Influence of Age on Leukemia Mortality Associated with Exposure to γ rays and 2-MeV Fast Neutrons in Male C3H Mice.

The relative biological effectiveness (RBE) of densely ionizing radiation can depend on the biological context. From a radiological perspective, age is an important factor affecting health risks of radiation exposure, but little is known about the modifying impact of age on the effects of densely ionizing radiation. Herein, we addressed the influence of age on leukemogenesis induced by accelerator-generated fast neutrons (mean energy, ∼2 MeV). Male C3H/HeNrs mice were exposed to 137Cs γ rays (0.2-3.0 Gy) or neutrons (0.0485-0.97 Gy, γ ray contamination 0.0105-0.21 Gy) at 1, 3, 8, or 35 weeks of age and observed over their lifetimes under specific pathogen-free conditions. Leukemia and lymphoma were diagnosed pathologically. Hazard ratio (HR) and RBE for myeloid leukemia mortality as well as the age dependence of these two parameters were modeled and analyzed using Cox regression. Neutron exposure increased HR concordant with a linear dose response. The increase of HR per dose depended on age at exposure, with no significant dose dependence at age 1 or 3 weeks but a significant increase in HR of 5.5 per Gy (γ rays) and 16 per Gy (neutrons) at 8 weeks and 5.8 per Gy (γ rays) and 9 per Gy (neutrons) at 35 weeks. The RBE of neutrons was 2.1 (95% confidence interval, 1.1-3.7), with no dependence on age. The development of lymphoid neoplasms was not related to radiation exposure. The observed increasing trend of radiation-associated mortality of myeloid leukemia with age at exposure supports previous epidemiological and experimental findings. The results also suggest that exposure at the susceptible age of 8 or 35 weeks does not significantly influence the RBE value for neutrons for induction of leukemia, unlike what has been documented for breast and brain tumors.

Two Cases of Metastatic Parathyroid Carcinoma in Male C3H Mice Following Irradiation.

White nodules were observed in the thyroid in two male C3H mice (at 99 and 122 weeks of age) exposed to fast neutrons at the age of 8 weeks. Histopathologically, in both cases, tumors were developed in the region corresponding to the parathyroid gland, and the tumor cells were arranged in a solid sheet or nest-like structures. Necrosis, cell debris and/or hemorrhage were sometimes seen in the center of the tumor structures. Tumor cells were small and uniform with scanty cytoplasm, cell margins were indistinct, and basally located tumor cells were aligned along the vascular stroma. Mitotic figures were frequently observed. Metastasis to the renal cortex was observed in both cases. These cases were diagnosed as parathyroid carcinoma. A parathyroid tumor is an extremely rare endocrine tumor in mice, regardless of whether the tumor is spontaneous or experimentally induced. These cases may have been induced by neutron-exposure; however, how radiation induces parathyroid carcinoma in mice is not clear.

Neurobehavioral changes in mice exposed to fast neutrons in utero.

Epidemiological studies have revealed that radiation causes brain development abnormalities in atomic bomb survivors exposed in utero. Rat and mouse studies have also shown that prenatal exposure to low-linear energy transfer radiation induces developmental brain anomalies. Because the effects of prenatal irradiation on adult behavior patterns remain largely unknown, the present study investigated the effects of neutron exposure in utero on postnatal behavior patterns in mice. [C57BL/6J × C3H/He] hybrid (B6C3F1) mice were exposed to cyclotron-derived fast neutrons with peak energy of 10 MeV (0.02-0.2 Gy) or Cs-137 gamma-rays (0.2-1.5 Gy) on embryonic day 13.5. At 5.5-8 months of age, the neurobehavior of male offspring was examined by Rota-rod treadmill and locomotor activity. The accumulation of radio-labeled drug at muscarinic acetylcholine and serotonin receptors in mice from control and neutron-irradiated groups was determined by the tracer method. Locomotor activity during the dark period increased in the 0.02 Gy neutron-irradiated group. Furthermore, at 5.5 months of age, tracer binding in vivo to the muscarinic acetylcholine increased and to the serotonin receptors decreased in the 0.02 Gy neutron-irradiated group. In conclusion, the present study reveals that a certain "low-dose window" may exist for radiation-induced changes in neurobehavior and binding to neurotransmitter receptors, because there was correlation in neurobehavior and binding to neurotransmitter receptors in the 0.02 Gy neutron-irradiated group though there was not correlation in the neutron-irradiated groups more than 0.05 Gy.

