Ionizing radiation-inducible miR-27b suppresses leukemia proliferation via targeting cyclin A2.

PURPOSE: Ionizing radiation is a common carcinogen that is important for the development of leukemia. However, the underlying epigenetic mechanisms remain largely unknown. The goal of the study was to explore microRNAome alterations induced by ionizing radiation (IR) in murine thymus, and to determine the role of IR-inducible microRNA (miRNA/miR) in the development of leukemia. METHODS AND MATERIALS: We used the well-established C57BL/6 mouse model and miRNA microarray profiling to identify miRNAs that are differentially expressed in murine thymus in response to irradiation. TIB152 human leukemia cell line was used to determine the role of estrogen receptor-α (ERα) in miR-27b transcription. The biological effects of ectopic miR-27b on leukemogenesis were measured by western immunoblotting, cell viability, apoptosis, and cell cycle analyses. RESULTS: Here, we have shown that IR triggers the differential expression of miR-27b in murine thymus tissue in a dose-, time- and sex-dependent manner. miR-27b was significantly down-regulated in leukemia cell lines CCL119 and TIB152. Interestingly, ERα was overexpressed in those 2 cell lines, and it was inversely correlated with miR-27b expression. Therefore, we used TIB152 as a model system to determine the role of ERα in miR-27b expression and the contribution of miR-27b to leukemogenesis. ß-Estradiol caused a rapid and transient reduction in miR-27b expression reversed by either ERα-neutralizing antibody or ERK1/2 inhibitor. Ectopic expression of miR-27b remarkably suppressed TIB152 cell proliferation, at least in part, by inducing S-phase arrest. In addition, it attenuated the expression of cyclin A2, although it had no effect on the levels of PCNA, PPARγ, CDK2, p21, p27, p-p53, and cleaved caspase-3. CONCLUSION: Our data reveal that ß-estradiol/ERα signaling may contribute to the down-regulation of miR-27b in acute leukemia cell lines through the ERK1/2 pathway, and that miR-27b may function as a tumor suppressor that inhibits cell proliferation by targeting cyclin A2.

Live cell detection of chromosome 2 deletion and Sfpi1/PU1 loss in radiation-induced mouse acute myeloid leukaemia.

The CBA/H mouse model of radiation-induced acute myeloid leukaemia (rAML) has been studied for decades to bring to light the molecular mechanisms associated with multistage carcinogenesis. A specific interstitial deletion of chromosome 2 found in a high proportion of rAML is recognised as the initiating event. The deletion leads to the loss of Sfpi, a gene essential for haematopoietic development. Its product, the transcription factor PU.1 acts as a tumour suppressor in this model. Although the deletion can be detected early following ionising radiation exposure by cytogenetic techniques, precise characterisation of the haematopoietic cells carrying the deletion and the study of their fate in vivo cannot be achieved. Here, using a genetically engineered C57BL/6 mouse model expressing the GFP fluorescent molecule under the control of the Sfpi1 promoter, which we have bred onto the rAML-susceptible CBA/H strain, we demonstrate that GFP expression did not interfere with X-ray induced leukaemia incidence and that GFP fluorescence in live leukaemic cells is a surrogate marker of radiation-induced chromosome 2 deletions with or without point mutations on the remaining allele of the Sfpi1 gene. This study presents the first experimental evidence for the detection of this leukaemia initiating event in live leukemic cells.

Membrane transport and apoptosis-related proteins in radiation-associated acute myeloid leukemia following the Chornobyl accident.

We report on the results of multidrug-resistance transporters (P-glycoprotein, LRP, and MDR1), and apoptosis-related proteins (Fas, Bcl-2, Bax, p53, and Bcl-X(L)) expression analysis of 56 acute myeloid leukemia (AML) patients by flow cytometry. Of these, there were 21 persons exposed to ionizing radiation due to the Chornobyl accident with radiation-associated and 35 patients with spontaneous AML. Leukemic cells in patients with radiation-associated AML more often overexpressed antiapoptotic protein Bcl-2 (12/21 vs. 6/35, p < 0.005) and less often demonstrated expression of Fas receptor (12/21 vs. 30/35, p < 0.05). Moreover, leukemic cells were simultaneously Fas negative and Bcl-2 positive in 4 out of 21 patients exposed to ionizing radiation but none of spontaneous cases had similar phenotype (p < 0.05). Patients with radiation-associated AML compared to spontaneous cases more often were P-glycoprotein positive (12/20 vs. 9/31, p < 0.05). P-glycoprotein overexpression significantly correlated with the resistance of the disease to chemotherapy in patients with radiation-associated AML (p < 0.05).

