LONG/TERM GENETIC AND EPIGENETIC DISORDERS IN PERSONS EXPOSED TO IONIZING RADIATION AND THEIR DESCENDANTS (review). / VIDDALENI GENETYChNI TA EPIGENETYChNI PORUShENNIa U OPROMINENYKh OSIB TA ÏKhNIKh NAShchADKIV (ogliad).

The review is devoted to long-term genetic and epigenetic disorders in exposed individuals and their descendants,namely to cytogenetic effects in the Chornobyl NPP accident clean-up workers and their children, DNA methylation as an epigenetic modification of human genome. Data presented in review expand the understanding of risk of the prolonged exposure for the present and future generations, which is one of key problems posed by fundamental radiation genetics and human radiobiology.

THE EXPRESSION OF THE MAIN AND ALTERNATIVE TRANSCRIPT (SORL1 Delta2) OF THE SORL1 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS AFFECTED BY THE CHORNOBYL ACCIDENT. / DOSLIDZhENNIa OSNOVNOGO I ALTERNATYVNOGO TRANSKRYPTU (SORL1 Delta2) GENA SORL1 U KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEAKEMIIu, POSTRAZhDALYKh VNASLIDOK ChORNOBYLSKA KATASTROFY.

OBJECTIVE: to study clinical-hematological data and expression of the main and alternative transcripts of SORL1 genein chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophe. METHODS: Analysis was performed in the main group of 34 CLL patients irradiated due to the Chornobyl NPP acci-dent (30 clean-up workers, and 4 evacuees) and in the control group of 27 non-irradiated CLL patients. Groups ofpatients were comparable by age, sex, stage of disease, mutational status of IGHV genes. Expression of the main andalternative transcripts of SORL1 gene was evaluated by Quantitative Real-time polymerase chain reaction (PCR). TheIGHV gene mutational status, TP53 and SF3B1 mutations were studied by PCR followed by direct sequencing. Data wereanalyzed with the SPSS software package, version 20.0. RESULTS: Relative expression level of the main transcript of SORL1 gene was low (mean 1.71 ± 0.55, median 0.57),did not correlate with the IGHV gene mutational status, TP53 and SF3B1 mutations, stage of disease. The expressionof B transcript was not detected, F transcript was expressed at a very low level in 9 patients. The average relativeexpression level of SORL1-Δ2 transcript was 14.1 ± 6.04 (median 3.48; range 0.01-90.51). The expression of SORL1-Δ2transcript above the median was more frequent among patients on C stage (p = 0.001), and in patients with unmu-tated IGHV genes was associated with an extremely negative course of CLL (median of overall survival 9 months vs61 months at low expression). Relative expression levels of the main and alternative transcripts of SORL1 gene inpatients of the main and the control groups did not differ. CONCLUSIONS: Our preliminary data suggest that increased expression of SORL1-Δ2 transcript in CLL patients withunmutated IGHV genes can be considered as a negative prognostic marker.

THYROID DISEASE IN THE LATE OBSERVATION PERIOD UPON CHEMO AND RADIOTHERAPY IN CHILDREN/SURVIVORS OF ACUTE LYMPHOBLASTIC LEUKEMIA. / KhVOROBY ShchYTOPODIBNOÏ ZALOZY PISLIa KhIMIO TA PROMENEVOÏ TERAPIÏ U DITEI, IaKI PERENESLY GOSTRU LIMFOBLASTNU LEIKEMIIu, U VIDDALENOMU PERIODI SPOSTEREZhENNIa.

