Placenta-mediated pregnancy complications are not associated with fetal or paternal factor V Leiden mutation.

OBJECTIVE: Maternal thrombophilia is a risk factor for adverse pregnancy outcomes. The aim of this study was to elucidate the controversial role of fetal and paternal thrombophilia in the development of severe placenta-mediated pregnancy complications. STUDY DESIGN: The study group comprised 126 mothers, 72 fetuses and 58 fathers. 111 mothers, 50 fetuses and 91 fathers acted as controls. 106 couples were selected to study the thrombophilias of paternal inheritance, 58 from the study group and 48 from the control group. The prevalence of factor V Leiden mutation, prothrombin G20210 A mutation and homozygous 10-methylenetetrahydrofolate reductase C677 T mutations were compared between the study and control groups to study whether maternal, fetal or paternal thrombophilias increase the risk of severe preeclampsia, intrauterine growth restriction, placental abruption and stillbirth. RESULTS: The total prevalence of fetal thrombophilic mutations was 8.3% in the study group and 14.0% in the control group. Paternal prevalence of thrombophilic mutations was 6.8% and 4.3%, respectively. There were no statistical differences between fetal or paternal thrombophilic mutations between the study and control groups. CONCLUSION: Fetal or paternal factor V Leiden mutation is not associated with severe placenta-mediated pregnancy complications.

Novel non-neutral mitochondrial DNA mutations found in childhood acute lymphoblastic leukemia.

Mitochondria produce adenosine triphosphate (ATP) for energy requirements via the mitochondrial oxidative phosphorylation (OXPHOS) system. One of the hallmarks of cancer is the energy shift toward glycolysis. Low OXPHOS activity and increased glycolysis are associated with aggressive types of cancer. Mitochondria have their own genome (mitochondrial DNA [mtDNA]) encoding for 13 essential subunits of the OXPHOS enzyme complexes. We studied mtDNA in childhood acute lymphoblastic leukemia (ALL) to detect potential pathogenic mutations in OXPHOS complexes. The whole mtDNA from blood and bone marrow samples at diagnosis and follow-up from 36 ALL patients were analyzed. Novel or previously described pathogenic mtDNA mutations were identified in 8 out of 36 patients. Six out of these 8 patients had died from ALL. Five out of 36 patients had an identified poor prognosis genetic marker, and 4 of these patients had mtDNA mutations. Missense or nonsense mtDNA mutations were detected in the genes encoding subunits of OXPHOS complexes, as follows: MT-ND1, MT-ND2, MT-ND4L and MT-ND6 of complex I; MT-CO3 of complex IV; and MT-ATP6 and MT-ATP8 of complex V. We discovered mtDNA mutations in childhood ALL supporting the hypothesis that non-neutral variants in mtDNA affecting the OXPHOS function may be related to leukemic clones.

Discovery of novel drug sensitivities in T-PLL by high-throughput ex vivo drug testing and mutation profiling.

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase-signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.

Kinetics of leukocyte CD11b and CD64 expression in severe sepsis and non-infectious critical care patients.

BACKGROUND: Leukocyte surface molecules may improve sepsis diagnostics. Our aim was to study whether monocyte and neutrophil CD11b and CD64 expression differs between patients with severe sepsis (including septic shock) and intensive care unit (ICU) controls, and also to investigate the expression kinetics in patient groups. METHODS: Monocyte and neutrophil CD11b and CD64 expression was analyzed in 27 patients with severe sepsis, 7 off-pump coronary artery bypass (OPCAB) patients, and 8 ICU patients without systemic inflammation in the beginning of the treatment using quantitative flow cytometry. Blood samples were collected within 48 h of the beginning of severe sepsis, at admission to the ICU for non-systemic inflammatory response syndrome patients, and on the day of surgery before the skin incision for OPCAB patients, and on 2 consecutive days for all patients. Ten healthy individuals served as controls. RESULTS: Monocyte and neutrophil CD11b and neutrophil CD64 expression was higher in severe sepsis patients compared with the other groups (P < 0.05). In severe sepsis, the expression decreased over time (P < 0.05). In OPCAB patients, the monocyte and neutrophil CD64 expression increased after surgery (P < 0.05). Neutrophil CD64 expression had the highest and statistically significant area under curves (AUC) values for identification of severe sepsis during 3 consecutive days, the highest AUC being 0.990 on D0. CONCLUSION: Neutrophil CD64 as well as neutrophil and monocyte CD11b expressions were highest in severe sepsis compared with non-infectious conditions, and thus analyses of their expression may be promising approach for sepsis diagnosis in ICU population.