Comparison of murine gene expression profiles between spontaneous and radiation-induced myelogenous leukemias: stochastic and probabilistic expression variances in the former vs radiation-specific expression commonalities in the latter.

OBJECTIVE: To elucidate the common characteristics of murine radiation-induced myelogenous leukemias, global gene-chip expression profiles were compared with age-matched steady-state bone marrow tissue profiles and spontaneous myelogenous leukemia profiles. MATERIALS AND METHODS: Six each of C3H/He mice-derived radiation-induced and spontaneously developed myelogenous leukemias were analyzed. Bone marrow cells from five each of 2- and 21-month-old mice were used to subtract nonleukemic information in the analysis. mRNAs from individual mice were analyzed separately using 45,101 gene chips followed by computational biological analysis. RESULTS: First, principal component analysis (PCA) was performed to discriminate the gene expression profiles of individual mice with radiation-induced myelogenous leukemia from those of bone marrow cells from 2- or 21-month-old mice. Discriminant union genes for individual leukemias were then selected, which finally yielded 242 genes, among which six are radiation-related genes including Hus-1, Edf1a2, andVegf-c; 16 are apoptosis/cell-death-related genes, 13 are cell-cycle/cell-growth-related genes, and 50 are suppressor/promoter genes. PCA of these 242 genes consistently enabled the discrimination of the radiation-induced leukemias from the spontaneous leukemias. Second, the other components of the same PCA provided four different eigenvector clusters in an unsupervised manner representing four histopathological findings, with which the differential diagnosis in molecular taxonomy was significant as determined by analysis of variance of the global gene expression profiles. CONCLUSION: Discriminant union genes in radiation-induced myelogenous leukemias against spontaneous myelogenous leukemias and age-matched nonleukemic bone marrow profilings generated by unsupervised computational analysis essentially represent probabilistic biomarkers for radiation-induced myelogenous leukemias, which may contribute to developing a model for risk of secondary carcinogenesis in patients treated by whole-body irradiation.

Ongoing activation of p53 pathway responses is a long-term consequence of radiation exposure in vivo and associates with altered macrophage activities.

The major adverse consequences of radiation exposure, including the initiation of leukaemia and other malignancies, are generally attributed to effects in the cell nucleus at the time of irradiation. However, genomic damage as a longer term consequence of radiation exposure has more recently been demonstrated due to untargeted radiation effects including delayed chromosomal instability and bystander effects. These processes, mainly studied in vitro, are characterized by un-irradiated cells demonstrating effects as though they themselves had been irradiated and have been associated with altered oxidative processes. To investigate the potential for these untargeted effects of radiation to produce delayed damaging events in vivo, we studied a well-characterized model of radiation-induced acute myeloid leukaemia in CBA/Ca mice. Haemopoietic tissues of irradiated CBA/Ca mice exhibit enhanced levels of p53 stabilization, increased levels of p21(waf1), and increased amounts of apoptosis, as expected, in the first few hours post-irradiation, but also at much later times: weeks and months after the initial exposure. Because these responses are seen in cells that were not themselves directly irradiated but are the descendants of irradiated cells, the data are consistent with an initial radiation exposure leading to persistently increased levels of ongoing DNA damage, analogous to radiation-induced chromosomal instability. To investigate the potential source of ongoing oxidative processes, we show increased levels of 3-nitrotyrosine, a marker of damaging nitrogen/oxygen species in macrophages. Not all animals show increased oxidative activity or p53 responses as long-term consequences of irradiation, but increased levels of p53, p21, and apoptosis are directly correlated with increased 3-nitrotyrosine in individual mice post-irradiation. The data implicate persistent activation of inflammatory-type responses in irradiated tissues as a contributory bystander mechanism for causing delayed DNA damage.

