BDIX Rats Research Articles

Expression of c-sis and platelet-derived growth factor in in vitro-transformed glioma cells from rat brain tissue transplacentally treated with ethylnitrosourea.

Long-term culturing of brain cells from neonatal BD-IX rats after transplacental treatment with N-ethyl-N-nitrosourea (ENU) results in malignantly transformed cells after a lag period of about 250 days. During culturing, the brain cells undergo a sequence of morphological changes. We examined oncogene expression in cultured cells from ENU-treated animals and found that transformed glioma cells differ from premalignant glial cells by containing high levels of c-sis transcripts. We also report that the transformed cells synthesize functional platelet-derived growth factor. Because glial cells have receptors for platelet-derived growth factor, we propose that an autocrine mechanism plays an important role in ENU-induced brain tumorigenesis.

Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185

The neu oncogene, which is frequently activated in neuro- and glloblastomas of BDIX rats, was originally identified in the NIH 3T3 focus-forming assay. cDNA clones of the normal and transforming alleles of neu have been isolated. When these clones are inserted into the expression vector pSV2, they direct the synthesis of p185, the neu gene product. The transforming cDNA clone yields foci when transfected onto a NIH 3T3 monolayer, but the normal cDNA does not. The construction of in vitro recombinants between the normal and transforming cDNAs has allowed the determination of the mutation responsible for the activation of the neu proto-oncogene. A single point mutation changes a valine in the transmembrane domain of the predicted protein product insert to a glutamic acid. The DNAs from four independent cell lines containing activated neu oncogenes contain the identical mutation at this position.

Immunocytochemical characterization of the A15 A5 transplantable brain tumour model in vivo.

A comparative immunocytochemical study was carried out on intracerebral and extracranial gliomas of the rat produced by intracerebral injection of low (10th) and high (40th) in vitro passages of neoplastic glial cells. The cells injected were a neoplastic astrocytic clone-A15 A5-derived from a mixed glioma induced transplacentally by N-ethyl-N-nitrosourea (ENU) in a BD-IX rat. An inverse relationship was seen between the expression of the astrocytic markers glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) at low and high passage: GFAP decreased with increasing passage while GS increased. The distribution of vimentin, the major cytoskeletal component of immature glia, was constant, irrespective of passage--a feature consistent with previous in vitro findings. The expression of laminin by both reactive and neoplastic astrocytes increased with increasing passage, while high magnification examination revealed the presence of the glycoprotein fibronectin on the cell-surfaces of A15 A5-derived tumour cells. Both neoplastic and reactive astrocytes expressed S-100 protein with a higher proportion of positive cells in extracranial tumours. Occasional cells, probably actively phagocytizing populations of reactive astrocytes and macrophages, were positive for alpha-1-antitrypsin. None of the neoplastic cells expressed the oligodendrocyte marker carbonic anhydrase II. This immunocytochemical study supports previous morphological findings in differences in differentiation between the cells of tumours produced by high and low passage cells.

Characteristics of the karyotype in chemically transformed rat glial cells.

The karyotypes of two malignant cell lines, GA1 and GA2, derived from chemically transformed glioblasts of WKA and BDIX rats, respectively, were investigated by G-banding at early passage numbers. In GA1, the karyotype 43; XY, 2q-, 4q+ and 12 trisomy, predominated and the cells with an additional 10p+ were less frequently observed. These consistent chromosomal changes were exactly identical with those previously found in virus-induced glioblast lines. GA2 had the normal male diploid stemline although a minor population of cells having the GA1-karyotype was also present. Thus, the present results combined with prior findings indicated that the in vitro transformed rat glial cells showed the common and specific changes in the karyotype regardless of the type of the oncogenic agent and rat strain.

Lack of mast cell reactivity during leukemic infiltration of the rat mesentery under syngeneic and allogeneic conditions. A study by transmission electron microscopy (TEM).

The ultrastructural morphology of mast cells localized in rat mesenteries was studied after intraperitoneal implantation of L5222 rat leukemia cells in syngeneic and allogenic hosts. It became evident that the mast cells in the syngeneic (BDIX rat) as well as the allogeneic system (BN rat) showed nor morphological alterations. Degranulation was never observed. This is in contrast to the behavior of macrophages which displayed a strong phagocytotic activity in allogeneic hosts. Thus, it seems that mast cells, under the present experimental conditions, remained inactive during a phase of intense tumor rejection.