Comparison of the use of minimized cardiopulmonary bypass with conventional techniques on the incidence of retinal microemboli during aortic valve replacement surgery.

OBJECTIVES: Minimized cardiopulmonary bypass (MCPB) circuits have been shown to reduce cerebral and retinal microembolisation during coronary artery bypass graft (CABG) surgery compared to conventional CPB (CCPB) circuits. Our aim was to evaluate whether the reduction of microembolisation is sustained in aortic valve surgery, as well as to evaluate the effects of MCPB on inflammatory, endothelial, and platelet activation markers. MATERIAL AND METHODS: Patients were randomized to undergo aortic valve replacement (AVR), with or without CABG, with MPCB (n=20) or CCPB (n=20). After anaesthesia induction and termination of CPB, standardized digital retinal fluorescein angiography images were obtained on both eyes and analyzed in a blinded fashion. Blood samples were collected at eight time points until the third postoperative day. RESULTS: Fewer patients in the MCPB group showed evidence of microembolic perfusion defects on postperfusion retinal fluorescein angiographs compared to the CCPB group (37% vs. 63%, absolute difference 26%, 95% CI -5% -51%, P = 0.194). Polymorphonuclear leukocyte (PMN) elastase and von Willebrand factor release were statistically significantly reduced in the MCPB group, but there were no significant differences in other markers of inflammation, coagulation or endothelial activation. A significantly higher three-fold increase in the amount of shed blood was collected to the cell saver with a higher rate of intraoperative platelet transfusion in the MCPB group compared to CCPB. CONCLUSIONS: The use of MCPB was associated statistically insignificantly with less retinal microemboli compared to CCPB. MCPB was complicated by excess bleeding and need for transfusion. The feasibility of MCPB techniques in valve surgery requires further studies.

Polymorphism of the manganese superoxide dismutase gene but not of vascular endothelial growth factor gene is a risk factor for diabetic retinopathy.

BACKGROUND: In diabetic retinopathy, the vascular endothelium is damaged due to oxidative stress and inflammation, and vitreous VEGF concentration becomes elevated. The association of diabetic retinopathy with single nucleotide polymorphisms (SNPs) was studied on two genes: VEGF, an important mediator of neovascularisation, and MnSOD, a major antioxidant enzyme. METHODS: The study population was 755 individuals consisting of 131 diabetic (type 1 or type 2) patients with diabetic retinopathy (DR group), 98 diabetic controls without retinopathy (DC group) and 526 non-diabetic controls. VEGF SNPs rs699947, rs2010963, rs2146232, rs3025033, rs3025039 and Ala16Val polymorphism of the MnSOD gene were genotyped. RESULTS: The frequencies of allele and genotype of the single genotyped VEGF SNPs or reconstructed haplotypes of these single SNPs did not differ between DR and DC groups. A higher frequency of the AlaAla genotype (p = 0.03) and Ala16 allele (p = 0.04) of the MnSOD gene in the DR group was found when compared with the DC group. CONCLUSIONS: In conclusion, the studied VEGF SNPs were not associated with the risk of diabetic retinopathy, and so it is unlikely that the VEGF gene is a major locus determining the risk of diabetic retinopathy. A statistically significant association of MnSOD Ala16Val polymorphism with diabetic retinopathy was found.

Cord compression may rapidly influence the expression of placental angiogenic genes in pre-eclampsia.

Gene expression studies have demonstrated the altered expression level of placental angiogenesis related genes in severe pre-eclampsia (PE). In cord compression, the transportation of oxygen from the placenta to the fetus is blocked, and it is speculated that during blockade the originally hypoxic placenta may become hyperoxic. We compared the placental gene expression profiles of one pre-eclamptic patient with cord compression (the index patient) to the profiles of patients with PE and those of normal pregnancy controls (including one woman with cord compression). The gene expression of the cord compression PE patient resembled that observed in the normal pregnancies. We hypothesize that umbilical blockade may in a short period of time lead to placental hyperoxia, which in turn has an effect on angiogenic gene expression profile.

Development of permanent national register of blood component use utilizing electronic hospital information systems.