Implications of hemopoietic progenitor cell kinetics and experimental leukemogenesis: Relevance to Gompertzean mortality as possible hematotoxicological endpoint.

OBJECTIVE: The aim of this study is to investigate a possible implication in cell kinetics of the hematopoietic progenitors to the experimental leukemogenesis to elucidate the relevance of various leukemic mode of action to Gompertzean survival curves, a new parameter based on the lifespan. MATERIALS AND METHODS: Mice, C3H/He, and C57BL/6 strain, male and female, with or without genetic modifications, e.g., p53-deficiency or thioredoxin overexpression were used in the present hemopoietic stem/progenitor research, radiation- or benzene-induced leukemogenesis followed by histopathological examination. A lethal dose of radiation for bone marrow transplantation, and a graded increased dose up to 5 Gy of x-rays for induction of hematopoietic malignancies were given. For caloric restriction studies, 77 kcal/week was maintained in accordance to different restriction-timing. For assays of hematopoietic colonization, colony-forming unit spleen and colony-forming unit granulocyte macrophage were evaluated. Hematopoietic progenitor cell-specific kinetics were studied by continuous labeling of bromodeoxyuridine for cycling cells, followed by ultraviolet (UV) exposure and hemopoietic colonization (bromodeoxyuridine UV [BUUV] method). Various experimental survival curves were applied to a mathematical analysis by Gompertz-Makeham law of mortality. RESULTS: Referring current authors' studies on leukemogenesis induced by ionizing radiation and benzene exposure, implications of hematopoietic progenitor cell kinetics to the experimental leukemogenesis were evaluated by means of a novel experimental tool, the BUUV method. Comparative studies to elucidate relevancies of these data, including two prevention studies, one on caloric restriction and the other on antioxidative thioredoxin overexpression, to those Gompertzean survival curves of experimental animals were analyzed. CONCLUSION: The Gompertzean expression may elucidate an appropriate toxicological endpoint for evaluating the effect of radiation and/or benzene-exposure on the lifespan and its modification by various experimental preventive measures.

Expression changes of ERK1/2, STAT3 and SHP-2 in bone marrow cells from gamma-ray induced leukemia mice.

The aim is to clarify expression changes of ERK1/2, STAT3 and SHP-2 in bone marrow cells from gamma-ray induced leukemia mice. A mouse model of gamma-ray induced leukemia was produced, and by means of quantitative real-time PCR, immunoprecipitation, Western blotting and electrophoretic mobility shift assays (EMSA), the expression of mRNA and protein, phosphorylation level, and protein activity of ERK1/2, STAT3 and SHP-2 in bone marrow cells were investigated in these mice. The results indicated that mRNA and protein expressions of ERK1/2 were upregulated, with significant increase of phosphorylation level and protein activity, but with insignificant differences in mRNA and protein expressions, phosphorylation level and protein activity of STAT3 and SHP-2 in bone marrow cells from gamma-ray induced leukemia mice compared to the radiation/tumor-free or control mice. It is concluded that in the pathogenesis of gamma-ray induced leukemia in Balb/C mice, activated ERK1/2 pathway may play a role, without involving STAT3 pathway; meanwhile, SHP-2 exerts no regulative effect on pathways of Ras-ERK1/2 and JAK-STAT.

Do magnetic fields cause increased risk of childhood leukemia via melatonin disruption?

Epidemiological studies have reported associations between exposure to power frequency magnetic fields and increased risk of certain cancer and noncancer illnesses. For childhood leukemia, a doubling of risk has been associated with exposures above 0.3/0.4 microT. Here, we propose that the melatonin hypothesis, in which power frequency magnetic fields suppress the nocturnal production of melatonin in the pineal gland, accounts for the observed increased risk of childhood leukemia. Such melatonin disruption has been shown in animals, especially with exposure to electric and/or rapid on/off magnetic fields. Equivocal evidence has been obtained from controlled laboratory magnetic field exposures of volunteers, although the exposure conditions are generally atypical of neighborhood exposures. In contrast, support for the hypothesis is found in the body of studies showing magnetic field disruption of melatonin in human populations chronically exposed to both electric and magnetic fields associated with electricity distribution. Further support comes from the observation that melatonin is highly protective of oxidative damage to the human haemopoietic system. Aspects of the hypothesis are amenable to further investigation.

