WF Rats Research Articles

Serial Circulating Immune Complex Levels and Mitogen Responses During Progressive Tumor Growth in WF Rats<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Inbred male WF rats were given im injections of one of two antigenically and histologically distinct syngeneic tumor isografts, adenocarcinoma DMH-W 163 or spontaneous renal cell carcinoma SPK. Serum and peripheral blood lymphocytes were harvested from tumor-bearing and normal age-matched controls before and after isograft challenge at weekly intervals. Serial circulating immune complex (CIC) levels were quantitated by polyethylene glycol (PEG) insolubilization. T-cell mitogen responses to phytohemagglutinin (PHA) and concanavalin A (Con A) were followed serially. Tumor growth was measured at least weekly. PEG-CIC values rose early after tumor injection, increased with tumor growth, and declined in some animals just before death. Mitogen response to PHA was significantly decreased in isografted tumor-bearing rats, particularly at later stages of tumor development, compared to normal uninoculated controls. Responses to Con A were variable, and suppression was not always seen in tumor bearers. In animals that did not have progressive tumor growth after isograft injection, PEG-CIC levels did not change and responses to PHA were not suppressed. Patterns of CIC change and responses to PHA were not affected by differences in tumor histology or growth rates. Thus serial CIC levels measured by the PEG assay correlate with tumor growth and precede nonspecific suppression of T-cell mitogenic response in these animal tumor models.

Induction of Hepatic Tumors by Diethyistilbestrol Alone or in Synergism With <italic>N</italic>-Nitrosobutylurea in Castrated Male WF Rats<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Inbred male WF rats were castrated at 40 days of age and divided into 5 groups. Group I was given no further treatment. Groups III, IV, and V received pellet implants of 5.0 mg diethylstilbestrol (DES) concurrently with castration. At 50-55 days of age, groups II, IV, and V were given drinking water containing 5.0 mg N-nitrosobutylurea (NBU) per day for 30 days (subthreshold dose). At the termination of NBU treatment, group V further received daily sc injections of 2-bromoergocryptine (CB-154; 0.4 mg/100 g body wt) four times a week throughout the experiment. None of castrated rats or rats castrated and treated with NBU alone developed hepatic tumors (HT) and pituitary tumors (PT). Incidences of HT and PT in groups III, IV, and V were 4/9 (44%) and 7/9 (78%), 15/17 (88%) and 12/17 (71%), and 17/20 (85%) and 4/20 (20%), respectively. The treatment of DES alone resulted in the concurrent development of HT and PT in castrated male rats (group III), and further NBU treatment significantly increased the incidence of HT (group IV). CB-154 treatment did not change the incidence of HT, the number of HT per rat, and the liver weight, although it significantly reduced the incidence of PT, the pituitary weight, and the serum prolactin level in castrated male rats given DES and NBU (group V). These results indicate that DES itself had a direct carcinogenic effect on the liver; this effect was not mediated by prolactin, and NBU increased the effect of DES in this process.

Androgen Control of Cytosol Progesterone Receptor Levels in the MT-W9B Transplantable Mammary Tumor in the Rat<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

The effect of androgen on the levels of the cytosol progesterone receptor was examined in the transplantable rat mammary tumor MT-W9B and in the normal uteri of inbred WF rats. Progesterone receptor levels were barely detectable in tumors grown in male WF rats but were increased after castration or administration of 17 beta-estradiol. Both effects were blocked by testosterone. In tumors grown in intact female rats, both testosterone and dihydrotestosterone decreased progesterone receptor levels in a dose-dependent manner, and testosterone completely blocked the estradiol-induced increase in progesterone receptor levels in tumors from ovariectomized rats. The inhibitory effect of testosterone in female rats was blocked by the antiandrogen flutamide, suggesting an androgen receptor-dependent mechanism. Neither dihydrotestosterone nor testosterone had any effect on basal levels of progesterone receptor in tumors from ovariectomized rats. In uterus, up to 5 mg dihydrotestosterone/kg did not affect progesterone receptor levels, and a dose of 5 mg/kg was also uterotropic. This fact plus the finding that testosterone only partially blocked the estradiol-induced increase in uterine progesterone receptor levels suggested stimulation of different cell types by testosterone and estradiol. This did not appear to be the case in the tumor, however. Androgen is suggested to act as a negative modulator of progesterone receptor levels, which might have clinical relevance in terms of hormone therapy of breast cancer.