Existence of a threshold-like dose for gamma-ray induction of thymic lymphomas and no susceptibility to radiation-induced solid tumors in SCID mice.

Severe combined immune deficiency (SCID) mice exhibit limited repair of DNA double-strand breaks and are sensitive to ionizing radiation due to a mutation of the DNA-dependent protein kinase catalytic subunit gene. To elucidate the effects of deficient DNA double-strand break repair on radiation-induced carcinogenesis, the dose-response relationship for the induction of all tumor types was examined in wild-type and SCID mice. In wild-type mice, the incidence of thymic lymphomas at gamma-ray doses up to 1 Gy was almost equal to the background level, increased gradually above 1 Gy, and reached a maximum of 12.5% at 5 Gy, which is indicative of a threshold dose of less than 1 Gy. SCID mice were extremely susceptible to the induction of spontaneous and radiation-induced thymic lymphomas. The incidence of thymic lymphomas in SCID mice irradiated with 0.1 Gy or less was similar to the background level; that is, it increased markedly from 31.7% at 0.1 Gy to 51.4% at 0.25 Gy, and reached a maximum of 80.6% at 2 Gy, suggesting the presence of a threshold-like dose at low gamma-ray doses, even in radiosensitive SCID mice. As the average latency for the induction of thymic lymphomas at 0.1 Gy was significantly shortened, the effect of 0.1 Gy gamma-rays on thymic lymphoma induction was marginal. The high susceptibility of SCID mice to develop thymic lymphomas indicates that thymic lymphomas are induced by a defect in DNA double-strand break repair or V(D)J recombination. Excessive development of tumors other than thymic and nonthymic lymphomas was not observed in SCID mice. Furthermore, our data suggest that the defective double-strand break repair in SCID mice is not a major determinant for the induction of nonlymphoid tumors.

Dose-response and large relative biological effectiveness of fast neutrons with regard to mouse fetal cerebral neuron apoptosis.

To evaluate the relative biological effectiveness (RBE) of low doses of neutrons on fetal nervous development, [C57BL/6J x C3H/He] hybrid (B6C3F1) mice were exposed to cyclotron-derived fast neutrons with peak energy of 10 MeV (0.02-1.0 Gy) or 137Cs-generated gamma-rays (0.1-2.0 Gy) on embryonic day 13.5. We then evaluated the incidence of neuronal apoptosis in the cerebral cortex 24 hours after irradiation. Neuronal apoptosis increased in a dose-dependent manner in both neutron- and gamma-ray-irradiated groups: even at the lowest dose, a minimal increase in the apoptotic index was noted in response to both types of radiation. The dose-response curves were best fitted to linear quadratic models, and the evaluated RBE was 9.8, which was considered to be large for a prenatal effect and acute tissue injury induced by a low dose of neutrons.

Defective repair of radiation-induced DNA damage is complemented by a CHORI-230-65K18 BAC clone on rat chromosome 4.

The Long Evans cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is thus a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three ESTs (TC523872, TC533727, and CB607546) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat.

Tumor induction in mice locally irradiated with carbon ions: a retrospective analysis.

Tumor induction in mice legs that were locally irradiated with carbon ions was compared to tumor induction by gamma rays after single and fractionated irradiation. A total of 250 tumors were induced in 1104 mice that received carbon-ion doses of 5 through 65 Gy. A total of 77 tumors were induced in 371 mice that received gamma-ray doses of 45 through 95 Gy. Of 91 carbon-ion induced tumors examined histologically, 97 percent were malignant, and sarcomas such as malignant fibrous histiocytoma (47%) and fibrosarcoma (32%) were most frequently observed. Malignant fibrous histiocytoma was also the most frequently observed tumor (12 out of 20 tumors; 60%) after gamma-ray irradiation, followed by carcinomas (25%) such as adenocarcinoma and squamous cell carcinoma. Neither dose fractionation nor linear energy transfer affected tumor induction for carbon ions and gamma rays. Dose responses were linear for carbon ions and gamma rays, and showed no saturation up to 65 Gy of carbon ions and 95 Gy of gamma rays. The relative biological effectiveness of carbon ions was 2.2 for tumor induction and 1.9 for early skin reaction. We conclude that risk of secondary tumor induction by carbon-ion radiotherapy would not be seriously higher than anticipated.

