Telomerase activity in mouse myeloid leukemic cells and in cells from normal hematopoietic systems.

The telomeric repeat amplification protocol (TRAP) assay was used to measure telomerase activity in radiation-induced mouse myeloid leukemic (ML) cells and in several populations of normal cells. A detectable level of telomerase activity was found in normal hematopoietic tissues, i.e., bone marrow (BM) cells, day 9 colony-forming unit spleen (CFU-S) colonies, peripheral blood (PB) lymphocytes, and spleen. The level of telomerase activity in normal BM cells was used as a background level. Nine of the 12 cases of ML had higher levels of activity than that of the normal BM cells and therefore they were scored as ML with positive telomerase. The other three cases were considered as ML with negative telomerase because the levels of the enzyme were equivalent to that of normal BM cells. The data indicate that cellular differentiation may suppress telomerase activity in mouse ML cells. In summary, the results suggest that the CBA/Ca mouse model should be a useful animal system for future studies on the assessment of telomerase activity in both malignant and normal hematopoietic cells.

Characterization of genetic instability in radiation- and benzene-induced murine acute leukemia.

This study, using the CBA/Ca mouse as a model, compares genetic lesions associated with radiation- and benzene-induced acute leukemias. Specific types of leukemia included in the analyses are radiation-induced acute myeloid leukemia (ML), and benzene-induced lymphoblastic leukemias, lymphomas, or mix-lineage leukemias. These leukemias have histopathological characteristics similar to those seen in human acute leukemias. G-band cytogenetic analysis showed that specific deletions involving regions D-E of one copy of mouse chromosome 2 [del(2)(D-E)] were frequently associated in both radiation- and benzene-induced acute leukemias. In addition, translocations of chr2(D-E) were also observed in some cases. These results suggest an important role of chr2 (D-E) deletions and translocations in the development of radiation- and benzene-induced murine acute leukemias. Fluorescence in situ hybridization with DNA probes specific for 2(D-E), constructed in our laboratory by means of chromosomal microdissection and PCR amplification, also demonstrate 2(D-E) deletions and/or translocations in these leukemic cells. Aneuploidy of chromosomes 3, 15, 16, and Y were also frequently detected in benzene-induced leukemic cells with or without lesions on chr2. These cytogenetic findings support the previous observations that metabolites of benzene lead to spindle-fiber disruption or abnormal cytokinesis in exposed animals. In summary, genetic instabilities observed in leukemic cells isolated from mice that had developed leukemia after exposure to radiation or benzene are syntenic with those frequently detected in patients with myelodysplastic syndrome, acute ML, and acute lymphoblastic leukemia. Thus, the CBA/Ca mouse has several characteristics that make it an excellent model for the study of radiation or benzene leukemogenesis in humans.

Advantages of the CBA mouse in leukemogenesis research.

The objectives of this review are to: (a) demonstrate that the male CBA/Ca mouse has several characteristics that make it an excellent animal for the study of leukemogenesis, (b) show that several of the genetic abnormalities observed in the male CBA/Ca mouse during the development of radiation induced acute myeloid leukemia (AML) are syntenic with those frequently detected in patients with myeloid disorders such as myelodysplastic syndrome and AML, (c) illustrate that leukemia-related chromosomal lesions are the indicators for high risk individuals.

Historical events associated with fallout from Bravo Shot--Operation Castle and 25 Y of medical findings.

The events prior to Bravo Shot-Operation Castle that led to a decision not to evacuate the Marshallese prior to testing the thermonuclear bombs are presented as are the actions taken after the fallout incident in evacuating the exposed Marshallese and the military personnel. The initial medical effects (findings during first 6 wk after exposure) are briefly described and are followed by description of long term effects, namely, induction of one case of fatal acute myeloid leukemia and a large number of thyroid tumors (benign and malignant) in addition to hypothyroidism in adults and children and two cases of cretinism. The hypothyroidism and cretinism responded well to administration of oral thyroxine. During the first 25 y, there was also much unrest and political agitation initiated by exposed and unexposed Marshallese who were very unhappy as a result of relocation and inability to return to their homelands and feeling that all illness and deaths were due to the mysterious radiation, which they understandably did not understand. The difficulties in part were ameliorated by financial aid from the U.S. Congress. In view of one of us (EPC), no one agency or person in the U.S. Government was willing to take the responsibility for care of the Marshallese and its financing. The exposed and nonexposed Marshallese had their lifestyle changed, some of their homelands made uninhabitable for several years and could aptly be called "nuclear nomads," an expression coined by others.