Effect of selenium on the induction of breast fibroadenomas by adenovirus type 9 and 1,2-dimethylhydrazine-induced bowel carcinogenesis in rats.

Selenium in its organic and inorganic forms has been shown to inhibit the development of chemically induced, spontaneous and transplanted tumors. The present investigation was performed to study the effect of selenium (4 micrograms per ml of drinking water) on tumorigenesis of adenovirus-type-9-induced breast fibroadenomas and on 1,2-dimethylhydrazine-induced bowel carcinogenesis in WF rats. It was found that identical treatment with Se under identical conditions and with no obvious toxic effects on the rats (1) resulted in inhibition of DMH-induced large-bowel carcinogenesis; (2) facilitated induction of small-bowel cancer by the same carcinogen in the same animals, and (3) greatly facilitated induction of breast fibroadenoma by adenovirus type 9 in the same strain of rats. The effect of Se treatment on DMH-induced large-bowel carcinogenesis confirms previous findings and proves that the opposite effect on fibroadenoma development is not due to differences in e.g. effective dose, animal strains or condition of the animals. It is not yet clear through which mechanisms Se exerts these effects.

Thy-1 Antigen on Normal and Neoplastic Rat Mammary Tissues: Changes in Location and Amount of Antigen During Differentiation of Cultured Stem Cells<xref ref-type="fn" rid="fn2">2</xref>

With the use of immunofluorescent and immunocytochemical techniques on histologic sections of mammary glands from inbred WF rats, the thymocyte differentiation antigen (Thy-1) was identified partially on and immediately adjacent to the myoepithelial cells of ducts and alveoli. This antigen was absent from epithelial cells lining such structures. Some fibroblastic cells external to these structures also bore Thy-1. During glandular development the intensity of fluorescence due to the binding of Thy-1 antibodies increased as the myoepithelial cells matured. Similarly, mammary adenocarcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) and N-methyl-N-nitrosourea contained elongated, presumptive myoepithelial cells that demonstrated varying degrees of Thy-1 immunofluorescence. This phenomenon was qualitatively mimicked by cultured cell lines from normal and DMBA-induced tumors. Elongated myoepithelial-like and fibroblast-like cells possessed Thy-1, whereas the cuboidal epithelial cell lines failed to express this antigen on the cell surface. Measurement of the relative number of Thy-1 molecules per cell by antiserum absorption techniques suggested that a neoplastic stem cell line, rat mammary (Rama) 25, contained about 3 X 10(5) Thy-1 molecules per cell in a form not directly accessible at the cell surface to anti-Thy-1 antibodies; the number of cryptic Thy-1 molecules was reduced when this cell line differentiated to alveolar-like cells in culture. However, when this cell line differentiated to myoepithelial-like cells, approximately 5 X 10(6) molecules per cell were exposed to anti-Thy-1 antibodies with a concomitant reduction of the cryptic pool. Morphologic maturation of elongated, myoepithelial-like cell line variants was also accompanied by increased surface Thy-1 fluorescence. Thus some of the myoepithelial cells in normal glands and tumors may arise by differentiation of epithelial stem cells and a spectrum of maturation states of the myoepithelial cell may exist as monitored by cellular morphology and surface Thy-1 expression.

Enzymatic changes in rat erythrocytes with increasing cell and donor age: Loss of superoxide dismutase activity associated with increases in catalytically defective forms

Erythrocytes from young (6–8 month old) and old (28–31 month old) WF rats were separated into four age fractions by centrifugation on a discontinuous, isotonic, arabinogalactan density gradient. Specific activities of super-oxide dismutase (SOD) with respect to activity per unit hemoglobin (Hb) and activity per cell were determined for each cell age group. SOD activity was found to diminish with respect to erythrocyte age. More significantly, it was found that young erythrocytes of old animals already contain considerably reduced SOD activity as compared to cells of young animals. The level of SOD catalytic activity per unit enzyme antigen was also found to decrease with both increasing cell and animal age. Young cells of old animals contain significant amounts of catalytically altered molecules.

