Susana Merani

PROTECTION OF RADIATION-INDUCED DAMAGE TO THE HEMATOPOIETIC SYSTEM, SMALL INTESTINE AND SALIVARY GLANDS IN RATS BY JNJ7777120 COMPOUND, A HISTAMINE H4 LIGAND

Based on previous data on the histamine radioprotective effect on highly radiosensitive tissues, in the present work we aimed at investigating the radioprotective potential of the H4R ligand, JNJ7777120, on ionizing radiation-induced injury and genotoxic damage in small intestine, salivary glands and hematopoietic tissue. For that purpose, rats were divided into 4 groups. JNJ7777120 and JNJ7777120-irradiated groups received a daily subcutaneous JNJ7777120 injection (10 mg/kg) starting 24 h before irradiation. Irradiated groups received a single dose of 5 Gy on whole-body using Cesium-137 source and were sacrificed 3 or 30 days after irradiation. Tissues were removed, fixed, stained with hematoxylin and eosin or PAS staining and histological characteristics were evaluated. Proliferative and apoptotic markers were studied by immunohistochemistry, while micronucleus assay was performed to evaluate DNA damage. Submandibular gland (SMG) function was evaluated by methacholine-induced salivation. Results indicate that JNJ7777120 treatment diminished mucosal atrophy and preserved villi and the number of crypts after radiation exposure (240±8 vs. 165±10, P<0.01). This effect was associated to a reduced apoptosis and DNA damage in intestinal crypts. JNJ7777120 reduced radiation-induced aplasia, preserving medullar components and reducing formation of micronucleus and also it accelerated bone marrow repopulation. Furthermore, it reduced micronucleus frequency in peripheral blood (27±8 vs. 149±22, in 1,000 erythrocytes, P<0.01). JNJ7777120 completely reversed radiation-induced reduced salivation, conserving glandular mass with normal histological appearance and reducing apoptosis and atrophy of SMG. JNJ7777120 exhibits radioprotective effects against radiation-induced cytotoxic and genotoxic damages in small intestine, SMG and hematopoietic tissues and, thus, could be of clinical value for patients undergoing radiotherapy.

Establishment of two hormone-responsive mouse mammary carcinoma cell lines derived from a metastatic mammary tumor.

We report the establishment of two mouse mammary cancer cell lines, MC7-2A and MC7-2B obtained from a mouse mammary carcinoma induced by medroxyprogesterone acetate (MPA) and maintained by syngeneic transplantation in BALB/c mice. They are epithelial (express cytokeratins) and express both estrogen receptors alpha (ERα) and progesterone receptors (PRs) isoforms A and B (western blots). In vitro, MPA inhibited 3H-thymidine uptake, starting from concentrations as low as 10−13 M in MC7-2A and 1010 M in MC7-2B; the antiprogestin RU 486 exerted a stimulatory effect at 10−14 M in both cell lines; 17-β-estradiol (E2) also exerted a stimulatory effect starting at 10−10 M in MC7-2A and at 10−13 M in MC7-2B. When transplanted in syngeneic mice, both cell lines originated adenocarcinomas that gave rise to lung metastases within 3 months. In in vivo studies, in MC7-2A, the antiprogestin inhibited completely tumor growth, E2 induced a slight although significant (p 9 0.05) stimulatory effect and MPA stimulated tumor growth while MC7-2B cells were unresponsive to all treatments. ER and PR were also expressed in tumors as assessed by immunohistochemistry. Two marker chromosomes were identified by FISH as translocations between chromosomes 4 and 7, and between chromosomes X and 2; the third marker chromosome remains unidentified. All these markers were also present in the parental tumor. A new marker, a centric fusion of chromosomes 2, was acquired in both cell lines. Considering that there are very few murine breast carcinoma responsive cell lines, these cells represent new tools in which the regulatory effect of hormones can be studied.

Karyotypic evolution of four novel mouse mammary carcinoma cell lines. Identification of marker chromosomes by fluorescence in situ hybridization

We studied the karyotypes of four different mammary carcinoma cell lines derived from a medroxy-progesterone acetate (MPA)-induced mouse mammary carcinoma using G-banding and fluorescence in situ hybridization. All the cell lines showed the same four marker chromosomes (M1-M4) as the parental tumor and also acquired new markers. M1 and M2 are Robertsonian translocations between chromosomes 1 and 10 and 2 and 17. M3 is an acrocentric marker derived from chromosomes 4, 5, and 12; M4 is derived from chromosomes 6 and 8. The parental tumor disclosed a modal number of 39, with a trisomy of chromosomes 3, 4, 10, and 11 and monosomies of 9, 13, and 16. MC4-L1 and MC4-L3 lines had a chromosome number similar to that of the parental tumor in early passages, which increased to the triploid range in late passages. MC4-L5 showed a near-diploid modal number in both early and late passages. MC4-L2 cells had a high chromosome number even in early passages. To our knowledge, this is the first study in which a complete characterization of the cytogenetics of murine mammary carcinoma cell lines and of their parental tumor is described. No associations between changes in ploidy, invasiveness, or hormone dependence were found. Conversely, the presence of one exclusive marker chromosome, a translocation between chromosomes 1 and 18 (M5), in the most aggressive and in vivo hormone-independent line suggests that this rearrangement may be associated with these biologic features. The constant presence of common marker chromosomes in both the parental tumor and the derived cell lines suggests that they are involved in the maintenance of this tumor phenotype.