Lifetime treatment of mice with azidothymidine (AZT) produces myelodysplasia.

AZT has induced a macrocytic anemia in AIDS patients on long term AZT therapy. It is generally assumed that DNA elongation is stopped by the insertion of AZT into the chain in place of thymidine thus preventing the phosphate hydroxyl linkages and therefore suppresses hemopoietic progenitor cell proliferation in an early stage of differentiation. CBA/Ca male mice started on AZT 0.75 mg/ml H2O at 84 days of age and kept on it for 687 days when dosage reduced to 0.5 mg/ml H2O for a group, another group removed from AZT to see recovery, and third group remained on 0.75 mg. At 687 days mice that had been on 0.75 mg had average platelet counts of 2.5 x 10(6). Histological examination on 9 of 10 mice with such thrombocytopenia showed changes compatible with myelodysplastic syndrome (MDS). A variety of histological patterns was observed. There were two cases of hypocellular myelodysplasia, two cases of hypersegmented myelodysplastic granulocytosis, two cases of hypercellular marrow with abnormal megakaryocytes with bizarre nuclei, one case of megakaryocytic myelosis associated with a hyperplastic marrow, dysmyelopoiesis and a hypocellular marrow and two cases of myelodysplasia with dyserythropoiesis, hemosiderosis and a hypocellular marrow. Above mentioned AZT incorporation may have induced an ineffective hemopoiesis in the primitive hemopoietic progenitor cells, which is known to be seen commonly in the myelodysplastic syndrome.

Evidence for an uncommon microsatellite instability on mouse chromosomes 2 and 4 and its possible role in radiation leukemogenesis.

Although microsatellite instability (MSI), usually detected by DNA length polymorphisms, has been implicated in the induction of solid tumors in both humans and animals, its role in leukemogenesis is unclear. The goal of this study was to investigate whether there is an association between MSI and radiation leukemogenesis in CBA/Ca mice. Microsatellite lengths at 55 loci, mapped to eight different mouse chromosomes, were examined in two groups of DNA samples: 1) 10 normal DNA samples collected from the bone marrow cells of control male CBA/Ca mice, and 2) 17 DNA samples isolated from the spleens of mice that developed myeloid leukemia (ML) after exposure to neutrons, or X rays, or gamma rays. Microsatellite markers were amplified using the non-radioisotopic multiplex-touchdown PCR protocols developed in our laboratory, and the sizes of amplicons were examined on 6% non-denaturing polyacrylamide gels. Although no correlation between microsatellite length polymorphisms and radiation leukemogenesis was observed at the 55 CBA/Ca mouse loci tested in this study, an uncommon MSI, manifested as the absence of DNA bands after PCR amplification at 2 loci (D2MIT140 and D4MIT104), was observed in both control and ML samples. However, the frequency of ML samples showing this type of MSI is statistically significant (p<0.05). Although there is no direct evidence that this type of MSI predisposes mice to the development of leukemia, the results suggests that genes flanking the D2MIT140 and D4MIT104 are susceptible to spontaneous mutation and perhaps to damage caused by ionizing radiation.

Effects of irradiation of CBA/CA mice on hematopoietic stem cells and stromal cells in long-term bone marrow cultures.