[P-glycoprotein expression in radiation-associated acute myeloid leukemia].

The article presents findings of the analysis on P-glycoprotein expression of leukemic cells in 52 acute myeloid leukemia (AML) patients. Of these, there were 20 persons exposed to ionizing radiation due to the Chernobyl accident and 32 patients with spontaneous disease. Leukemic cells in patients with radiation-associated AML compared to spontaneous cases more often were P-glycoprotein positive (12/20 vs 9/32, P<0,05). P-glycoprotein overexpression significantly correlated with resistant disease in patients with radiation-associated AML, but was not a prognostic variable for treatment outcome in terms of overall survival.

Transient modifications of respiratory capacity in thymic cells during murine radioleukemogenesis.

The evolution of mitochondrial oxidative phosphorylation was studied during cancer induction in a model of thymic radiolymphomagenesis in C57BL/Ka mice. During the preneoplastic period, thymuses displayed an increase of the cytochrome c oxidase activity and oxygen consumption together with oxidative DNA damage assessed by the presence of the 8-hydroxydeoxyguanine DNA base modification. These transient changes in mitochondrial functional activity were not observed in thymuses of mice rescued from lymphoma development by a bone marrow graft, suggesting an important role of mitochondria for neoplastic transformation in this model, which might therefore be of interest to test the utilization of antioxidants for the prevention of radiation-induced malignancies.

Genetic imbalances in preleukemic thymuses.

To understand the molecular mechanisms involved in preleukemia, the suppression subtractive hybridization method was used in a murine radiation-induced thymic lymphoma model. Seventeen mRNAs overexpressed in preleukemic thymuses were identified: mouse laminin binding protein (p40/37LBP), E25 protein, Rattus norvegicus clone BB.1.4.1, profilin, poly(A) binding protein (PABP), mouse high mobility group protein 1, topoisomerase I, clusterin, proteasome RC1 subunit, rat prostatein C3 and C1 subunits; two ESTs and four unknown genes. The overexpression of PABP, clusterin, profilin, and the p40/37LBP mRNAs was confirmed in preleukemic thymuses and can be related to some cellular events observed during the preleukemic period, i.e., alterations of cell cycle and apoptosis properties. The p40/37LBP and 67-kDa laminin receptor proteins were upregulated during the preleukemic period. The data suggest that additional studies on p40/37LBP and 67-kDa laminin receptor regulation are required to evaluate their potential role in the lymphoma prevention by TNF-alpha and IFN-gamma.

Production of murine leukemia RLmale1 rejection antigen peptide pRL1a by proteolysis of natural precursor pRL1b.

In this study, we demonstrated that NH2-terminal Ser and Ile residues of pRL1b (SI-pRL1a) (SIIPGLPLSL) are not involved in the recognition by RLmale 1-specific cytotoxic T lymphocyte. The sensitization activity observed with pRL1b (SI-pRL1a) was not greater than that of peptides substituted with irrelevant amino acids at these positions. In serum-free medium, pRLla retained sensitization activity, but pRL1b (SI-pRL1a) did not. Furthermore, addition of bestatin to serum-containing medium blocked sensitization by pRL1b (SI-pRL1a). On the other hand, the addition of captopril enhanced it, probably by inhibiting the degradation of pRL1a by ACE. pRL1a-D peptide with D-Ile in place of the L-Ile residue of pRL1a (IPGLPLSL) showed sensitization, but SI-pRLla-2,3D peptide, which has D-Iles in place of the L-Ile residues of pRLlb (SI-pRL1a), and which was not cleaved between the two D-Iles, did not. The findings suggest that pRL1a is the antigenic peptide bound to L(d) molecules and pRL1b (SI-pRL1a) peptide is its natural precursor, which generates pRL1a via proteolysis.