BACKGROUND AND OBJECTIVES: We wanted to establish a permanent national database system, which can be utilized to study transfusion recipients and blood use in Finland. MATERIALS AND METHODS: A regularly updated register for permanent use was developed. To study the usability of the database, years 2002 and 2003 were further analysed. Database included all transfused patients in major blood-transfusing hospitals from four university and five central hospital districts managing altogether 63% of Finnish inpatient hospital episodes. RESULTS: Audit of gathered data reveal 96.8% match in adult blood components with Finnish Red Cross, Blood Service sales figures. Model data set includes 59,535 transfused patients (44.3% men and 55.7% women) having received 529,104 blood components. Half of all blood units were transfused in connection with surgical operations. Most of the blood recipients were elderly (51.6% are over 64 years of age). Blood-component use and transfusion-related costs varied widely between hospitals. CONCLUSION: Hospital data managing systems can be useful for creating a population-based database system to monitor and compare transfusion practices. This record provides information about transfusion epidemiology for transfusion professionals, hospital management, and hospital administration.

Evaluation of factor V Leiden, prothrombin and methylenetetrahydrofolate reductase gene mutations in patients with severe pregnancy complications in northern Finland.

BACKGROUND: Thrombosis in placenta may lead to severe pregnancy complications. Most important inherited thrombophilias are factor V Leiden mutation, prothrombin mutation, and methylenetetrahydrofolate reductase mutation. The aim of our research was to evaluate the prevalence of inherited thrombophilias in severe pregnancy complications and in normal pregnancies. MATERIAL AND METHODS: The study subjects with severe preeclampsia, intrauterine growth restriction, placental abruption or fetal death were collected during the period 1999-2004 from Oulu University Hospital. We also collected during the same period voluntary parturients with normal pregnancy outcome as the control group. FVL, FII, and MTHFR gene mutations of the patients and controls were analyzed. RESULTS: We found a significant difference in the prevalence of FVL mutation between the groups. There were 9.5% FVL mutations in the study group compared to 1.8% in the control group; the observed difference between prevalences was 7.7% (95% CI 2.0-13.4). No statistical difference was found in the FII or MTHFR mutations between the groups. All FV and FII mutations were heterozygous and all the MTHFR mutations homozygous. CONCLUSION: Women with thrombophilia have a risk for severe pregnancy complications. Randomized controlled trials are needed to assess the influence of low-molecular-weight heparin in pregnant women with thrombophilia.

The -1C to T polymorphism in the annexin A5 gene is not associated with the risk of acute myocardial infarction or sudden cardiac death in middle-aged Finnish males.

OBJECTIVE: A common polymorphism (-1C to T) in the translation initiation sequence of annexin A5 (ANV) gene has recently been associated with a decreased risk of acute myocardial infarction (AMI). The aim of the present study was to analyze the association between the ANV genepolymorphism and the risk of AMI and ischemic sudden cardiac death (SCD) in middle-aged Finnish males. MATERIAL AND METHODS: A case-control study involving three distinct groups of subjects was carried out: (1) victims of SCD (n=98), (2) survivors of AMI (n=212), and (3) randomly selected control subjects without any history of coronary heart disease (n=243). The ANV polymorphism was genotyped in each study group. RESULTS: Among the control group of healthy Finnish males the prevalence rates of the CC, CT, and TT genotypes were 83.1%, 15.2%, and 1.6%, respectively. Among the survivors of AMI, the prevalence rates of CC, CT, and TT were 79.7%, 20.3%, and 0%, respectively, and among the victims of SCD 83.7%, 16.3%, and 0%, respectively. No significant differences in the genotype or allele distributions were observed between the study groups. CONCLUSION: The -1C to T polymorphism in the ANV gene is not associated with the risk of AMI or SCD in middle-aged Finnish males.

A multicentre study of reference intervals for haemoglobin, basic blood cell counts and erythrocyte indices in the adult population of the Nordic countries.