Following 200 cGy total body irradiation, 20-25% of CBA/Ca mice and their CBA/B and CBA/H sublines develop myeloid leukemia. To determine whether hematologic changes in vitro were detectable, long-term marrow cultures (LTBMCs) were established from the right and left hind limbs of 11 individual control and 11 CBA/B mice 100-114 days after 200 cGy total body irradiation. Individual cultures were studied weekly for cumulative production of nonadherent cells and colony-forming, hematopoietic progenitor cells. Control cultures produced significantly more nonadherent cells over 25 weeks in long-term marrow culture compared to those from irradiated (treated) mice. Permanent stromal cell lines were established from control and irradiated CBA/B mouse LTBMCs and clonal sublines were established. The stromal cell lines from LTBMCs of in vivo irradiated CBA/B mice had uniformly lower plating efficiencies, and only one formed a permanent clonal subline at 100-fold lower frequency compared to stromal cell lines from control mouse LTBMCs. The irradiation sensitivity of both uncloned and clonal sublines was similar by single-hit, multi-hit or by linear quadratic formula. Cocultivation of an IL-3 dependent hematopoietic progenitor cell line established from a control CBA/B, LTBMC with control of irradiated stromal cell lines derived from either a control (CC3) or the one successfully cloned in vivo irradiated (CT4) LTBMC, produced few cobblestone islands in the presence of IL-3. In contrast, formation of cobblestone islands in the presence of L cell-condition medium as a source of M-CSF was significantly greater, and these persisted for 21 days on both CC3 and CT4 stromal lines. The data provide evidence for irradiation induced changes in the bone marrow stromal cell compartment of CBA/B mice which persist and are detectable in vitro 6 months after explant of the cells to culture. These marrow stromal cell lines may provide valuable resources for analyzing the molecular biologic changes in the hematopoietic microenvironment during irradiation leukemogenesis.

N-ras mutations in radiation-induced murine leukemic cells.

N-ras mutations were examined in DNA samples extracted from the spleen of CBA/Ca mice that developed myeloid leukemia (ML) following exposure to radiations of different qualities. A total of 17 ML cases, i.e. 5 cases of neutron-induced and 12 cases of photon- (3 gamma-ray and 9 x-ray) induced ML were included in the study along with 12 DNA samples from the bone marrow cells of control mice. Polymerase chain reaction-single strand conformational polymorphisms (PCR-SSCP) and the direct sequencing of PCR products were used to analyze three regions of the N-ras gene: (i) a 120 base-pair (bp) long portion of exon I (codons 2-37); (ii) a 103 bp long portion of exon II (codons 48-82); and (iii) a 107 bp long portion of exon III (codons 118-150). PCR-SSCP mobility shifts indicated mutations within only exon II of the N-ras gene. Such mutations were more prevalent in samples from mice exposed to fast neutrons. The exact type and location of these mutations were then determined by direct DNA sequencing. Silent point mutations, i.e. base transitions at the third base of codons 57 (GAC-->GAT), 62 (CAA-->CAC), or 70 (CAG-->CAA) were present only in mice that developed ML after exposure to fast neutrons. A base transversion at the third base of codon 61 (CAA-->CAC) was also observed in some ML cases. DNA sequencing demonstrated that ML samples contained normal as well as mutated DNA sequences. The higher frequency of N-ras mutations in neutron-induced ML suggested that fast neutrons are more effective in inducing genomic instability at the N-ras region of the genome. More importantly, N-ras mutations are not the initiating event in radiation leukemogenesis. This conclusion was supported by the finding that N-ras mutations were detected only in mice with an overt leukemic phenotype but not in mice with minimal tissue infiltration of leukemic cells, suggesting that the disease may be present prior to the presence of N-ras mutations. Alternatively, N-ras may be present in these mice but a large number of normal spleen cells in these mice interferes with the detection of mutation in a small population of leukemic cells.

Survival of spleen colony-forming units (CFU-S) of irradiated bone marrow cells in mice: evidence for the existence of a radioresistant subfraction.