Steroid receptors in dimethylhydrazine-induced colon carcinogenesis.

To investigate whether gonadal hormones are involved in the tumorigenesis of dimethylhydrazine (DMH)-induced colonic neoplasms, the authors measured steroid receptors in the neoplasms. 30- or 60-day-old BD-IX rats were injected with 20 mg of 1,2-DMH per kg of body weight once a week for 20 weeks. Fifty-seven rats were sacrificed at 40 to 45 weeks after the initial injection. Androgen receptor (AR), estrogen receptor (ER), and progesterone receptor (PR) were measured in colonic neoplasms. The total number of colonic neoplasms was 274 among 57 rats, 65.8% in male rats and 34.2% in female rats. The mean number of colonic neoplasms per rat was higher in male rats, i.e., 5.6, compared with 3.5 in female rats. A slightly higher number of colonic neoplasms per rat was seen in the rats that had the initial injection at 30 days of age. The number of large colonic neoplasms with a diameter of more than 1 cm was 77 (28.1%), 74% of which were seen in male rats. Thus, a higher incidence of tumors that were also larger were seen in male rats. Histologic findings showed that 53.6% of the neoplasms were carcinomas. The highest incidence of colonic neoplasms was in the distal colon in both sexes. Most of the well-differentiated adenocarcinoma were seen in the distal colon (82.2%), whereas mucinous carcinoma and undifferentiated adenocarcinoma were prominent in the proximal colon or cecum (56.1%). In rats with a normal colon, low levels of AR and PR were determined; but ER was not found in any regions of the colon. In DMH-induced colonic cancer, the incidence as well as the concentration was higher in male rats (60.6%, 16.9 +/- 3.6 fm/mg protein), compared with female rats (40.0%, 4.6 +/- 0.8 fm/mg protein). Similar incidences and levels of ER and PR were seen in both sexes. There was no relationship between steroid receptors and histologic findings in colonic neoplasms. These results suggest that the gonadal hormones, especially androgens, appear to be involved in DMH-induced colon tumorigenesis in male rats.

Expression of monoclonal antibody-defined cell surface antigens during rat brain development

Using single-cell suspensions of mechanically dissociated, prenatal BDIX-rat brain cells (13th, 15th, and 21st days after fertilization) for immunization, we have established a collection of 37 monoclonal antibodies (Mabs) directed against neural cell surface determinants. The developmental-stage-dependent expression of cell-surface antigens recognized by these Mabs was analyzed both on plasma membranes isolated from whole brains of BDIX rats (prenatal days 13–22 and adults) using an indirect 125I so-lid-phase radioimmunoassay, and on intact BDIX-rat brain cells (prenatal days 13-22) using a fluorescence-activated cell sorter. Different types of developmental stagedependent profiles of Mab binding were found, these being indicative of the presence of neural cell surface determinants whose expression increases, decreases, or does not change with brain development. Some of the Mab-binding profiles showed transient changes as a function of developmental stage. These Mabs are currently being used for the characterization, reproducible identification, and isolation of neural cell subpopulations of the developing rat brain, with the aim of investigating the cell type dependence and developmental (differentiation) stage dependence of malignant transformation following pulse exposure to the carcinogen N-ethyl-N-nitrosourea at defined stages of brain development.

Radiation therapy of rat brain tumor using misonidazole as hypoxic cell sensitizer.

Rat brain tumor was used as a model to evaluate radiation therapy with and without misonidazole. BD-IX rats were implanted intracerebrally with an ethylnitrosourea-induced glioma. Three series of experiments were performed, with radiation given 14 days after inoculation of the glioma clone. In each series, the following radiation doses were given: 500 rads once, 1,000 rads once; and 1,000 rads twice, every time with or without two different doses of misonidazole. Radiation therapy significantly prolonged survival when compared to the longevity of the control group. The dose of 1,000 rads given twice was highly effective and the life-span of tumor-bearing rats increased from 72% to 121%. Misonidazole plus irradiation negated the prolongation of survival, achieved with radiation therapy alone.