Eight haematological quantities were measured in EDTA anticoagulated venous blood specimens collected from 1826 healthy male and female individuals between 18 and 90 years of age in the Nordic countries (Denmark, Finland, Iceland, Norway and Sweden). The samples, collected between November 1999 and November 2001 as part of the Nordic Reference Interval Project (NORIP), were analysed on 12 different types of modern automated haematology instruments currently in use among the 60 laboratories participating in the study. Non-parametric reference intervals (between 2.5 and 97.5 percentiles) have been calculated for B-Haemoglobin (females 117-153 g/L, males 134-170 g/L), B-Erythrocytes (females 3.94-5.16 x 10(12)/L, males 4.25-5.71 x 10(12)/L), B-EVF (females 0.348-0.459, males 0.395-0.500), B-MCV (82-98 fL), Erc-MCH (27.1-33.3 pg), Erc-MCHC (317-357 g/L), B-Trc (females 165-387 x 10(9)/L, males 145 x 348 x 10(9)/L) and B-Lkc (3.5-8.8 x 10(9)/L). Partitioning of data according to age and gender was done according to a standardized procedure. For most variables the calculated reference intervals corresponded well with older and less well-defined reference intervals. The mean concentration of B-Haemoglobin increased by 0.08 g/L per year of age in women, and decreased by 0.1 g/L per year of age in men. B-Haemoglobin increased with body mass index in both men and women. Smoking increased the mean of B-Lkc by 1.1 x 10(9)/L and regular use of alcohol increased the mean of B-MCV by 0.8 fL. The influence of these factors was small overall and did not promote specific reference intervals.

Deletion of the Ink4-locus (the p16ink4a, p14ARF and p15ink4b genes) predicts relapse in children with ALL treated according to the Nordic protocols NOPHO-86 and NOPHO-92.

Inactivation of the Ink4 gene locus locus on 9p comprising the tumour suppressor gene p16ink4a and its neighbours p14ARF and p15ink4b is common in childhood acute lymphoblastic leukaemia (ALL), but the prognostic significance is controversial. DNA from 230 patients was retrospectively analysed by Southern blotting, single strand conformation polymorphism (SSCP) and sequencing techniques. The results were correlated with clinical characteristics and outcome. One hundred and ninety-four fully analysed patients, similarly treated using the Nordic NOPHO-86 or the current NOPHO-92 protocols, were included in the outcome analysis. Deletions approached a minimally deleted region between the p16ink4a and p15ink4b genes, making the p14ARF gene the most commonly deleted coding sequence. Bi-allelic deletion was associated with high white blood cell count (WBC) (P < 0.001), T cell phenotype (P < 0.001) and mediastinal mass (P < 0.001). Patients with Ink4 locus bi-allelic deletions had an inferior pEFS (P < 0.01) and multivariate analysis indicated that bi-allelic deletion of the p16ink4a and the p14ARF genes was an independent prognostic risk factor (P < 0.05). Sub-group analysis revealed a pronounced impact of deletion status for high-risk patients, ie with high WBC. Deletion-status and clinical risk criteria (WBC) could thus be combined to further differentiate risk within the high-risk group. The analysis of the Ink4 locus adds independent prognostic information in childhood ALL treated by Nordic protocols and may help in selection of patients for alternative treatment.

p53 and redox state in etoposide-induced acute myeloblastic leukemia cell death.

We investigated whether p53, being a redox-sensitive protein, has a role in the responsiveness of AML cells to etoposide. Two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), were used as models. Sensitivity to etoposide was measured by trypan blue and annexin V assays. Etoposide-induced peroxide formation was associated with the induction of cell death. Evident expression of mutated p53 was observed in both subclones in basal growth conditions as analysed by Western blotting and flow cytometry. After etoposide exposure for up to 24 hours, some nuclear accumulation of p53 was observed in the ER subclone, as analysed by Western blotting. The conformation of p53, however, was not changed from mutated toward wild-type during exposure in either of the subclones as analysed by flow cytometry. In conclusion, etoposide-induced change in cellular redox state was associated with apoptosis, but was not a sufficient stimulus for p53 to make its conformation active. Thus, mutated p53 seems to have no role in etoposide-induced apoptosis.

Regulation of the acute myeloid leukemia cell line OCI/AML-2 by endothelial nitric oxide synthase under the control of a vascular endothelial growth factor signaling system.