Because of increasing evidence of heterogeneity in the hematopoietic stem cell compartments, the radiosensitivity of spleen colony-forming units (CFU-S) was reevaluated to ascertain whether the classical single exponential curve for a graded dose of radiation is applicable at higher doses of radiation, 400-600 cGy. Bone marrow cells (BMC) removed from mice immediately after death under anesthesia were irradiated in vitro. Great care was taken to exclude anoxic effects during irradiation and to avoid any possible effects in the recipient mice from injection of excessive numbers of BMC. By estimating the number of cells to be injected to produce numbers of colonies within the evaluation range of the assay, we obtained a radiation survival curve that appeared to have a multiphasic concave shape; the D0 value for the 400-600 cGy range was estimated to be about 275 cGy, whereas the D0 for the lower doses was 95 cGy, the same value as previously reported. The reason a single exponential survival curve was previously obtained after graded doses of radiation is discussed, and a comparison of those results with the present data from in vitro radiation is made. Lacking experimental evidence, we speculate that the major factor that determines the slope of the survival curve is the degree to which the stem cells are in their normal hematopoietic environment during the irradiation. The probable existence of a fraction surviving after an exposure to 600 cGy, estimated by the limiting dilution assay, was about 1 per 2 x 10(6) BMC. Such radio-insensitive CFU-S appear to be primitive CFU-S, which can contribute materially to the long-term survival of lethally irradiated bone marrow recipients.

Physiological and pathophysiological aspects of the immune system contributing to a biomathematical model of lymphocytes.

The effects of chronic low-dose irradiation on the immune system and the lymphocytes are largely unknown. The uranium miners in the former German Democratic Republic (GDR) were exposed mainly to a local low-dose irradiation in the lung by radon and its progeny, but also to some whole-body gamma irradiation. The local irradiation led to an increased rate of lung cancer and perhaps to some increase in extrapulmonary neoplasms. But little is known about the effects on the lymphocytes circulating and recirculating to the lung. As a prerequisite for the establishment of a biomathematical model to estimate lymphocyte fluxes, and to assess the radiation effects on the lymphocytic (and stem cell) populations passing through the lung, it was necessary to establish the current knowledge with respect to the physiology and pathophysiology of the lymphocytic cell renewal systems. The data concerning lymphopoiesis and lymphocyte kinetics, which are important for the development of this model, are summarized. The distribution of lymphocytes between different compartments including the lung is taken into consideration, as well as the effects of acute and chronic irradiation on the immune system. The extracorporeal irradiation of the blood (ECIB) may serve as a model of irradiation of blood in the lung. This review shows that many data necessary for development of a detailed biomathematical model are still missing, especially data concerning details on lymphocyte production rates of their different subsets and regulatory mechanisms of the lymphocytic system.

Medical effects of exposure of human beings to fallout radiation from a thermonuclear explosion.

On March 1, 1954, after detonation of a thermonuclear device on Bikini atoll, an unexpected wind shift resulted in the deposition of radioactive fallout on inhabited atolls. The fallout radiation caused fleeting systemic effects, dose-dependent depression of hematopoiesis and skin burns primarily due to the beta ray component of the fission radionuclides. Within a few weeks, hematopoietic recovery was substantial but slight depression of blood counts was maintained for several years. One case of fatal acute myeloblastic leukemia developed in a boy receiving 1.9 Gy as an infant. Cretinism developed in two boys exposed as infants with estimated thyroidal dose in excess of 50 Gy. Chemical hypothyroidism was detected in several persons. Thyroid adenomas and cancer commenced appearance ten years after exposure and became a major long-term medical problem. There have been no late effects attributable to the beta burns 40 years after exposure. Internal contamination from ingestion and inhalation of radionuclides is detectable. The doses are comparable to background levels in the U.S. There is no detectible decrease in longevity of the exposed Marshallese compared to an unexposed Marshallese population.

Hypermutability of mouse chromosome 2 during the development of x-ray-induced murine myeloid leukemia.