OBJECTIVE: to assess the thyroid disease in the late observation period in children who had received chemo- andradiotherapy for the acute lymphoblastic leukemia (ALL) taking into account gender, age period and disease sub-type. MATERIALS AND METHODS: The incidence and nature of thyroid disease (hypothyroidism, thyroiditis, and thyroid can-cer) were studied in children-survivors of acute lymphoblastic leukemia (ALL) being in remission from 6 to 25 years.The distribution of patients by leukemia subtypes was as follows: «common¼ - 67.4 %, pre-B - 23.9 %, pro-B andT-cell - 4.3 %. Children had been receiving chemo- and radiotherapy according to the protocol. Regarding the ageof patients at the time of ALL diagnosis the prepubertal, pubertal and postpubertal periods were taken into account.The endocrine diseases in family history, body weight at birth, serum content of free thyroxine, pituitary thyroid-stimulating hormone, cortisol, iron, ferritin and thyroperoxidase antibodies were evaluated and assayed. RESULTS: Thyroid disease in children was emerging in the first 2-3 years after the ALL treatment with an incidenceof 22.8 % (hypothyroidism - 14.1 %, autoimmune thyroiditis - 7.6 %, papillary cancer - 1.1 %). Seven children inthis group had received radiotherapy (12-18 Gy doses) on the central nervous system (CNS). No correlation wasfound between the radiation exposure event itself, radiation dose to the CNS and thyroid disease in the long-termfollow-up period. Thyroid cancer had developed in a child 11 years upon chemo- and radiotherapy. Hypothyroidismwas more often diagnosed in the patients of prepubertal age (rs = 0.49). There were endocrine diseases in thefamily history in about a half of children, being significantly higher than in the general sample (р < 0.05). The bodyweight at birth of a child who had later developed hypothyroidism was less than in children having got thyroiditis(rs = 0.57). CONCLUSIONS: Disorders in endocrine regulation and of thyroid in particular can affect the prognosis of blood can-cer course in the long-term follow-up in children, especially in prepubertal age, which requires systematic supervi-sion by hematologist and endocrinologist.

Assessing the potential leukemogenic effects of 50 Hz magnetic fields and their harmonics using an animal leukemia model.

To answer the still unresolved question of the possible leukemogenic effects of extremely low frequency magnetic fields (ELF-MFs) and of their harmonics on the incidence of B acute lymphoblastic leukemia in children, we used an animal model to explore the possible co-initiating or co-promoting effects of ELF-MFs on the development of leukemia. We used a rat model in which B acute lymphoblastic leukemia is chemically induced by a nitrosurea derivative. From the onset of the chemical treatment, the animals were also exposed to ELF-MFs (100 microT, sinusoidal 50 Hz MFs), with or without harmonics. The experiment was conducted on 280 rats. We compared body weight and survival time, percentage of bone marrow blast cells, cumulative incidence of leukemia and type of leukemia in the unexposed groups and in the groups exposed to 50 Hz MFs, with and without harmonics. The results showed no significant differences between exposed and unexposed rats for any of these parameters (p > 0.05). Significant changes in the leukemia type obtained after gamma-irradiation of the leukemia model, showed its sensitivity to a physical agent. Our results do not support the hypothesis that ELF-MFs, with or without harmonics, affect the development of B acute lymphoblastic leukemia in children.

p53 (TRP53) deficiency-mediated antiapoptosis escape after 5 Gy X irradiation still induces stem cell leukemia in C3H/He mice: comparison between whole-body assay and bone marrow transplantation (BMT) assay.

Mice exposed to a lethal dose of radiation were repopulated with heterozygous p53(+/-) (TRP53(+/-)) bone marrow cells and then exposed to doses of 1, 3 and 5 Gy 1 month later. This resulted in the transplanted bone marrow-specific diseases other than competitively induced nonhematopoietic neoplasms. Interestingly, the present study showed a high frequency of stem cell leukemia, i.e., leukemias characterized by a lack of differentiation due also to p53 deficiency, even after 5 Gy irradiation. The frequencies of stem cell leukemias (and those of total hematopoietic malignancies) were 16% (24%) at 1 Gy and 45% (75%) at 3 Gy. Furthermore, markedly high incidences of stem cell leukemias were observed at 5 Gy in p53(+/-) mice, i.e., 87% (100%) in the transplantation assay and 60% (83.3%) in the whole-body assay, whereas a conventional whole-body assay induced only 14% in wild-type mice. The high incidence of stem cell leukemias observed in this study using heterozygous p53-deficient mice agrees with results of a previous study of homozygous p53-deficient mice and is consistent with the high frequency of loss of heterozygosity in the p53 wild-type allele observed in leukemias. This suggests that the target cells for radiation-induced stem cell leukemias may be p53-deficient hematopoietic stem cells.