Hormone, Serum, and Methylxanthine Interactions in the Regulation of DNA Synthesis in Organ-Cultured Rat Mammary Tumors<xref ref-type="fn" rid="FN2">2</xref>

Previous studies have shown that the growth of MTW9, a hormone-dependent rat mammary tumor, is stimulated by the presence of a mammosomatotropic tumor coimplant (MtTW10) but is inhibited by the administration of dibutyryl cyclic AMP (DBcAMP). This study examines the direct effect of serum taken from MtTW10-bearing WF rats (MtT serum) and of substances that increase cellular levels of cyclic AMP (cAMP) on DNA synthesis in organ-cultured MTW9. Explant DNA synthesis was significantly greater in the presence of MtT serum than in either normal female or male rat serum. Preparations of hormones known to be present in MtT serum failed to simulate the activity of MtT serum. Whereas 1 mM cAMP 1 mM did not alter DNA synthesis in MTW9 explants, inhibitors of cyclic nucleotide phosphodiesterases substantially suppressed the rate of [3H]thymidine incorporation into DNA. However, addition of MtT serum to cultures completely prevented the effects of these substances. This activity of MtT serum could not be duplicated by any of the following additions to the culture medium: normal female or male rat serum, National Institutes of Health (NIH) rat prolactin (2.5 micrograms/ml), NIH rat growth hormone (2.5 micrograms/ml), insulin (5 micrograms/ml), 17 beta-estradiol (5 micrograms/ml), or a hormone combination simulating the known endocrine constituents of MtT serum. The results suggested that the serum of the MtT-bearing animal contained an activity, unidentified at present, that could antagonize the growth-inhibitory action of the cAMP system and that may participate in the regulation of mammary tumor growth.

Synergism of Diethylstilbestrol and Other Carcinogens in Concurrent Development of Hepatic, Mammary, and Pituitary Tumors in Castrated Male Rats<xref ref-type="fn" rid="FN2">2</xref>

Castrated male WF rats, given implants of pellets containing 5.0 mg diethylstilbestrol (DES), were given N-butyl-N-nitrosourea (NBU) in small amounts, which alone produced no mammary tumors in intact female rats. Treatment resulted in the high yield of hepatic tumors (HT), mammary tumors (MT), and pituitary tumors (PT) concurrently in each rat. If animals were further treated with prolactin, the development of HT and MT was accelerated, whereas that of PT was suppressed. None of the intact or castrated rats receiving NBU and/or prolactin developed tumors in any tissues if DES treatment was omitted. Exposure of male rats, preconditioned similarly to NBU treatment, to 200 rads of 14.1-MeV fast-neutron radiation also elicited HT, MT, and PT with an efficiency comparable to that of NBU-treated rats. These findings indicate that DES played an essential role in the whole carcinogenic process in each tissue and that castrated male rats, if conditioned properly with estrogens, are useful for the study of the carcinogenesis mechanism in these tissues.

Effect of zinc deficiency on the weight, cellularity and zinc concentration of different skeletal muscles in the post-weanling rat.

Zinc-deficient (ZD), weight-restricted (WR), pair-fed (PF) andad lib.-fed (AL) Sprague-Dawley male rats were killed after feeding the respective Zn-deficient and Zn-supplemented diets from 3 to 8 weeks of age. Animals killed at the start of the experiment served as a baseline control (BC).Four different skeletal muscles – biceps brachii, soleus, plantaris and extensor digitorum longus (EDL) – were studied for changes in weight, the number and diameter of muscle fibres and Zn concentration.The soleus muscle had the highest concentration of Zn. It was the only muscle to reduce its Zn concentration due to Zn deficiency.There was a loss of muscle fibres during normal growth (groups BCv.AL) in the soleus muscle (P< 0.05). The estimated length of muscle and the diameter of the muscle fibres in all four muscles increased significantly (P< 0.001). Therefore postweanling growth appears to occur as a result of longitudinal and transverse increases in the dimensions of these muscles.The reduction in muscle fibre number in ZD rats compared to BC animals may occur within the range of expected fibre loss during normal growth. Fibre loss in ZD rats may be more affected by feeding-pattern-dependent metabolic changes than by a deficiency of Zn per se (groups ZDv.WR). Soleus fibre loss in ZD rats may be related to the high Zn concentration in this muscle.The effect of Zn deficiency per se on muscle fibre diameter may be inaccurately interpreted by comparing the ZD animals with their PF and AL controls. There was no significant difference in fibre diameter in any of the four muscles when ZD and WF rats were compared.

