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.

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.

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.

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.

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.

Synthesis of structurally unstable type III procollagen in patients with cerebral artery aneurysm.

The biosynthesis of collagen was studied in skin fibroblast cultures established from 11 patients with cerebral artery aneurysms. Six patients had familial subarachnoid hemorrhage (SAH), while five patients were considered as sporadic cases. The structural stability of the triple-helical medium procollagen was studied by measuring the thermal denaturation temperature (Tm) of type I and type III procollagen molecules. Structural instability of type III procollagen was demonstrated in two patients with familial SAH. The Tm of type III procollagen was 39.0 degrees C and 39.5 degrees C in two of the cell lines, while the control value was 40.3 degrees C. The stability of type I procollagen did not differ from that of the controls, and the main features of the biosynthesis of collagen were similar in the aneurysm patient cell lines and in the controls. The results suggest that a structural defect of type III procollagen may serve as an etiological factor in the formation of cerebral artery aneurysms.

Expression of the c-kit proto-oncogene in myeloproliferative disorders and myelodysplastic syndromes.

The c-kit proto-oncogene is the receptor gene for the stem cell growth factor. Little is known about the distribution and role of this gene product in malignant hematopoiesis. We analysed here the expression of c-kit in myeloproliferative disorders (MPDs), including chronic myelogenous leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), and idiopathic myelofibrosis (IMF) and in the myelodysplastic syndromes (MDS). The c-kit expression of peripheral blood mononuclear cells was measured both at the messenger RNA level using Northern analysis, the RNA dot blot technique with densitometric quantification, the sensitive reverse transcription polymerase chain reaction, and at the protein level using immunofluorescence with monoclonal antibodies. There was a statistically significant increase in c-kit messenger levels in CML, ET, PV, IMF, and MDS as compared with controls (healthy volunteers). The percentage of c-kit protein expressing cells was also higher than in the controls in these disorders. There was a significant correlation of the c-kit protein expression with the CD34 antigen of the cells. Expression correlated with the phase of the disease, being highest in the blast crisis of CML and in the RAEB/RAEBt phases of MDS. The data suggest that increased amounts of circulating stem/progenitor cells with c-kit receptor are found in MPDs and MDS. It is possible that elevated c-kit expression could maintain the affected clone in MPDs and MDS.

Calcitonin gene methylation in chronic myeloproliferative disorders.

Alterations in DNA methylation appear to be an integral part of the malignant transformation. For example, the p15 region of chromosome 11 with multiple genes related to cell growth regulation exhibits different methylation patterns in the 5' area of the calcitonin A gene in healthy bone marrow cells, and in leukemic cell populations. In this work the methylation status of the 5' area of the calcitonin gene in myeloproliferative disorders (MPD) other than chronic myeloid leukemia (CML) is studied. A total number of 37 patients with polycythemia vera, essential thrombocythemia, or myelofibrosis were studied. A control group of 18 healthy persons and patients with reactive hematologic changes was included. The DNA isolated from peripheral blood or bone marrow cells was digested with the methylation-sensitive HpaII restriction enzyme. A Southern blot was hybridized with a 1.7 kb probe specific to the 5' area of the calcitonin gene. The result was visualized autoradiographically and analyzed with a densitometer. The results have been expressed as ratios between the abnormal and normal autoradiography band intensities, referred to as the calc-value or CALC. An increase in the calc-value signifies increasing methylation. In the control group the calc-value had a mean of 0.274. The myelofibrosis patients exhibited very strong hypermethylation in the calcitonin gene 5' area, with a mean calc-value of 11.1 (median 2.6). The polycythemia vera patients showed considerable variation in their methylation status, with a mean value of 1.52. The essential thrombocythemia patients exhibited weak hypermethylation, with a mean calc-value of 0.58. A correlation between karyotypic abnormalities and hypermethylation was observed. Complicated forms of MPD exhibited higher levels of methylation than the uncomplicated disease forms.

Hypermethylation of the calcitonin gene in the myelodysplastic syndromes.

It is well documented that the calcitonin gene area in the short arm of chromosome 11 is hypermethylated in most acute leukemias as well as in chronic lymphatic leukemia. In contrast, the gene is normally methylated during the chronic phase of the chronic myeloid leukemia but turns hypermethylated as the disease escalates. As the methylation of the calcitonin gene correlates with the disease activity in chronic myeloid leukemia, it seemed worthwhile to study the gene methylation in other premalignant hematologic conditions with a potential to terminate in fulminant acute leukemia. We report here on the calcitonin gene methylation in patients with myelodysplastic syndromes (MDS) using a methylation sensitive restriction enzyme HpaII and standard Southern blotting techniques. Bone marrow aspirates from a total of 26 MDS patients were studied. In 24 of these patients, the calcitonin gene was hypermethylated. There was no correlation between the methylation status and the morphological stage of the disease. All six patients with a blast count < 5% had a hypermethylated gene. Of the 19 patients with a blast count > 5%, 17 were hypermethylated only two having normal methylation status of the gene. It appears that the hypermethylation of the calcitonin gene area in the short arm of chromosome 11 may be an early event in the pathogenesis of the myelodysplastic syndromes. The methylation analysis may thus be of value as a diagnostic tool in MDS but an abnormal methylation pattern does not seem to have a direct relation with the degree of blast infiltration.

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.

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.

Effects of long-term verapamil treatment on blood pressure, cardiac hypertrophy and collagen metabolism in spontaneously hypertensive rats.