Strain dependence of rat macrophage natural tumoricidal capacity and its stimulation by endotoxins

Without known stimulation in vivo and in vitro, resident peritoneal macrophages from 5 conventional or specific pathogen-free (SPF) rat strains [Hairless (H), BDIX, Wistar (W), Sprague-Dawley (SD) and Long-Evans (LE)] exhibited an in vitro strain-dependent cytolysis against DHD-K12/TS cancer cells. This natural cytolysis was also observed when polymyxin B was added to the culture medium. The percentage of natural cytolysis varied from one rat to another but was significantly different according to the strain. In the presence of 10 μg endotoxin/ml, macrophages from BDIX, W, SD and LE rats were always cytolytic, whilst those of H rats were irregularly cytolytic. Endotoxins induced or increased macrophage-mediated cytolysis from H, BDIX, W and SD rats, but they were without effect for LE rats. The endotoxin effect depended on the level of natural cytolysis. In contrast to mouse resident peritoneal macrophages, which were not naturally cytolytic and not activated in vitro by endotoxins, these results show that rat resident peritoneal macrophages can be naturally cytolytic. This cytolysis can be enhanced by endotoxins as the sole in vitro stimulus. Rat macrophage natural cytolytic activity is strain-dependent.

The vasculature of experimental brain tumours: Part 3. Permeability studies

The aim of this work was to elucidate the direction and time-course of transport processes which may affect the accumulation of oedema associated with experimental brain tumours. Astrocytomas were produced in BD-IX rats by intracerebral injection of cultured neoplastic glial cells. The cell line used was cloned from a culture of a primary mixed glioma induced by transplacental administration of N-ethyl-N-nitrosourea (ENU). At various times after cell injection the protein tracer horseradish peroxidase (HRP) was given to tumour-bearing rats, either intravenously or into the lateral ventricles of the brain. The movement of the HRP into tumours and surrounding brain either from blood or from ventricular cerebrospinal fluid (CSF) was studied by light and electron microscopy at various intervals after the injection of the tracer. The time-course of subsequent clearance of the HRP from the tumours and surrounding brain was also investigated. After intravenous injection, HRP rapidly penetrated all vascularised tumours and became evenly distributed within 10–20 min. The HRP remained present in sufficient quantity within the tumours to maintain this intensity for several hours, after which it gradually disappeared, showing no reaction product after 12 h. After intraventricular injection, HRP penetrated periventricular brain tissue up to a maximal distance 1–2 mm within 2 min, and the reaction product remained visible in this region for at least 20 min. In all tumour-bearing animals, HRP penetrated further into periventricular tumour tissue than into adjacent brain tissue. In large tumours HRP reaction product was seen up to 7 mm from the ventricular ependymal lining, although permeation to this distance took up to 10 min.

Immunologic enhancement of experimental metastasis in the rat.

Using a series of immunologically cross-reactive metastatic tumor variants, we demonstrate that serum from animals bearing pulmonary tumor colonies possesses enhancing properties in the experimental metastasis (lung colony) assay. Enhancement is produced by chronic serum administration and promotes the growth of tumor cells arrested in the lungs which would not otherwise proliferate to form grossly detectable lung nodules. Tumor-bearer serum from animals with lung colonies derived from the most highly metastatic variant examined is shown to possess enhancing properties in both BD-IX(H-1d) and BD-IV(H-1d) rat strains, while tumor-bearer serum from animals with lung colonies derived from the less metastatic parent tumor cell line possesses enhancing properties in the BD-IX rat strain only. Removal of immunoglobulin from enhancing serum by affinity column chromatography simultaneously removes the enhancing factor(s), and enhancing activity correlates with the presence of increased levels of Clq-binding immune complexes in the serum. Serum levels of immune complexes are shown to be more elevated in serum from animals bearing lung colonies derived from the most highly metastatic variant. The enhancing moieties are shown to bind to concanavalin A, but not to staphylococcal protein A, and the active fraction elutes from concanavalin A-Sepharose with alpha-methyl-mannoside. Consideration of immunoprecipitation studies on whole and fractionated enhancing sera, along with studies on affinity purified isotype fractions reveals that the activity resides with antibodies of IgG2b subclass.

