducing, colony-stimulating-factor-producing tumor.

Although mice possessing two mutant genes at the W locus have a defect in multipotential hematopoietic stem cells that form macroscopic colonies in the spleen of irradiated mice, the number of neutrophils in the blood of these mutant mice is normal or nearly normal. We investigated neutrophil production using the NFSA fibrosarcoma of C3H mouse origin, which induces neutrophilia accompanied by production of a neutrophil-macrophage colony-stimulating factor by the tumor. When the NFSA tumor was transplanted to (C57BL/6 X C3H/He)F1-Wv/Wx or to congenic +/+ mice, neutrophilia developed in mice of both genotypes. However, there was a significant difference between the degree of neutrophilia that developed in them; there was a 107-fold increase in the +/+ mice, but only a 28-fold increase in the Wv/Wx mice four weeks after tumor transplantation. This result is consistent with the concept that doubly heterozygous W mice have multipotential stem cells with diminished ability to respond to stimulation. The unperturbed condition may not provide a sufficient stimulus to demonstrate the defect in neutrophil production in doubly heterozygous W mutant mice.

Acute ischaemic preconditioning and chronic hypoxia independently increase myocardial tolerance to ischaemia.

OBJECTIVE: Myocardial adaptation has been reported to result from mild but chronic ischaemia in the hearts of patients with coronary artery disease. The aim of this study was to test the hypothesis that the responses of the chronically hypoxic myocardium to an episode of severe ischaemia, or the effects of acute ischaemic preconditioning on myocardial function after subsequent fatal ischaemia, may differ between the normoxic and the chronically hypoxic myocardium. METHODS: A rat model of three week hypoxia (10% O2) was used to simulate tissue hypoxia caused by chronic ischaemia. In isolated isovolumetrically contracting hearts perfused with oxygenated erythrocyte-containing Tyrode solution, systolic and diastolic functions during a 15 or 20 min period of ischaemia and reperfusion were measured in the normoxic control and chronically hypoxic groups. RESULTS: Increases in diastolic pressure during ischaemia were smaller and the recovery of developed pressure during reperfusion was greater in the chronically hypoxic group than in the normoxic group. The hearts of the normoxic group never recovered from ischaemic damage after 20 min ischaemia. The beneficial effects of acute preconditioning with 5 min ischaemia on myocardial function were observed after 15 min ischaemia in the normoxic group, and during and after 20 min ischaemia in the chronically hypoxic group. Changes in lactate production and high energy phosphates could not explain the increased tolerance to ischaemia in the chronically hypoxic group. CONCLUSIONS: Chronic hypoxia increased myocardial tolerance to ischaemia, and acute ischaemic preconditioning increased the tolerance further. Thus chronic hypoxia and acute ischaemic preconditioning independently activate protective mechanisms against ischaemia; the mechanisms may differ between the two types of insult.

Roles of stem cell factor in the in vitro growth of blast clonogenic cells from patients with acute myeloblastic leukemia.

We investigated the effects of stem cell factor (SCF) on the growth of blast clonogenic cells from 27 patients with acute myeloblastic leukemia (AML) and 3 patients with chronic myelocytic leukemia in myeloid crisis. SCF alone showed a significant stimulatory activity in 15 of 30 patients (50%). A marked reduction in the number of blast cell colonies supported by SCF alone was noted by the addition of neutralizing antibody (Ab) against granulocyte-macrophage colony-stimulating factor (GM-CSF). Ab against interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) also moderately reduced the number of colonies, whereas Ab against granulocyte CSF (G-CSF) failed to do so. All four Ab together completely abolished the growth in 5 of 6 patients tested. c-kit antisense oligonucleotides reduced the colony formation supported by IL-3 or G-CSF or, in the absence of growth factor, in only 2 of 10 patients tested. SCF caused stimulation by acting synergistically with G-CSF, GM-CSF, IL-3, IL-6, IL-9, IL-11, and IL-12 in 20 of 27 (74%), 17 of 27 (63%), 14 of 28 (50%), 9 of 28 (32%), 1 of 15 (7%), 3 of 28 (11%), and 2 of 15 (13%) patients, respectively. Thus, SCF alone or in combination with some other factor stimulated the growth in 27 of 30 (90%) patients. Of 3 nonresponders, 2 were AML, M3 at presentation. G-CSF at the optimal concentration increased the sensitivity of blasts to SCF. Taken together, SCF acting in combination with other factors, but not alone, stimulates the growth of blast clonogenic cells. GM-CSF, IL-6, and TNF-alpha may be produced endogenously, whereas G-CSF and SCF may be supplied exogenously. Autocrine regulation of the growth of blasts seems to increase the responsiveness of the cells to any of these factors, allowing them to achieve a highly active growth state.