Yasuhiko Takamori

Enhancement of estrogen receptor gene expression in the mediobasal hypothalamus during anestrus in the beagle bitch.

Estrogen receptor mRNA (ER mRNA) levels were measured in the mediobasal hypothalamus (MBH) of beagle bitches at different stages of the estrous cycle, and compared with levels in ovariectomized (OVX) estrogen-treated bitches. In cyclic bitches, the level of hypothalamic ER mRNA increased during the progression of anestrus and declined thereafter. Hypothalamic ER mRNA and plasma luteinizing hormone (LH) levels during anestrus and proestrus were positively correlated (r = 0.94, P < 0.001). In OVX bitches, levels of hypothalamic ER mRNA were low, and increased significantly after treatment with a low dose of estradiol benzoate. These results suggest that, during the course of anestrus in the bitch, hypothalamic ER mRNA expression increases, and may be up-regulated by estradiol.

Putative tumor suppressor gene region within 0.85 cM on chromosome 12 in radiation-induced murine lymphomas.

Analyses of genetic alterations in tumors from F1 hybrid mice produced by inter‐subspecific crosses between genetically well‐characterized inbred strains provide precise and comprehensive evidence for genetic abnormalities such as allelic loss. We performed loss of heterozygosity (LOH) analyses of 125 radiation‐induced lymphomas of (BALB/cHeA × MSM/Ms)F1 hybrid mice by polymerase chain reaction (PCR) analysis of microsatellite DNA polymorphic markers. Very frequent LOH was found at a distal region on chromosome 12. To precisely define the most common region of LOH, we first determined the order of and distances between the available microsatellite loci around the region by using 586 (CXSD × MSM/Ms)F2 hybrid mice (1172 meiosis). The locus order and distances were [centromere]—D12Mit132—(0.34 cM)—D12Mit50—(2.05 cM)—[D12Mit122, D12Mit53]—(0.85 cM)—D12Mit233—(0.43 cM)—D12Mit279—(0.17 cM)—D12Mit181—[telomere]. We then investigated the features of LOH at these loci. The highest frequency of LOH (83 of 125, 66%) was found at D12Mit233. The LOH patterns of individual lymphomas indicated that the most common region of LOH was within the 0.85 cM between D12Mit53 and D12Mit233, a region homologous to human chromosome 14q32.1. These results suggest that a putative novel tumor suppressor gene exists within this region. Mol. Carcinog. 22:175–181, 1998.

Highly sensitive assay of DNA abasic sites in mammalian cells-optimization of the aldehyde reactive probe method

We have recently developed a novel method for detection and quantitation of abasic (AP) sites in DNA, in which the biotinylated reagent, called the aldehyde reactive probe (ARP) specifically reacts with aldehyde groups of AP sites and biotin-tagged damage is detected by an ELISA-like assay. The present study has been carried out to improve the feasibility and the sensitivity of ARP assay. For immobilization of DNA, a protamine sulfate-coated plate was used instead of the conventional UV-irradiated plate to enhance DNA binding. As the result, the time for immobilization was shortened to 1 h without any loss of signal. The amount of [ 3 H]-labeled DNA bound to the plate was proportional to the DNA concentration employed. When DNA containing AP sites was treated with ARP in solution prior to coating the protamine-plate, the sensitivity of the assay was greatly increased. A linear relationship between the DNA concentration and the signal intensity was also observed. Thus, ∼0.1 fmol of AP sites (0.5 sites per 10 5 nt) could be detected in DNA isolated from HeLa cells after treatment with a sublethal dose (0.5 mM) of methylmethanesulfonate (MMS). Using this system, the number of total methylpurines generated by MMS in the cellular DNA was estimated after heat treatment, which converted methylated base lesions to AP sites. It was shown that the number of AP sites was about 140 sites per 10 4 nt with 25 mM MMS and 10% of total methylated bases were already released without heat depurination.

Increased lh pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.

Presence of B220 within thymocytes and its expression on the cell surface during apoptosis

B220 is the full‐length splicing isoform of a tyrosine phosphatase CD45 and is predominantly expressed as a transmembrane protein on B cells. Other splicing isoforms of CD45 are yielded by alternative splicing of exons 4, 5 and 6. Recently, the expression of B220 on peripheral T cells during activation‐induced cell death has been reported. To investigate whether B220 is implicated in apoptosis of immature T cells, we analysed (by flow cytometry using the anti‐B220 monoclonal antibody, RA3‐6B2) the expression of B220 on mouse thymocytes undergoing X‐irradiation‐ and dexamethasone (DEX)‐induced apoptosis. The expression of B220 on thymocytes positive for Thy‐1 was induced by X‐irradiation or DEX treatment and increased with length of incubation. The expression of B220 was pronounced on the apoptotic hypodiploid cells in the fraction showing lower forward scattering values. Reverse transcription–polymerase chain reaction detected mRNA containing exons 4, 5 and 6 of CD45 in normal thymocytes as well as those exposed to X‐rays or DEX. Surprisingly, cytoplasmic B220 antigens were detected in a considerable fraction of normal thymocytes. Moreover, the expression level of the 220 000‐MW protein in normal thymocytes was similar to that in the thymocytes undergoing apoptosis. During apoptosis, the expression level of B220 antigen was reduced in the cytoplasm but, conversely, up‐regulated on the surface of thymocytes. These results suggest that B220 is constitutively expressed as a cytoplasmic form within thymocytes and possibly translocated to the cell membrane during apoptosis.