Natural killer cell activity in fawn-hooded rats.

Fawn-hooded (FH) rats were shown to lack the genetically conditioned defect of natural killer (NK) activity hypothesized to be present by analogy with the Chediak-Higashi syndrome (CHS) in mice and human beings. In 4-h 51Cr release assays, splenic NK cells from FH rats killed YAC-1, RL male l and G1-TC tumor targets without deficiency based upon comparison with cells from BD-IV, BD-IX and NBR inbred rat strains. Progeny of BD X FH F1 rats backcrossed to FH failed to reveal a correlation of reduced NK activity and dilute coat color. From these, and other data presented, it is concluded that despite similarities in coat color dilution and platelet storage pool deficiency. FH rats do not closely resemble CHS mice or human beings in having deficient NK activity and cannot be considered the rat homolog of the CHS.

Conditions controlling long-term proliferation of Brown Norway rat promyelocytic leukemia in vitro: Primary growth stimulation by microenvironment and establishment of an autonomous Brown Norway ‘leukemic stem cell line’

Conditions for in vitro long-term maintenance and proliferation of the Brown Norway (BN) rat myelocytic leukemia cell (BNML) are described. During a primary culture of leukemic rat marrow, a few leukemic cells proliferated and were initially dependent on an adherent cell population but later acquired the capability of independent growth. A wild BN leukemic stem cell line has been maintained in vitro for several months, without noticable phenotypic alterations. The doubling time of the cultured cells was 40 h. The cells were promyelocytes. The cytochemical markers of the original BN leukemia cells were preserved. The cultured cell line transferred leukemia exclusively to BN rats. Wistar and BDIX rats were resistant. The virulence of cultured leukemic cell was measured by shortened survival times after transplantation in animals of a fixed number of leukemic cells. The role of bone marrow microenvironment in the initiation of long-term growth is discussed.

Sensitization of clonogenic malignant cells to hyperthermia by glucose-mediated, tumor-selective pH reduction.

The fraction of clonogenic cells (as assayed by colony formation in semisolid agar medium) in neurogenic TV1A tumor transplants carried subcutaneously by hyperglycemic BDIX rats (serum glucose concentration 50 mmoles/1; average intratumoral pH 6.1) was reduced by a factor of approximately 2.5 X 10(3) after in vivo exposure to 42.2 degrees C for 1 h. The corresponding reduction factor for TV1A tumors in normoglycemic rats was about 20 (serum glucose concentration 6 mmoles/1; average intratumoral pH 6.9). Hyperglycemia without hyperthermic treatment reduced clonogenicity by a factor of about 18 ("glucose-mediated suicide").

Inhibition of natural killer cell activity [formula omitted] by alcohols

The effect of the addition of small quantities of alcohols to cocultures of natural killer effector cells from C57BL 6 mice and BDIX rats with YAC-1 tumor cell targets has been studied. The order of inhibition of NK cell-mediated killing is 1-butanol > 1-propanol > 2-propanol > ethanol > methanol. The inhibition of killing due to the addition of alcohol correlates with decreases of effector-target cell binding. Therefore caution should be exercised in interpreting results of cellular experiments in which these alcohols have been used to solubilize inhibitors.

Responsiveness of fetal rat brain cells to glia maturation factor during neoplastic transformation in cell culture.

The effect of partially purified extracts from adult pig brains containing a glia maturation protein factor (BE) has been investigated on neural cells during carcinogenesis. Pregnant BD IX-rats were given a single transplacental dose of the carcinogen ethylnitrosourea (EtNU) on the 18th day of gestation. The brains of the treated fetuses were transferred to cell culture and underwent neoplastic transformation with a characteristic sequence of phenotypic alterations which could be divided into five different stages. During the first 40 days after explantation (stage I & II) BE induced morphological differentiation of epitheloid neural cells into astrocytes. This occurred in carcinogen treated cells as well as in untreated control cultures. At the same time cells with astrocyte morphology showed accumulation of glial fibrillary acidic protein (GFA) as tested by indirect immunofluorescence with monospecific antibodies against GFA. Thereafter, in the EtNU pre-treated cultures an increased number of cells with astrocyte morphology was seen, and BE further increased the number of cells with long cytoplasmic processes. Control cells were GFA negative, while some few strongly, as well as many weakly, positive cells were seen after treatment with BE (stage III). At the later stages of neoplastic transformation the effect of BE became gradually less, and in tumourigenic cells which occurred after about 200-300 days, only a slight morphological change took place in a few cell lines. No appreciable effect on GFA-content was seen any longer, although some few weakly GFA positive cells could be observed in all permanent cell lines. Fetal rat brain cells therefore seem to become less responsive to this differentiation inducer during neoplastic transformation in cell culture.