In an effort to identify the precise role of a deletion at regions D-E of mouse chromosome 2 [del2(D-E)] during the development of radiation-induced myeloid leukemia, we conducted a serial sacrifice study in which metaphase chromosomes were examined by the G-banding technique. Such metaphase cells were collected from x-irradiated mice during the period of transformation of some of the normal hematopoietic cells to the fully developed leukemic phenotype. A group of 250 CBA/Ca male mice (10-12 weeks old) were exposed to a single dose of 2 Gy of 250-kilovolt-peak x-rays; 42 age-matched male mice served as controls. Groups of randomly selected mice were sacrificed at 20 hr, 1 week, and then at intervals of 3 months up to 24 months after x-irradiation. Slides for cytogenetic, hematological, and histological examination were prepared for each animal at each sacrifice time. An expansion of cells with lesions on one copy of chromosome 2 was evident in 20-25% of treated mice at each sacrifice time. The majority of such lesions were translocations at 2F or 2H, strongly suggesting hypermutability of these sites on mouse chromosome 2. No lesions were found in control mice. The finding leads to the possibility that genomic lesions close to 2D and 2E are aberrants associated with radiation leukemogenesis, whereas a single clone of cells with a del2(D-E) may lead directly to overt leukemia. The data also indicate that leukemic transformation arises from the cumulative effects of multiple genetic events on chromosome 2, reinforcing the thesis that multiple steps of mutation occur in the pathogenesis of cancer.

Influence of radiation fractionation on survival of mice and spleen colony-forming units.

C57Bl/6 mice were given 10 Gy X rays fractionated in several ways. There was a cyclical pattern of animal survival which was correlated to the fractionation interval and which indicated a periodicity of 6 h. Ten grays given in a single dose is fatal to 100% of the mice and depresses the CFU-S to about one per leg with no evidence of proliferation during the remaining life. Ten grays given in 2.5-Gy increments at 24-h intervals causes no fatalities and results in a similar CFU-S depression but is followed by an exponential increase in CFU-S over the ensuing 12 days. Although bone marrow from survivors of such treatment was comparable to control marrow in its capacity for short-term rescue, it was clearly inferior in its capacity for long-term rescue. The periodicity of 6 h suggests that the cells responsible for survival of the mice have been synchronized into more or less radiosensitive and radioresistant stages of the cell cycle as a result of the time between the 2.5-Gy increments. Implications for the CFU-S and long-term repopulating cells are discussed.

Self-renewal of the long-term repopulating stem cell.

Self-renewal implies maintenance of all attributes of the original in the offspring and is considered characteristic of the hemopoietic stem cells. Yet, it has been questioned whether one of the most primitive hemopoietic stem cells, the long-term repopulating cell (LTRC), has that capacity. The present experiments demonstrate that single LTRCs can repopulate the lymphohemopoietic system of a lethally irradiated mouse and that the progeny of a single LTRC in a primary recipient again contains LTRCs capable of repopulating lethally irradiated secondary recipients. The transfusion of very small numbers of marrow cells (10,000-20,000 cells containing one or no LTRCs) unexpectedly provided insight into competitive marrow repopulation. At these low levels of stem cells, irradiated host stem cells or their progeny competed successfully with unirradiated donor cells. This parallels the known reemergence and marrow repopulation by host cells when the number of nonirradiated donor stem cells is reduced by serial transplantation.

Are stem cells exposed to ionizing radiation in vivo as effective as nonirradiated transfused stem cells in restoring hematopoiesis?