Radiation-induced leukemia: lessons from history.

Beginning in 1895, with the discovery of x-rays, alpha and beta radiation, uranium, radium, thorium, and polonium, the fascinating story of the beginning of knowledge concerning the existence of ionizing radiation unfolds. This brief history of radiation and leukemia is divided into two main parts: the first 50 years, which deals with the confusion regarding radiation effects and the failure to clearly recognize that exposure to ionizing radiation may induce leukemia. The second part focuses on the last 60 years, when the radiation induction of leukemia was accepted and some progress achieved in understanding the clinical and pathophysiological characteristics of radiation-induced leukemia. Particular attention in this is paid to the effects of radiation on the survivors of Hiroshima and Nagasaki. The discussion in this section also covers some concepts of radiation-induced cell damage and ruminations on unanswered questions.

Aetiology of childhood leukemia.

Leukemia is the most common cancer to affect children, accounting for approximately a third of all childhood cancers. The major morphological subtypes of leukemia, acute lymphoblastic leukemia (ALL), and acute myeloblastic leukemia (AML), are characterized by chromosomal translocations involving over 200 genes including mixed lineage leukemia (MLL), TEL, and AML1. Chromosomal translocations involving the MLL gene at 11q23 are a common feature of infant acute leukemia, found in up to 80% of all cases, and there is strong evidence that rearrangements involving the MLL gene or the TEL-AML1 gene fusion can originate in utero. As with most other cancers, the mechanism by which leukemia arises is likely to involve gene-environment interactions. Accordingly, it is important to identify exposures that cause DNA damage and induce chromosome breaks which are inadequately repaired, ultimately leading to the initiation and disease progression. Exposures acting before birth and early in life has long been thought to be important determinants of leukemia, and the list of suspected chemical, physical, and biological agents continues to increase. Unfortunately, the evidence regarding the majority of suggested exposures is limited and often contradictory, and there are areas, which clearly warrant further investigation in order to further our understanding of the aetiology of childhood leukemia.

[Estimation of the influence of physical and biological factors on the development of the hematopoietic type of radiation sickness in dogs and two types of monkeys].

In this investigation, the analysis of radiobiological experiments on 532 dogs and two types of monkeys (101 animals), irradiated totally in the 1.0 to 6.0 Gy dose range at different irradiation facilities, has been carried out. LD50 values at X-ray and gamma-neutron exposure were close to each other (2.35 and 2.83 Gy, respectively) while at gamma-radiation exposure LD(50/45) increased to 3.09 Gy. Comparison of LD(50/45) values for different kinds of animals allowed us to draw a conclusion of approximately equal radiosensitivities of dogs and Macaca fascicularis monkeys (LD(50/30-45) - 3.09 Gy and 3.17 Gy, respectively); Macaca rhesus monkeys revealed higher radioresistance (LD(50/30-45) - 5.03Gy). Analysis of the influence of several biological factors has not displayed any significant differences in the values of LD(50/45) and average lifespan of male and female dogs. Higher radiosensitivity of dogs with body weight less than 12 kg and lower radiosensitivity of dogs in summer time compared to other seasons have been shown. Dogs at the age of 2 to 3 years appeared to be more radioresistant than animals of the other age.

Ionizing radiation and chronic lymphocytic leukemia.