The vasculature of experimental brain tumours: Part 1. A sequential light and electron microscope study of angiogenesis

The process of vascularisation was studied in transplanted astrocytomas in BD-IX rats. The development of blood vessels was followed from the earliest signs of angiogenesis throughout tumour growth. On the basis of tumour vasculature, 3 consecutive stages of tumour growth could be distinguished; avascular, early vascular and late vascular. The tumours grew to a diameter of about 1 mm during the avascular stage after which new capillary sprouts began to penetrate the tumours. This resulted in an homogeneous vasculature of small immature capillaries up to about 5 μm in diameter characteristic of the early vascular growth stage. During this stage the tumours reached a diameter of about 4 mm and their vasculature consisted of capillaries similar to those seen in embryological cerebral vascularisation. During the subsequent late vascular stage of growth, continued endothelial proliferation led to an increase in blood vessel diameter up to 40 μm in some cases. The vessels varied in shape and size; this vascular pleomorphism and the abnormal morphological features associated with glioma vasculature were typical of the late vascular stage.

The development of brain tumours produced in rats by the intracerebral injection of neoplastic glial cells: a fine structural study.

Tumours were produced by the intracerebral injection of a clone of glial cells derived from a glioma induced transplacentally by N-ethyl-N-nitrosourea in a BD-IX rat. The injection of 5 X 10(5) cells into the left frontal lobe resulted in a 100% incidence of tumours. To follow the development of the neoplasms, the brains were studied from 1 day to 4 weeks after injection. The tumours maintained their glial characters throughout, but their features changed with time. Ultrastructurally, they were pleomorphic: the proportion of fibrillary astrocytes, undifferentiated and intermediate cell types varied according to tumour size. When smaller (1 and 2 weeks), fibrillary astrocytes predominated, but when larger (3 and 4 weeks), the number of undifferentiated astrocytes considerably increased. A reproducible brain tumour model with a short latency has thus been established and characterized, which may be of use for chemo- and radiotherapeutic studies and for examining the mechanisms of cerebral oedema.

Experimental brain tumors and edema in rats. I. Histology and cytology of tumors.

In this study an experimental intracerebral tumor has been investigated with special consideration of structures, which may be involved in edema production and/or resolution. For this purpose a cloned tumor cell line (RG1 2.2) has been injected stereotactically into the brain of BD-IX rats. The tumor has some characteristics in common with low differentiated oligodendroglioma in men. A honeycomb architecture may be seen in the center of the tumor. It is built up by rounded or elongated cells, which can be impregnated in parts. In the central area, cells exhibit a voluminous digestive apparatus, composed of dictyosomes, vesicles, and some vacuoles with a membranaceous or homogeneous content. Tumor cells in the periphery show large processes and a small digestive apparatus. The sinusoidal tumor vessels are composed of an endothelium with many vesicles but no openings.

Volume increase in L5222 leukemic cells following chemotherapy: Manifestation of leukemic cell damage

The L5222 leukemia of the BDIX rat provides an important model for the study of the effects of chemotherapy on leukemic cell proliferation. In this model system, damage done to leukemic cells by chemotherapeutic agents manifests itself as an increase in cell volume within a few hours following exposure. This effect is seen in vivo following intraperitoneal injection of drug or in vitro if the leukemic cells are removed and exposed to drug in short-term culture. The normal mature peripheral white blood cells of the animal when measured separately do not show this volume increase reflecting cell damage. The method of measuring cell volume as a means of assessing damage to leukemic cells and the lack of such an effect in normal blood cells is a means of more closely monitoring chemotherapeutic drug schedules. The mechanism producing this volume effect in leukemic cells but not in normal mature cells remains to be elucidated.