Constitutive overexpression of the c-fos gene in radiation-induced granulocytic leukemia in mice.

Among various myeloid leukemias which were induced by X rays in C3H/He mice (Seki et al., Radiat. Res. 127, 146-149, 1991), the three most frequent types were analyzed for myeloperoxidase, c-myc, c-myb, and c-fos mRNAs. It was shown by in situ hybridization that all the component cells were positive for myeloperoxidase mRNA in granulocytic leukemia, whereas only half the cells were positive in myelomonocytic leukemia and none in monocytic leukemia. Granulocytic leukemia was also characterized by a persistently heightened expression of c-fos, while the other two types of leukemia showed negligibly low expression of the c-fos message. By contrast, both c-myc and c-myb were expressed to a similar extent in all three types of leukemia. When fresh granulocytic leukemia cells were transferred to culture in a medium containing 0.5% fetal calf serum, c-fos mRNA was decreased rapidly during incubation. The decay of c-fos mRNA was inhibited by cycloheximide markedly but was not changed significantly by actinomycin D. In the culture containing 10% fetal calf serum, the rate of decay of c-fos mRNA was slowed down significantly. Addition of dibutyryl cyclic AMP rapidly restored the c-fos expression and kept it elevated for at least 2 h in the cultured granulocytic leukemia cells. Phorbol ester (TPA) and calcium ionophore A23187 also caused a rapid but transient c-fos expression. A transient c-fos expression was inducible by TPA in the other two types of leukemia cells and in the granulocytic leukemia cells. The results suggest that the persistent expression of c-fos is distinguished from its transient expression and is characteristic for granulocytic leukemia cells as it is for normal mature granulocytes.

Production of a negative regulator of IL-3 by bone marrow cells in response to the supernatant of IL-3-producing STIL-3 leukemia cells.

When spleen cells of mice grafted with STIL-3 C5 cells, a leukemic T-cell line producing IL-3, are cultured in vitro, a high IL-3 activity is detectable in the culture supernatant. However, when bone marrow cells of the same grafted mice are cultured under similar conditions, hardly any IL-3 activity is detectable. To elucidate the mechanism of this difference, we examined whether the bone marrow cells either suppress IL-3 production by STIL-3 C5 cells or produce an IL-3 inhibitor. When STIL-3 C5 cells were cultured in the presence of normal bone marrow cells, the culture supernatant showed a significantly reduced IL-3 activity as assessed by growth stimulatory effects on IL-3-dependent DA-1 cells and mast cells. The conditioned medium (CM) did not inhibit the growth of IL-3-independent cell lines. Heat treatment of the CM resulted in a recovery of the IL-3 activity, indicating that the effect was mediated by a heat-labile inhibitor rather than by suppression of IL-3 production. CM of bone marrow cells alone did not inhibit the IL-3 activity. The inhibitor was produced by a stem cell-enriched fraction of the bone marrow, and not by fractions of T cells, granulocytes, or adherent cells including macrophages. Stimulation of the stem cell-enriched fraction of bone marrow with STIL-3 C5-CM also induced the production of the IL-3 inhibitor, which was recovered in a MW 50-100 kD fraction after ultrafiltration. These results suggest a possible presence of a feedback mechanism against the IL-3 effect on hemopoietic stem cells and progenitors in the bone marrow.

Definition and estimation of lifetime detriment from radiation exposures: principles and methods.

Although the lifetable methodology is a standard tool in epidemiology and risk assessment, there are a number of differences in the way it has been applied by various advisory committees that have attempted to estimate radiation risks. The most fundamental of these differences concerns the choice of parameter to be estimated: the "excess lifetime risk" is the difference in lifetime risks between exposed and unexposed populations; the "risk of exposure-induced death" is the lifetime risk of dying of a disease attributable to exposure. These two quantities are not the same, even at low doses. Although both quantities have some utility in risk assessment, the "risk of exposure-induced death" comes closer to capturing the total impact of exposure. Other differences between reported risk estimates include details of the calculations, the baseline rates and age distributions of the exposed population, the forms of the models for excess rates, handling of organ-specific doses, and the groupings of cancer sites. These issues are discussed theoretically and illustrated with comparisons of the BEIR V and UNSCEAR reports. Although the risk estimates from these two reports are similar for most cancer sites, it is shown that this happens to be the result of an approximate cancellation of a number of differences that could be quite large.