The U.S. government recently implemented rules for awarding compensation to individuals with cancer who were exposed to ionizing radiation while working in the nuclear weapons complex. Under these rules, chronic lymphocytic leukemia (CLL) is considered to be a nonradiogenic form of cancer. In other words, workers who develop CLL automatically have their compensation claim rejected because the compensation rules hold that the risk of radiation-induced CLL is zero. In this article we review molecular, clinical, and epidemiologic evidence regarding the radiogenicity of CLL. We note that current understanding of radiation-induced tumorigenesis and the etiology of lymphatic neoplasia provides a strong mechanistic basis for expecting that ionizing radiation exposure increases CLL risk. The clinical characteristics of CLL, including prolonged latency and morbidity periods and a low case fatality rate, make it relatively difficult to evaluate associations between ionizing radiation and CLL risk via epidemiologic methods. The epidemiologic evidence of association between external exposure to ionizing radiation and CLL is weak. However, epidemiologic findings are consistent with a hypothesis of elevated CLL mortality risk after a latency and morbidity period that spans several decades. Our findings in this review suggest that there is not a persuasive basis for the conclusion that CLL is a nonradiogenic form of cancer.

A graft-versus-tumor effect in a patient with ependymoma who received an allogenic bone marrow transplant for therapy-related leukemia. Case report.

Graft-versus-leukemia effect is an immune-mediated antitumor phenomenon associated with allogenic bone marrow transplants (BMTs) for hematological malignancies, and recent findings have indicated that a similar effect could occur in some solid tumors such as breast cancers. The authors report on a 42-year-old man with a recurrent ependymoma who received an allogenic BMT for therapy-related leukemia. After transplantation, the patient developed chronic graft-versus-host disease, which was controlled with steroid agents. Interestingly, the recurrent ependymoma regressed steadily over the next 21 months posttransplant, until the tumor became almost undetectable on magnetic resonance images. This case indicates that the graft-versus-tumor effect, mediated by cytotoxic T cells, may be able to target intraparenchymal neuroepithelial tumors, despite the brain's generally recognized status as an immunoprivileged organ.

Treatment-related leukaemia--a clinical and scientific challenge.

The development of a second tumour, including treatment-related leukaemia (TRL), is the most devastating complication of intensive cancer chemotherapy. This is especially relevant in the paediatric population as over 70% of children diagnosed with a malignancy will now live at least 5 years. Most TRLs are myeloid leukaemias and carry an overall poor prognosis when compared with their de novo counterparts. Despite the well known association with specific cytotoxic agents, improved understanding of the pathogenesis and risk factors of TRL is ultimately essential if we are to develop successful strategies for prevention and treatment. Here we review these aspects, together with the clinical and diverse biological features of this complication and the efficacy of current therapy.

Effect of the irradiated microenvironment on the expression and retrotransposition of intracisternal type A particles in hematopoietic cells.

We have previously demonstrated that the frequency of transformation of the factor-dependent hematopoietic cell line FDCP-1JL26 was dramatically increased when cells were cocultured with the irradiated bone marrow cell line D2XRII. In many of our factor-independent subclonal cell lines that we examined, transformation to factor independence appeared to be due to the retrotransposition of intracisternal type A particles (IAP) into the growth factor genes that are normally required for survival and growth of FDCP-1JL26 cells. To determine the role of the irradiated microenvironment in the evolution of factor-independent cells, we have examined the expression and retrotransposition of IAPs after exposure to the irradiated bone marrow stromal cell line D2XRII. Differential display and Northern blot analysis demonstrated that IAPs were overexpressed in a nonautocrine factor-independent subclonal cell line, FI7CL2. The frequency of retrotransposition was determined by the introduction of the IAP-neo(RT) plasmid into FDCP-1JL26 cells. The IAP-neo(RT) contains a neomycin resistance gene (neo) that only becomes active after retrotransposition, and thus the frequency of retrotransposition in FDCP-1JL26 cells was quantified by determining the frequency of neo-resistant cells.No significant increases in the expression of IAPs were observed after the cells were exposed to the irradiated stromal cells. This observation is in agreement with the observation that no increase in the frequency of retrotransposition could be detected. These results suggest that the irradiated bone marrow may have a passive role in the selection of factor-independent cells. During cocultivation, bone marrow stromal cells may provide a factor(s) to hematopoietic cells that allow it to survive in medium lacking IL-3. At random, a retrotransposition may occur that provides a selective advantage to the hematopoietic cells. In the absence of the irradiated stromal cells, the hematopoietic cells are perhaps more likely to die and therefore are not available for a random retrotransposition event to occur. This model is to be distinguished from an active role in which the irradiated microenvironment would synthesize or activate a factor(s) that promotes retrotransposition.