Fine mapping of radiation susceptibility and gene expression analysis of LEC congenic rat lines.

LEC rats constitute an animal model of high susceptibility to X-rays. We developed congenic LEC rat lines (recipient strain, Fischer 344 (F344)) and performed genome-wide genotyping to identify radiation susceptibility genes. We mapped seven positional candidate genes, Bmp10, Gpr73, Gp9, Cnbp, Copg, Rab7, and Rpn1, to an approximately 1.2-Mb region located between loci D4Got85 and D4Got148 on chromosome 4. None of the seven genes has been reported to be associated with radiation susceptibility. Comparison of the coding sequences for these seven genes in F344 and LEC rats showed no changes in deduced amino acid sequences. We determined gene expression differences in Gp73, Gp9, and Cnbp as well as strain-specific variations in upstream sequences of these genes. Our results suggest that radiation susceptibility in the LEC rat is primarily attributable to one of the genes within this approximately 1.2-Mb region; however, expression analysis gave no clear indication as to which gene is responsible.

Involvement of illegitimate V(D)J recombination or microhomology-mediated nonhomologous end-joining in the formation of intragenic deletions of the Notch1 gene in mouse thymic lymphomas.

Deregulated V(D)J recombination-mediated chromosomal rearrangements are implicated in the etiology of B- and T-cell lymphomagenesis. We describe three pathways for the formation of 5'-deletions of the Notch1 gene in thymic lymphomas of wild-type or V(D)J recombination-defective severe combined immune deficiency (scid) mice. A pair of recombination signal sequence-like sequences composed of heptamer- and nonamer-like motifs separated by 12- or 23-bp spacers (12- and 23-recombination signal sequence) were present in the vicinity of the deletion breakpoints in wild-type thymic lymphomas, accompanied by palindromic or nontemplated nucleotides at the junctions. In scid thymic lymphomas, the deletions at the recombination signal sequence-like sequences occurred at a significantly lower frequency than in wild-type mice, whereas the deletions did not occur in Rag2(-/-) thymocytes. These results show that the 5'-deletions are formed by Rag-mediated V(D)J recombination machinery at cryptic recombination signal sequences in the Notch1 locus. In contrast, one third of the deletions in radiation-induced scid thymic lymphomas had microhomology at both ends, indicating that in the absence of DNA-dependent protein kinase-dependent nonhomologous end-joining, the microhomology-mediated nonhomologous end-joining pathway functions as the main mechanism to produce deletions. Furthermore, the deletions were induced via a coupled pathway between Rag-mediated cleavage at a cryptic recombination signal sequence and microhomology-mediated end-joining in radiation-induced scid thymic lymphomas. As the deletions at cryptic recombination signal sequences occur spontaneously, microhomology-mediated pathways might participate mainly in radiation-induced lymphomagenesis. Recombination signal sequence-mediated deletions were present clonally in the thymocyte population, suggesting that thymocytes with a 5'-deletion of the Notch1 gene have a growth advantage and are involved in lymphomagenesis.

Filamentous actin binding ability of cortactin isoforms is responsible for their cell-cell junctional localization in epithelial cells.