The effects of long-term treatment with verapamil on blood pressure, cardiac hypertrophy and collagen content, collagen concentration and prolyl hydroxylase activity were studied in spontaneously hypertensive rats (SHR). Verapamil administration (0.75 mg . ml-1 in drinking water) was commenced: to pregnant SHR 3 to 5 days before delivery and continued to the mothers and offspring during the nursing period; or to SHR at 10 weeks of age. Both groups were maintained on verapamil treatment up to the age of 45 weeks. Verapamil treatment significantly decreased blood pressure, heart rate and the ratio of ventricular weight to body weight in treated SHR. Verapamil did not significantly change the cardiac collagen concentration and prolyl hydroxylase activity. Since, however, the cardiac muscle mass was diminished by verapamil administration, treatment actually slightly reduced the collagen content of the heart. In the aorta collagen concentration was increased by verapamil treatment. Contrary to these results, minoxidil treatment was observed to increase the cardiac collagen concentration, content and prolyl hydroxylase activity in SHR. These results suggest that the factors governing myocardial connective tissue proliferation and regression may be independent of those governing muscle fibre hypertrophy and that particular drug actions on myocardial collagen metabolism must be taken into account.

Collagen gene expression in keloids: analysis of collagen metabolism and type I, III, IV, and V procollagen mRNAs in keloid tissue and keloid fibroblast cultures.

Regulation of collagen gene expression was studied in keloids and fibroblast cultures established from keloid biopsies from 9 patients. The collagen concentration in keloid tissue was not different from that in normal skin. The activities of 2 enzymes catalyzing intracellular collagen biosynthesis, prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT) were significantly elevated in the keloids, the mean increase in the former enzyme being 5-fold and in the latter 3-fold with respect to the controls. The mean procollagen production rate in the keloid fibroblasts was at the control level, with only 1 keloid cell line showing a procollagen synthesis rate higher than the mean value + 2 SD of the controls. The mean PH and GGT activities of the keloid fibroblasts were not elevated, but PH activity in 2 cell lines and GGT activity in 1 cell line were higher than the mean + 2 SD for the controls. Cellular type I, III, IV, and V procollagen mRNAs were measured by slot blot hybridization using specific human cDNA clones for the various collagen types. The amounts of type I, III, and V procollagen mRNAs corresponded to the ratios in which these collagen types are produced by fibroblasts. No synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. The total amount of type I and III procollagen mRNAs correlated significantly (p less than 0.01) with the procollagen synthesis rate measured after radioactive labeling of the cells in the keloid and control fibroblasts, indicating that collagen production in these cells is mainly controlled by regulating the final steady state levels of collagen mRNA. The results suggest that fibroblasts isolated from keloids often synthesize normal amounts of collagen.

Necrobiosis lipoidica: ultrastructural and biochemical demonstration of a collagen defect.

Ten patients with necrobiosis lipoidica lesions were studied. Five patients had diabetes mellitus. The age of the patients varied from 15 to 73 years and the duration of the skin lesions was from 2 to 20 years. Histologically, the lesions were characterized by degeneration of collagen and elastin. In some lesions elastin fibers could be seen in areas devoid of normal-looking collagen. Electron microscopy revealed loss of cross-striation of collagen fibrils and a marked variation in the diameter of individual collagen fibrils. The concentration of collagen, measured by assay of hydroxy-proline, a collagen-specific amino acid, was markedly decreased in the lesional skin, but the ratio of type I/III collagen was unchanged in the affected skin. Fibroblasts established from affected skin synthesized less collagen than cells derived from healthy-looking skin. The decreased collagen synthesis was due to a decreased amount of messenger RNA for type I procollagen, measured by hybridization with a specific human cDNA clone. The production of collagenase by these fibroblasts was not increased. Our results thus indicate that in necrobiosis lipoidica lesions, collagen fibrils are defective and the amount of collagen is reduced, probably due to decreased synthesis of collagen by affected fibroblasts.

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.

All-trans retinoic acid combined with interferon-alpha effectively inhibits granulocyte-macrophage colony formation in chronic myeloid leukemia.

We investigated the effect of all-trans retinoic acid (ATRA) alone and in combination with interferon-alpha (IFN-alpha) on the granulocyte-macrophage (GM) colony formation of peripheral blood progenitors isolated from patients with chronic myeloid leukemia (CML) (n = 12) or other myeloproliferative disorders (n = 10) as well as from healthy controls (n = 7). The ATRA or IFN-alpha alone inhibited slightly, but not significantly, the GM colony growth in CML. Granulocyte-macrophage colony formation decreased significantly (P<0.05) when ATRA and IFN-alpha were combined (114 +/- 96 versus 74 +/- 53 colonies/10(4) mononuclear cells). The combination did not have any inhibitory effect on the other MPDs. In healthy controls, ATRA or IFN-alpha alone or their combination stimulated GM colony growth, the increase being from 22 +/- 9 to 39 +/- 16 colonies for ATRA (P<0.05), up to 47 +/- 12 colonies for IFN-alpha (P<0.05) and up to 50 +/- 19 colonies for the combination (P<0.05). In conclusion, ATRA combined with IFN-alpha inhibits GM colony growth in CML. This combination may be worth testing clinically as a treatment of CML.

Spontaneous granulocyte-macrophage colony growth by peripheral blood mononuclear cells in myeloproliferative disorders.

In the present study, the ability of peripheral blood (PB) progenitor cells to form granulocyte-macrophage (GM) colonies spontaneously in methylcellulose was investigated in healthy controls and patients with myeloproliferative disorders (MPDs). Spontaneous colony formation was observed in only one of the 18 control cases (6%), but in 22 of the 29 MPD patients (76%). The incidence of spontaneous GM colonies correlated both with the number of blast cells and the amount of c-kit positive cells present in the initial sample. Spontaneous GM colony growth in PB mononuclear cells isolated from patients with MPDs seems to be a frequent phenomenon in contrast to the healthy controls and may present a marker of malignancy.

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.