Secondary chronic myelogenous leukemia after chemotherapy followed by adjuvant radiotherapy for small cell lung cancer.

A 40-year old patient with small cell lung cancer (SCLC) was treated with combined modalities including high-dose chemotherapy with subsequent autologous peripheral blood progenitor cell transplantation plus adjuvant radiotherapy. He achieved complete remission with regards to the primary disease. After an interval of 28 months, he was diagnosed with chronic myelogenous leukemia (CML). Analysis of graft samples at time of primary treatment for SCLC using polymerase chain reaction (PCR) did not show the bcr-abl transcript characteristic for CML. This case supports the observation that CML can develop as a treatment-related malignancy and gives insight in the length of the preclinical phase of the disease.

Approaches for studying radiation-induced leukemia.

According to the conclusion of the International Programme on the Health Effects of the Chernobyl Accident (IPHECA) Haematology Pilot Project (1991-1995), there was no increase in the incidence of malignant disease in hematopoietic and lymphoid tissues after the Chernobyl accident. Nevertheless, since studies of A-bomb survivors indicate that the peak in morbidity may occur more than 10 years after radiation exposure, long-term studies of hemoblastoses and myelodysplastic syndromes are needed today. Study of these leukemias and lymphomas that are potentially induced by ionizing radiation must include both fundamental and applied approaches, i.e., A) epidemiological design; B) utilization of modern methods of diagnosis (cytomorphology, immunocytochemistry, cytogenetics); C) studies of gene mutations, mechanisms of apoptosis, and G1 delay; D) monitoring of oncogene and multidrug resistance gene expression, and E) tracking changes in cell-cell signaling in the bone marrow microenvironment.

Effect of human recombinant granulocyte colony-stimulating factor on induction of myeloid leukemias by X-irradiation in mice.

Hematopoietic suppression is one of the serious problems induced by whole body irradiation. Granulocyte colony-stimulating factor (G-CSF) stimulates the progenitors of granulocytes and accelerates their recovery from bone marrow suppression induced by cytotoxic chemotherapy or radiation. On the other hand, G-CSF stimulates proliferation of myeloid leukemia cells as well as normal granulocytes in vitro. We designed a method to determine if G-CSF affects the incidence of myeloid leukemias induced by irradiation and the types of leukemias induced according to the French-American-British (FAB) classification in RFM/MsNrs mice. Administration of G-CSF (2 micrograms/d for 7 days) after a single 3-Gy irradiation significantly increased the number of peripheral blood neutrophils as compared with those in control mice. Even after discontinuation of G-CSF, both the total leukocyte and neutrophil counts increased to day 10, and their levels remained elevated until day 14. The incidence of myeloid leukemia in mice exposed to a single 3-Gy irradiation was 18.6% (38 of 204), and treatment with G-CSF did not increase the incidence (15.7% [32 of 204]). In the mice with radiation-induced leukemia, those receiving G-CSF had a mean survival time of 357 days, whereas those not receiving the factor survived for 349 days. There was no significant difference of survivals between the two groups. Most of the radiation-induced leukemias in the two groups were M1 or M2, according to the FAB classification; no characteristic difference was observed among the types of leukemias. Although G-CSF stimulated the leukemia cells in vitro, G-CSF administration after irradiation did not increase the occurrence of radiation-induced myeloid leukemias. Our results show that administration of G-CSF effectively accelerates neutrophil recovery from irradiation-induced hematopoietic injury and does not enhance the induction of myeloid leukemia in RFM/MsNrs mice by irradiation.

Lethal graft-versus-host disease in mice directed to multiple minor histocompatibility antigens: features of CD8+ and CD4+ T cell responses.