It is generally accepted that the vascular endothelial growth factor (VEGF) signal system has no role in the maintenance of normal blood cell formation, although it obviously regulates the development of primitive hematopoiesis during an early stage of embryogenesis. The VEGF signaling pathway, however, might have some role in malignant hematopoiesis, since malignant hematopoietic cells, including acute myeloid leukemia (AML) cells, have been shown to express VEGF and its receptors. In endothelial cells, the VEGF/Flk-1/KDR signal system is a very important generator of nitric oxide (NO) through the activation of its downstream effectors phosphatidylinositol-3-OH-kinase (PI3-K), Akt kinase and endothelial NO synthase (eNOS). It is known that NO regulates hematopoiesis and modulates AML cell growth. The role of the VEGF signaling pathway in the control of AML cell growth through eNOS, however, has not been studied. By using the OCI/AML-2 cell line, which expresses VEGF receptor-2, ie Flk-1/KDR, eNOS and VEGF, as analyzed by flow cytometry, and produces VEGF into growth medium, as analyzed by ELISA, we showed that the Akt kinase and NOS activities in these cells were decreased by the inhibitors of VEGF, Flk-1/KDR and PI3-K, and NOS activity also by the direct inhibitor of NOS. The decreased NOS activity led to inhibition of clonogenic cell growth and, to some extent, induction of apoptosis. We also found that blast cells of bone marrow samples randomly taken from 14 AML patients uniformly expressed Flk-1/KDR and to varying degrees eNOS and VEGF, as analyzed by immunohistochemistry. We conclude that autocrine VEGF through Flk-1/KDR, by activating eNOS to produce NO through PI3-K/Akt kinase, maintains clonogenic cell growth in the OCI/AML-2 cell line. Since the patient samples did not express VEGF in all cases, it is possible that in vivo the regulatory connection between these two signal systems is also mediated via endocrine VEGF in addition to autocrine or paracrine VEGF.

HFE mutations do not account for transfusional iron overload in patients with acute myeloid leukemia.

BACKGROUND: Hereditary hemochromatosis (HH) is a HFE gene-linked disorder affecting 1 of 200 to 400 persons in white populations. It has been proposed that patients with a hematologic malignancy who are receiving frequent RBC transfusions should be screened for HFE mutations. This would identify C282Y homozygotes, who have a high risk of developing severe iron overload. STUDY DESIGN AND METHODS: DNA samples from 128 controls and 23 adult long-term survivors of acute myeloid leukemia (AML) treated at the Oulu University Hospital (Oulu, Finland) from 1987 to 2000 were examined for the presence of the C282Y and H63D mutations in HFE. All the patients were severely iron-overloaded, as determined from high serum ferritin values and/or increased storage iron in bone marrow. Phlebotomies were performed in five patients because of the symptoms of iron overload. DNA extracted from the blood was used to amplify HFE gene fragments by the PCR method, after which the amplification products were digested with restriction endonucleases SnaB I and Bcl I, and the restriction fragments were analyzed on agarose gels. RESULTS: No chromosomes with the C282Y mutation were found among the AML patients, and 5 patients (21.7%) were heterozygous for the H63D mutation. In the control group, 13 persons (10.2%) were heterozygous for the C282Y mutation and 26 (20.3%) for the H63D mutation, including 3 C282Y/H63D double heterozygotes. CONCLUSION: HFE mutations do not account for the harmful iron overload that develops in AML patients who receive large quantities of RBC concentrates after intensive chemotherapy.

c-erbB-2 positivity is a factor for poor prognosis in breast cancer and poor response to hormonal or chemotherapy treatment in advanced disease.

The aim of this work was to evaluate the prognostic and predictive values of c-erbB-2 in breast cancer. 650 patients were enrolled. The amplification/overexpression of c-erbB-2 from fresh frozen or paraffin-embedded breast tumour tissue samples was analysed by polymerase chain reaction (PCR) technique (75%), immunohistochemically (17%) or by Southern blot analysis (8%). 126 patients (19%) were positive for c-erbB-2. 148 patients developed metastatic disease, but only 35 were positive for c-erbB-2. Positivity for c-erbB-2 was significantly associated with node positivity, large tumour size, high grade of malignancy, low receptor status, postmenopausal status, and with a shorter overall survival. In multivariate regression analysis, only tumour size and nodal involvement were risk factors for poor survival when analysed separately together with c-erbB-2 and receptor status. Metastatic patients with c-erbB-2 positivity had a significantly shorter survival and disease-free survival (DFS) than the c-erbB-2-negative patients. 29 advanced patients with c-erbB-2 positivity showed a poor response rate to hormonal, non-anthracycline-based and anthracycline-based therapies. Positivity for the c-erbB-2 is a poor prognostic factor in breast cancer, but it also emerges as predictive of the response to hormonal or chemotherapy treatment once the disease has recurred.

MMP-2 and MMP-9 expression in adult and childhood acute lymphatic leukemia (ALL).