Repair Kinetics of Abasic Sites in Mammalian Cells Selectively Monitored by the Aldehyde Reactive Probe (ARP)

Human methylpurine N-glycosylase (MPG) activity was investigated by monitoring abasic (AP) sites resulting from removal of alkylated bases. The amount of AP sites in MMS-treated HeLa cells transiently increased at 3 h, then gradually decreased to 40% at 24 h. The presence of adenine, an inhibitor of AP endonucleases, in the repair incubation of MMS-treated cells induced moderate accumulation of AP sites, suggesting inhibition of the activities of MPG as well as AP endonucleases by adenine metabolites.

Endogenous type-C viral expression during lymphoma development in irradiated NFS mice.

The expression of the type-C retrovirus and the virus-related components in NFS mice were examined during preleukemic and leukemic phases after fractionated whole-body X irradiation. The NFS mice were highly susceptible to induction of thymoma by fractionated X irradiation. The leukemic tissues were negative for infectious type-C virus, as detected by both the XC-plaque test and mink S+ L- focus-inducing assays, but contained a substantially higher level of viral-specific RNA-dependent DNA polymerase activity and a major core protein p30 than the corresponding tissues from unirradiated age-control mice. In the preleukemic phase, the amount of p30-related antigen increased transiently in spleen. The leukemic cell lines established from radiation-induced lymphomas produced particulate entities with a buoyant density of about 1.15 g/ml. These virus-like particles lacked in vitro infectivity to mouse cells and mink lung cells and leukemogenicity in syngeneic mice. The p30-related antigens of these particles were immunologically similar to that of xenotropic virus derived from NZB mouse.

Lack of Evidence for the Involvement of Type-C and Type-B Retroviruses in Radiation Leukemogenesis of NFS Mice

Southern blot analysis revealed no difference between the DNA from radiation-induced thymic lymphomas and DNA from normal NFS mice. The probes used in the Southern blot analyses used a murine leukemia virus (MuLV) env DNA probe (pXenv), which specifically hybridizes with xenotropic and recombinant viral env genes, and mouse mammary tumor virus (MMTV) DNA probes (MMTV gag-pol, MMTV env, and MMTV LTR). This suggests that radiation leukemogenesis was not associated with gross alteration of the organization of these retroviral genomes. In DNA from radiation-induced thymic lymphoma, there was no indication of gross rearrangement in the common integration site of MuLV, pim-1, or in the common integration sites of MMTV, int-1 and int-2. Dot blot analysis of RNA from radiation-induced thymic lymphomas and normal thymuses demonstrated that there was no substantial difference between them in the expression of retroviral sequences, pim-1, pvt-1, int-1, or int-2, although transcripts that could be hybridized to the retroviral sequences were slightly elevated in some radiation-induced thymic lymphomas. These results show that radiation leukemogenesis does not appear to involve the activation of endogenous type-C and type-B retroviruses.

The Genesis of Thy-1- Lymphomas in NFS Mice Exposed to X Irradiation

The kinetics of the appearance of potentially leukemic cells (PoLCs) for radiation-induced lymphoma in NFS mice was investigated by the opposite sex (male----female) transplantation assay. The origin of the cells of the lymphomas that developed in the host was decided by sex chromosome markers. The bone marrow and the spleen cells collected from mice 30 days after fractionated irradiation (1.7 Gy X 4) gave rise, upon transfer to 4-Gy-irradiated hosts, to tumors of either donor or host origin. Most tumors of donor origin were thymine-1-negative (Thy-1-) and surface immunoglobulin negative and classified as nonthymic lymphoma, while the tumors of host origin were mainly Thy-1-positive thymic lymphoma. In contrast, neither the bone marrow nor the thymus contained any PoLCs for thymic lymphoma 30 days after split-dose irradiation. These results indicate that PoLCs for Thy-1-lymphoma were induced in the bone marrow and spleens of NFS mice by the split-dose regimen which developed exclusively T-cell lymphomas in the absence of cell grafting.

Host Sex-Dependent Growth of Potential Thy-1+ Lymphoma Cells That Appear in the Thymus of X-Irradiated NFS Mice

During the course of studies designed to detect potentially leukemic cells in radiation lymphomagenesis, using an opposite-sex (male----female) transplantation assay method, we previously found that potential Thy-1- lymphoma cells are generated in the bone marrow of NFS mice exposed to a split-dose irradiation (1.7 Gy X 4), while potential Thy-1+ lymphoma cells are not detectable. In this report, using a (female----male) intrathymic transplantation assay system we show that potential Thy-1+ lymphoma cells were generated in the thymus of female NFS mice exposed to split-dose irradiation, and reconfirm that such cells were not detected in the (male----female) transplantation system. These results demonstrate that the detection of potential Thy-1+ lymphoma cells strictly depends on the transplantation system.