Lethal graft-versus-host disease (GVHD) can be induced in MHC-matched strain combinations which differ in their expression of multiple minor histocompatibility (H) antigens. It has been shown that CD8+ T cells play an important role in the development of disease directed to the minor H antigens, and that initial indications were that highly purified preparations of these cells were capable of mediating GVHD, without apparent 'help' from mature donor-derived CD4+ T cells. To further strengthen this hypothesis, the current study was undertaken with the B10.BR----CBA strain combination in which irradiated recipient mice were additionally treated with an anti-CD4 monoclonal antibody, as a single or repeated injection, to minimize the presence of either residual host CD4+ cells or recently generated donor-derived CD4+ cells at later stages of disease development. The results indicate that these treatments do not affect the GVHD outcome and that the CD8+ cells are indeed capable of inducing disease independent of CD4+ 'help'. The addition of donor CD4+ T cells in the inoculum, however, does enhance the potential of these CD8+ cells, and is observed with both low and high dosages of CD4+ cells. CD4+ T cells, on their own, have also been observed to cause GVHD directed to minor H antigens in certain strain combinations, and their response has been further characterized in this study. Results indicate that CD4+ cells capable of mediating GVHD in the B10.D2----DBA/2 strain combination can do so over a wide range of recipient irradiation exposures. The transfer of high dosages of CD4+ cells only shortens survival times of the recipients and does not afford any apparent protection phenomenon as previously observed in CD4+ cell mediated anti-class II MHC GVHD. The study also indicates that neither CD4+ nor CD8+ cells responsible for GVHD directed to minor H antigens seem capable of targeting host stem cell elements.

Acute inflammation effects on in vivo granulopoiesis: comparative studies in healthy and leukaemic mice.

Mice undergoing an inflammatory reaction, induced by subcutaneous implantation of copper rods, elaborate two kinds of humoral stimulatory factors: the diffusible granulopoietic stimulator (DGS) that enhances diffusion chamber (DC) granulopoiesis, and the serum colony stimulating factor (CSF) that stimulates in vitro granulocyte-monocyte colony growth. We demonstrate here that mice suffering from acute myeloid leukaemia (AML) are unable to augment the production of these humoral stimulatory factors when acute inflammation is induced. Moreover, our results show that increased levels of normal humoral stimulatory factors (DGS and CSF) do not influence the proliferation and/or the differentiation of leukaemic cells implanted in DC.

Cellular aspects of the pathogenesis of radiation--induced thymic lymphomas in C57 BL mice (review).

Radiation-induced thymic lymphomas in C57Bl/Ka mice are interesting models for studying the successive steps of carcinogenesis. Irradiation initiates "preleukemic" cells, which are promoted to become neoplastic. Studies in mice in which lymphoma development is inhibited by a bone marrow transplantation after irradiation suggest that radiation--induced alterations to the T cell lineage, and particularly to thymic microenvironment, are critical for the promotion of preleukemic cells. It is proposed that the lack of physiological differentiation signals within the thymus, as a result of irradiation, allows these cells to escape the normal controls of thymocyte production and pushes them towards neoplastic transformation. A disturbance in the production of cytokines may be involved, since exogenous cytokines, such as Interferon gamma or Tumor Necrosis Factor a, can inhibit radiation-induced lymphomagenesis, reproducing the effects of bone marrow transplantation. The model is thus suitable for studying the mechanisms of carcinogenesis and designing biological manipulation devoted to cancer prevention in individuals who have been exposed to oncogenic agents.

[Interaction of radiation-induced preleukemia cells and bone marrow grafts in C57BL/Ka mice]. / Interactions de cellules préleucémiques radio-induites et de greffe de moelle chez la Souris C57BL/Ka.

The repopulation of thymus inoculated with radiation-induced preleukemia cells was studied in 400 R irradiated mice grafted with normal bone marrow cells. These marrow cells gave rise to an actively regenerating thymic progeny, as well as in 400 R treated mice receiving only a bone marrow graft. Moreover, the marrow graft did not prevent the progression of inoculated preleukemic cells towards lymphoma growth.