Cortactin is an F-actin binding protein that contributes to cytoskeleton remodelling. We identified five isoforms of mouse cortactin that differ in the number of tandem 37-amino acid repeats, named cortactin repeats. The transcription of minor isoforms with 4.5, 3.5 or 2.5 cortactin repeats was low in most adult tissues whereas an isoform with 4.5 cortactin repeats was highly transcribed in the adult brain. In accordance with the brain-specific upregulation of a minor isoform, a brain-specific novel 72-kDa cortactin protein was identified. Major isoforms with 6.5 or 5.5 cortactin repeats bound F-actin more robustly than minor isoforms in vitro. All isoforms were concentrated at cell-cell junction sites in epithelial cells. Deletion mutants lacking whole cortactin repeats did not bind F-actin and were not concentrated at cell-cell junction sites. Thus, the F-actin binding ability is mostly correlated with the number of cortactin repeats and is required for the cell-cell junctional localization.

Radiation-induced deletions in the 5' end region of Notch1 lead to the formation of truncated proteins and are involved in the development of mouse thymic lymphomas.

Notch1 protein is a transmembrane receptor that directs various cell fate decisions. Active forms of Notch1 consisting of a transmembrane domain and an intracellular domain (Notch1TM) or only an intracellular domain (Notch1IC) function as oncoproteins. To elucidate the effect of Notch1 abnormalities in radiation-induced lymphomagenesis, we determined the structure of the Notch1 gene and examined the frequency and the sites of Notch1 rearrangements in radiation-induced mouse thymic lymphomas. The Notch1 gene consists of 37 exons, including three exons upstream of the previously reported exon 1. The transcript starting from exon 1 was the major transcript whereas the transcripts read upstream from exon 1a, in which amino acid sequences in the N-terminal region were changed, were minor. More than 50% of radiation-induced thymic lymphomas exhibited Notch1 rearrangements, suggesting that Notch1 acts as a major oncogene in radiation-induced lymphomagenesis. We identified three rearranged sites: novel sites in the 5' end region encompassing exons 1 and 2, the previously identified juxtamembrane extracellular region, and the 3' end region. The 5' deletion and the insertion of murine leukemia virus in the juxtamembrane region led to the production of abnormal transcripts starting from cryptic transcription start sites located halfway through the Notch1 gene and resulted in transcripts lacking most of the extracellular domain. As a result of these rearrangements, truncated Notch1 polypeptides resembling Notch1TM or Notch1IC were formed. In contrast, the 3' deletion led to the production of a C-terminal PEST motif-deleted transcript. The downstream target gene Hes1 was transcribed in a lymphoma with insertion of murine leukemia virus, but not in a lymphoma with a 5' deletion. These results indicate that in addition to Hes1 expression, other Notch1 pathway(s) have a role in thymic lymphomagenesis and suggest the presence of a novel mechanism for oncogenic activation of Notch1 by 5' deletion.

Formation of an active form of the interleukin-2/15 receptor beta-chain by insertion of the intracisternal A particle in a radiation-induced mouse thymic lymphoma and its role in tumorigenesis.

Although many reports suggest that aberrant regulation of cytokine signaling pathways via the interleukin-2 receptor (IL-2R) induces tumorigenic transformation, constitutively active IL-2R in tumors has not been reported. We searched for genomic alteration of the IL-2/15R beta-subunit gene (IL-2/15R beta) in cytokine-independent cell lines established from radiation-induced mouse thymic lymphomas. In the TL34 cell line and its primary tumor, one of the IL-2/15R beta alleles was rearranged by the insertion of an intracisternal A particle (IAP) retrotransposon. The IAP-IL2/15R beta chimeric gene expressed chimeric mRNA in which IAP-coding Gag-Pol mRNA was fused to IL-2/15R beta mRNA and coded for Gag-Pol-IL-2/15R beta chimeric protein. Forced expression of the Gag-Pol-IL-2/15R beta chimeric cDNA in a mouse cytotoxic T-cell line (CTLL-2) converted IL-2-dependent cell growth to IL-2-independent growth, suggesting that the chimeric protein activates some of the IL-2 signaling pathways necessary for cell proliferation. Downregulation of the expression of the Gag-Pol-IL-2/15R beta chimeric protein in TL34 by antisense RNA inhibited cell growth, and concomitantly reduced the level of c-myc protein. These results suggest that the Gag-Pol-IL-2/15R beta is a constitutively active form that transmits proliferative signals by expressing downstream target genes, including c-myc. Thus, we demonstrated that the chimeric receptor gene produced by the insertion of an IAP functions as an oncogene by providing IL-2-independent autonomous growth potential.