In the present study the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in acute lymphatic leukemia (ALL) is studied with immunocytochemical staining in bone marrow aspirate smears from 75 patients. The material included 20 adult and 55 pediatric ALL patients. In 65% of the adult ALL cases the immunoreactive protein for either MMP-2 or MMP-9 or for both the enzymes was seen. A statistically significant correlation (P 0.027) with MMP-2 positivity and appearance of extramedullary infiltrates was found in adult ALL, suggesting that the gelatinase activity could be related with increased extravasation of the leukemic cells in ALL. This has to be confirmed in a larger patient population. In pediatric patients there were only seven cases (12.7%) out of 55 with positive immunostaining for either of these metalloproteinases. MMP expression correlated with L2 FAB class, high-risk tumor group and T-cell immunophenotype, but not with extramedullary presentation in pediatric patients with ALL indicating basic biological differences between adult and childhood ALL.

Induction of mitochondrial manganese superoxide dismutase confers resistance to apoptosis in acute myeloblastic leukaemia cells exposed to etoposide.

We investigated the possible roles of mitochondrial manganese superoxide dismutase (MnSOD) and bcl-2 in etoposide-induced cell death in acute myeloblastic leukaemia (AML) using two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), as models. Cell death after 24 h exposure to 10 micromol/l etoposide was about 60% and 70% in the ES subclone and about 20% and 25% in the ER subclone, when analysed by trypan blue and annexin V respectively. Cytochrome c efflux from mitochondria to cytosol was observed after 4 h of exposure in both subclones, whereas the activation of caspase-3 was not detectable until after 12 h of exposure in the ES subclone and 24 h of exposure in the ER subclone, using Western blotting. The decrease in mitochondrial membrane potential, when analysed by the JC-1 probe fluorocytometrically, also appeared to take place later in the ER than in the ES subclone. Both subclones showed evident basal expression of MnSOD and bcl-2 by Western blotting. Etoposide caused a potent induction of MnSOD, more than 400% at 12 h, in the ER but not in the ES subclone. No significant change in bcl-2 expression could be observed in either of the subclones during exposure to etoposide when analysed by Western blotting or flow cytometry. In conclusion, we suggest that MnSOD might have a special role in the protection of AML cells against etoposide-induced cell death. Although unable to influence the cytochrome c efflux to cytosol, MnSOD might prevent the disruption of mitochondrial membrane potential, which evidently leads to cell death by releasing various activators of apoptosis.

Cellular redox state and its relationship to the inhibition of clonal cell growth and the induction of apoptosis during all-trans retinoic acid exposure in acute myeloblastic leukemia cells.

BACKGROUND AND OBJECTIVE: All-trans retinoic acid (ATRA) induces growth arrest and apoptosis in acute myeloblastic leukemia (AML) cells. Since cellular redox state regulates these events, we were interested in studying whether it has any role in the responsiveness of AML cells to ATRA. DESIGN AND METHODS: Two human AML cell lines, the ATRA-sensitive OU-AML-3, and the ATRA-resistant OU-AML-7, were used as models. Clonogenic cell culture assay, annexin V method, and measurement of mitochondrial membrane potential were used for the determination of cell growth and apoptosis. Peroxide formation was analyzed by flow cytometry, glutathione and g-glutamylcysteine synthetase (g-GCS) activity was determined spectrophotometrically, and the expression of manganese superoxide dismutase (MnSOD) by Western blotting. RESULTS: ATRA inhibited clonogenic cell growth and induced apoptosis particularly in OU-AML-3 cells. The OU-AML-7 cells had a higher basal level of glutathione and g-GCS activity than the OU-AML-3 cells. ATRA enhanced the generation of peroxides after 24h exposure, which was more prominent in the sensitive than the resistant cell line and was not preventable by N-acetyl-L-cysteine. ATRA also increased the activity of g-GCS, which was associated with increased intracellular glutathione in the resistant cell line, while the glutathione level was maintained in the sensitive cell line. During ATRA exposure, MnSOD was induced in the sensitive cell line, but not until after 72 h. Buthionine sulfoximine significantly increased the inhibitory effect of ATRA on colony formation in both cell lines, but only marginally enhanced the effect of ATRA on the induction of apoptosis. INTERPRETATION AND CONCLUSIONS: The balance between oxidative and antioxidative actions of ATRA, as well as the basal redox state of the cells seem to have a definite influence on the responsiveness of AML cells to ATRA.