Mortality Induced by Adoptive Immunity in Junin Virus-Infected Athymic Mice

The effect of normal or sensitized spleen cell transfer from syngeneic euthymic mice to Junin virus-infected suckling athymic mice was studied. Transfer was performed 1 or 7 days after infection. In both cases, an acute lethal disease developed 6-11 days after transfer. The mortality reached 100% in all infected groups receiving normal or sensitized splenocytes, while it was negligible for different control groups of athymic mice. Transfer of normal or sensitized splenocytes was unable to significantly modify brain viral titers, as compared with infected nontransferred athymic mice killed after a 25-day observation period. Brain lesions were demonstrated in about half of the infected athymic mice transferred with sensitized splenocytes and in all euthymic infected mice. These results show that splenocyte transfer from immunocompetent donors is able to change the normal course of persistent Junin virus infection in nude mice to a lethal acute disease, thus pointing to a main role for T cells in its pathogenesis.

Induction of Junin Virus Persistence in Adult Athymic Mice

To determine the role of T lymphocytes in adult mice infected with Junin virus, 60-day-old athymic (nu/nu) mice and their immunocompetent (nu/+) littermates were inoculated intracerebrally with 10(3) TCD50 of the XJ strain. None of them exhibited neurologic illness during a 6-month observation period, and mortality was 3% for nu/nu and 7% for nu/+ animals. The main features in infected nu/nu mice were: high viral titers in brain, reaching a late peak (6.5 log/ml) 32 days postinoculation and persisting at least 6 months; low, late viremia appearing simultaneously with the viral peak in the central nervous system (CNS) and persisting up to 3 months after infection; absence of signs of neurologic disease or histologic lesions in brain and almost no mortality; and lack of detectable circulating antibodies either in IgM or in other immunoglobulins (Igs). Circulating anti-Junin antibodies were demonstrated in IgM and other Igs in immunocompetent mice, although no infectious virus or histologic lesions could be detected. These results show an important role for T lymphocytes in the clearance of Junin virus in the CNS, as demonstrated by viral persistence induced in adult athymic mice.

Chromosome studies of murine T-cell lymphoid leukemia and derived cell lines.

Several cell lines were previously established from a spontaneous murine T-cell leukemia (LB). The aim of this study was to analyze the G- and C-banded karyotypes of the parental LB tumor cells and the derived cell lines. A sensitive cell line (LBL) from which two sublines originated, as well as Vincristine (LBR-V160) and Doxorubicin (LBR-D160) resistant cell lines, were used. Our results showed that LB cells had a pseudo-diploid karyotype with 40 acrocentric chromosomes in which trisomy of chromosome 14 was the most relevant alteration. The sensitive cell line showed this alteration in all metaphases studied; no changes in karyotypes were observed in either subline, despite their dissimilar morphology and growth patterns. In contrast, both resistant lines displayed a more heterogeneous karyotype with no common markers, except for the finding that chromosome 5 was involved in a trisomy in LBR-V160 and in a translocation with chromosome 12 in LBR-D160. Taking into account that the mdr genes are located in chromosome 5, these results suggest a possible association between such alterations and the acquisition of drug resistance.

Abstract 329: Identification of the breakpoints of two chromosome translocations involving chromosomes 4 and 7 observed in two murine mammary carcinomas

The translocation between chromosomes 4 and 7, T(4;7), was observed in two different mammary carcinomas from the medroxyprogesterone acetate (MPA) mouse breast cancer model which have acquired a hormone independent (HI) phenotype (C7-2-HI and C4-HI). We had developed a cell line from the C7-2-HI tumor, the MC7-L2A cells, which retain the chromosome translocation T(4;7), and provide an easy tool to obtain metaphases. G-banding analysis showed that the breakpoint on chromosome 4 is located on the band 4E in the C7-2-HI tumor, whereas in the C4-HI tumor, it is located on the region 4C4-4D3. However, the breakpoint on chromosome 7 is located near the centromere in both tumors. Further experiments using fluorescence in situ hybridization (FISH) in MC7-L2A cells have shown that the breakpoint of the translocation involved the chromosome bands 4E2 and 7A1-A2. The aim of this project is to elucidate whether these breakpoints are involving new gene rearrangements that might participate in the acquisition of hormone independency. In this study we focused in determining if the break in chromosome 7 is located on the same band in both HI tumors. A detailed analysis in MC7-L2A cells using FISH with different DNA probes, spanning the region 7A1-7A2, showed that the breakpoint is located on chromosome 7A1 and not on band 7A2. However, a different picture was observed in metaphases obtained from C4-HI tumor cells, in which the probes corresponding to region 7A1-7B1 hybridized only to normal chromosome 7, but not to the translocation T(4;7). These results indicate that the breakpoints on chromosomes 4 and 7, although involving the same chromosomes, are located on different regions in the T(4;7) translocations, suggesting that they may be random breakpoints. Even though we could not associate this breakpoint with the hormone-independent phenotype, the identification of the sites of chromosome breaks may be relevant to understand the biology of each HI tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 329.