Involvement of DNA-dependent protein kinase in down-regulation of cell cycle progression.

The catalytic polypeptide of DNA-dependent protein kinase (p470) is encoded by the gene responsible for murine severe combined immunodeficiency (SCID) devoid of DNA double-strand break repair and V(D)J recombination. Here, we have characterized the role of p470 in cell proliferation using SCID mice and the cell lines. In accord with DNA histogram patterns, SCID cell lines (SD/SD-eA and SC3VA2) expressing extremely low level of DNA-PK activity grew faster than a normal mouse cell line (CB/CB-eB) and SC3VA2 complemented with human p470 gene (RD13B2). In regenerating liver after partial hepatectomy, de novo DNA synthesis determined by [(3)H]thymidine incorporation started at 30h in C.B-17/Icr-SCID (SCID) mice and at around 36h in C.B-17/Icr (C.B-17) mice. Compared with normal cells, SCID cells contained slightly higher levels of transcripts of cyclin A, cyclin E, B-Myb and dihydrofolate reductase, which are regulated by E2F-1. E2F-1 playing a key role in G1- to S-phase progression was phosphorylated in vitro by DNA-PK. Importantly, the E2F-1 promoter transcriptional activity in SCID cell lines (SD/SD-eA and SC3VA2) was 4-5-fold higher than that in CB/CB-eB and RD13B2. These results suggest that p470 is involved in down-regulation of cell cycle progression through E2F-1-responsible genes.

A new technique to prevent self-ligation of DNA.

The most widely used technique for preventing self-ligation (self-circularization and concatenation) of DNA is dephosphorylation of the 5'-end, which stops DNA ligase from catalyzing the formation of phosphodiester bonds between the 3'-hydroxyl and 5'-phosphate residues at the DNA ends. The 5'-dephosphorylation technique cannot be applied to both DNA species to be ligated and thus, the untreated DNA species remains capable of self-ligation. To prevent this self-ligation, we replaced the 2'-deoxyribose at the 3'-end of the untreated DNA species with a 2',3'-dideoxyribose. Self-ligation was prevented at the replaced 3'-end, while the 5'-phosphate remaining at the 5'-end permitted ligation with the 3'-hydroxyl end of the 5'-dephosphorylated DNA strand. We successfully applied this 3'-replacement technique to gene cloning, adapter-mediated polymerase chain reaction and messenger RNA fingerprinting. The 3'-replacement technique is simple and not restricted by sequence or conformation of the DNA termini and is thus applicable to a wide variety of methods involving ligation.

Independent variation in susceptibilities of six different mouse strains to induction of pepsinogen-altered pyloric glands and gastric tumor intestinalization by N-methyl-N-nitrosourea.

Strain differences in susceptibility regarding stomach carcinogenesis due to N-methyl-N-nitrosourea were examined in males of six strains of mice: BALB/cA (BALB), C57BL/6N (C57BL6), CBA/JN (CBA), C3H/HeN (C3H), DBA/2N (DBA/2), and CD-1 (ICR). The frequency of pepsinogen-altered pyloric glands (PAPGs), putative precancerous lesions, was highest (19.6+/-9.9%) in the BALB and lowest in the ICR (12.3+/-5.7%) mice (P<0.05). Incidences of adenocarcinomas at week 52 were 59.3% (16 of 27) and 18.5% (5 of 27), respectively (P<0.005). Invasion also tended to be deepest in BALB compared with the other strains. Intestinal alkaline phosphatase-positive intestinal type cells were observed heterogeneously in some hyperplasias, adenomas and adenocarcinomas consisting of gastric type cells. Thus, intestinalization appeared to occur at random in both non-neoplastic and monoclonal neoplastic lesions, making it unlikely that IAP-positive cells could be precursors of gastric tumors. In contrast, the data suggest a direct histogenetic role for the PAPG, a useful preneoplastic marker lesion in mouse strains.