Are the stem cells that survive graded doses of ionizing radiation as effective in restoring hematopoiesis in irradiated mice as transfused nonirradiated stem cells? This question was addressed by determining animal dose mortality and 10-day colony-forming units (CFU-S) survival curves and then replotting the percent animal survival against the number of CFU-S surviving the different doses of radiation, and by determining the number of nonirradiated CFU-S injected into fatally irradiated mice that result in a CFU-S dose mortality response curve. The number of CFU-S surviving per mouse after doses of radiation resulting in 95, 50 and 5% animal survival were calculated to be 520, 300 and 153, respectively. From the transfused CFU-S dose mortality curve of otherwise fatally irradiated mice (8.5 Gy), the number of transfused normal CFU-S required for 95, 50 and 5% animal survival was estimated to be 153, 24 and 3, respectively. The ratios of surviving CFU-S to nonirradiated, injected CFU-S are: at 95% survival (6.3 Gy), 3.4; at 50% survival (6.88 Gy), 12.5; and at 5% survival (7.4 Gy), 51.0. These data show that, in addition to a reduced number of CFU-S, as radiation dose increases, the quality of surviving CFU-S responsible for 30-day survival decreases. By implication, the long-term repopulating cell (LTRC) that is now known not to be the 10-day CFU-S must also decrease.

A specific chromosomal deletion in murine leukemic cells induced by radiation with different qualities.

G-banded metaphase chromosomes prepared from 14 male CBA/Ca mice with histologically confirmed myeloid leukemia (ML) were studied in an effort to identify specific chromosomal changes associated with radiation leukemogenesis. The chromosome studies were undertaken as part of a larger investigation of radiation carcinogenesis, in which mice were exposed to radiation of several different qualities, i.e., x-rays, gamma-rays and "monoenergetic" fast neutrons of 5 mean energies ranging from 0.2 to 14 MeV. The 14 ML cases showed no histologically phenotypic differences and they were transplantable in syngeneic mice. We detected a specific chromosomal deletion in 1 copy of mouse chromosome 2 at regions D-E in all radiation-induced ML cells, regardless of radiation quality. Our results strongly implicate the involvement of genes within or close to regions D-E of chromosome 2 in radiation leukemogenesis. In addition to the specific deletion in chromosome 2, loss or gain of the Y chromosome was also detected in some cells from 6 ML cases. Because this hypo- or hyperploidy occurred in only a small fraction of leukemic cells, a causative role in radiation leukemogenesis appears unlikely.

Anemia induced in splenectomized mice by administration of rhG-CSF.

Normal and splenectomized mice (SPLXM) were given rhG-CSF for 10 to 128 days and serial observations were made on blood counts for 128 days. After 10 days, mice were killed for histologic studies. All treatment schedules produced, in addition to elevated white blood counts, a macrocytic anemia which only partially responded to large doses of Epo. Stopping rhG-CSF treatment for 2 days resulted in the return of granulocytes, lymphocytes, monocytes, platelets and polychromatophilic erythrocytes to near normal levels, indicating a need for the continued presence of rhG-CSF to maintain peripheral blood increases. Treatment of normal and SPLXM with rhG-CSF induced marked granulocytic hyperplasia of the bone marrow with expansion of the granulocytic marrow into the adjoining muscle as in acute myelocytic leukemia. The hyperplasia is greater in the SPLXM than in the normal mouse where splenic hyperplasia occurs in all cell lines. The rhG-CSF also results in expansion of granulopoiesis into the normally fatty tail bone marrow in SPLXM. The rhG-CSF treatment produced marked increases in the assayable numbers of GM-CFU, G-CFU and M-CFU. The significance and mechanisms of induction of these changes are not clear. It is speculated that treatment with rhG-CSF has multicellular effects, suggesting that it initiates a cascade of molecular reactions that cause the effects observed.

Competitive repopulation in leukemic and normal bone marrow.

The success of chemotherapy in leukemias in which the marrow appears entirely replaced by leukemic cells must be due to the persistence of some normal stem cells. The implied competition between leukemic and normal stem cells is thus akin to the competition between donor and host cells in irradiated animals. A review of that competition points to the importance of the quantitative relationships between the competing stem cells, even when one of the competing stem cell clones has a proliferative advantage. Pursuing that analogy, it is suggested that stimulating the surviving normal stem cells by appropriate combinations of cytokines may be of therapeutic benefit, once the tumor load has been reduced by chemotherapy. Complete eradication of leukemic cells may not be necessary, if surviving normal cells could gain ascendancy over the residual leukemic cell clones.