Iron, radiation, and cancer.

Increased iron content of cells and tissue may increase the risk of cancer. In particular, high available iron status may increase the risk of a radiation-induced cancer. There are two possible mechanisms for this effect: iron can catalyze the production of oxygen radicals, and it may be a limiting nutrient to the growth and development of a transformed cell in vivo. Given the high available iron content of the western diet and the fact that the world is changing to the western model, it is important to determine if high iron increases the risk of cancer.

Highly restricted tumor-specific antigen of radiation leukemia virus-induced murine leukemias.

A tumor-specific Ag expressed by the radiation leukemia virus-induced BALB/c leukemia BALBRVC has been serologically and biochemically identified with mAb of rat origin--RH221 and RH16. These mAb recognize two distinct epitopes on a Mr 85,000 glycoprotein. Expression of the RH221/16 Ag was also detected on two radiation leukemia virus-induced C57BL/6 leukemias, B6RV1 and B6RV2. The Ag was not detected on 66 other leukemias, 16 sarcomas, or 19 normal tissues tested. The results of sequential immunoprecipitation and partial peptide analyses of the RH221/16 Ag indicate that this tumor-specific Ag is immunologically and structurally distinct from murine leukemia virus-related determinants expressed on the three RH221/16+ leukemias.

Physiopathological studies on granulocyte-macrophage colony stimulating factor and multi colony stimulating factor producing leukemia, L8313, induced by irradiation of C3H mice.

Radiation-induced L8313 leukemia bearing mice (L8313 mice) had marked granulocytosis with splenomegaly. Hemopoietic stem cells and progenitors increased in the spleen but not in the bone marrow. Spleen conditioned-medium and serum from L8313 mice induced the formation of granulocyte-macrophage colonies (CFU-GM), erythroid bursts (BFU-E) and mixed colonies (CFU-Mix). Bone marrow conditioned medium did not show such activity. A cell line (STIL-3) was established from the spleen cells of L8313 mice. Surface marker analysis showed that the established cells were suppressor T cell. The cells produced IL-3 and GM-CSF in vitro, and induce essentially the same "leukemic" response in recipient mice. Inoculation of STIL-3 in diffusion chamber also induced leukemoid reaction, i.e. a marked granulocytosis with splenomegaly. Therefore, L8313 leukemia may be linked to an abnormality of growth and production of hemopoietic factors in hemopoietic regulatory cells.

Functional heterogeneity in macrophages activated by Corynebacterium parvum: characterization of subpopulations with different activities in promoting immune responses and suppressing tumor cell growth.

Peritoneal cells (PEC) from mice injected i.p. with heat-killed Corynebacterium parvum (CP) showed enhanced immunostimulatory (accessor or A cell) activity as measured by their ability to restore the immune responsiveness of nonadherent spleen cells to sheep erythrocytes (SRBC) and polymeric flagellin (POL) of Salmonella adelaide in vitro. This was true whether the PEC and nonadherent spleen cells were in direct contact or separated by a cell-impermeable membrane which allowed the free passage of soluble mediators. CP-activated PEC also exhibited greatly increased cytostatic activity against the growth of syngeneic tumor cells in vitro. After fractionation of the PEC according to cell size by velocity sedimentation, a separation of A cell activity from anti-tumor activity was observed. Although both these functions were associated with phagocytic cells of the monocyte-macrophage series, the highest A cell activity was found in fractions containing small and medium-sized macrophages, whereas the anti-tumor activity increased with cell size to a maximum with the largest macrophages. Thus, there is a relative increase of suppressive activity over stimulatory activity with an increase in cell size. Cytochemical and morphologic evidence suggests that the A cell-rich fractions contained small and medium-sized macrophages which were derived from newly arrived monocytes, whereas the large tumor-suppressive macrophages were relatively more differentiated.