Technique for Preclinical Evaluation of Continuous Infusion Chemotherapy With the Use of WF Rat Acute Myelogenous<xref ref-type="fn" rid="FN1">2</xref>

A technique was developed for continuous iv infusion chemotherapy in an inbred rat model of acute myelogenous leukemia. Polyethylene tubing was inserted surgically into the internal jugular vein of adult WF rats, burrowed sc to the base of the tail, and connected to an infusion pump. A flexible spring was sutured at the base of the tail and fastened to the cage wall; it protected the infusion catheter and allowed movement of the rat within the cage. This technique was used to compare bolus with continuous infusion therapy with adriamycin, cytosine arabinoside, and neocarzinostatin. Only small differences were noted in host toxicity and in antitumor effect against tumor grown as a subcutaneous myeloblastoma. Nearly three times more neocarzinostatin was required by continuous infusion for an effect equivalent to that of bolus injection. In contrast, continuous infusion of methotrexate with concurrent thymidine infusion prevented toxicity, enhanced the antitumor effect, and prolonged survival. This infusion system should facilitate rapid preclinical evaluation of drugs considered for constant iv infusion therapy.

Natural Cytotoxicity of Mouse, Rat, and Human Lymphocytes Against Heterologous Target Cells

Natural cell-mediated cytotoxicity by a particular subpopulation of lymphocytes [designated natural killer (NK) cells] in NIH Swiss nude and CBA/N mice, WF rats, and humans was demonstrated against tumor cells in 4-hour 51Cr release assays. In most studies, only reactivity against target cells of the homologous species was examined. In the present studies, mouse NK activity also was found against a rat lymphoma tissue culture cell line and against human tissue culture lines. Rat NK cells reacted not only against syngeneic tumor cells but also against heterologous tumor cell lines. In contrast to the heterologous NK activity in rodents, no significant NK activity of human peripheral blood lymphocytes against heterologous targets was found in the present studies. In mice and rats the effector cells that mediated the cytotoxicity against heterologous target cells were indistinguishable from NK cells, the effector cells being nonadherent and nonphagocytic. In addition, the mouse effector cells for heterologous activity as well as mouse NK cells were sensitive to repeated treatment with anti-Thy 1.2 serum plus complement. The specificities of these reactions were indicated by a cold target inhibition assay. The results indicated a sharing of specificities between homologous and heterologous tumor cells recognized by mouse and rat NK cells. In contrast, only the human cell lines were able to appreciably inhibit the cytotoxicity of human peripheral blood lymphocytes.

Lymphoma Model: Correlation of In Vitro Findings With In Vivo Observations

Previous studies concerning the tumor-host relationship in the (C58NT)D rat tumor system indicated that some T-lymphocyte responses against tumor antigens were only transiently detectable after tumor inoculation; however, more sensitive procedures for the detection of proliferative responses in the mixed lymphocyte-tumor interaction (MLTI ) have been developed. Upon reexamination of the kinetics of this response, positive responses were detected as long as 650 days after inoculation. Cytotoxic effector cells could be generated in MLTI with lymphocytes from immune WF rats as long as 1 year after inoculation, and evidence was provided which indicated that the persistence of the responses observed in vitro correlated well with the resistance of immune rats to rechallenge with (C58NT)D tumor cells in vivo.

Effect of Unblocking Therapy and Levamisole on Primary Gastrointestinal Tumors in Rats: Immunologic and Histologic Correlation2

The effect of multimodal immunotherapy was studied in WF rats bearing primary gastrointestinal (GI) tumors induced by 1,2-dimethylhydrazine dihydrochloride. The alterations induced in antitumor immune responses of the treated rats were studied in vitro and were correlated with tumor status in vivo. Multimodal immunotherapy consisted of unblocking serum, unblocked lymphoid cells, and levamisole. Such immunologic intervention resulted in significant inhibition of tumor growth, inhibition of metastases, and prolonged survival of the host. Serum blocking activity could be completely counteracted in 6 rats, all of which showed complete tumor regression. Of 20 rats, 8 showed inadequate counteraction of serum blocking activity and transient appearance of cytotoxic antibodies. All 8 rats showed marked tumor inhibition and prolonged survival. Six remaining rats succumbed from either GI or extra-GI tumors, although they survived significantly longer than untreated rats; these 6 rats had only transient counteraction of their serum blocking activity. All 20 tumors in 14 rats of the therapy group showed histologic evidence of tumor rejection. Our studies suggested that a complete counteraction of blocking activity in conjunction with methods capable of improving the specific and nonspecific immune competence of the host may be important to achieve optimal antitumor effects.

Effect of Embryonic Tissue Immunization on Chemically Induced Gastrointestinal Tumors in Rats. I. Can Embryonic Antigens Act as Rejection Antigens?2

Inbred WF rats were inoculated with crude suspensions prepared from liver and gut tissue of 12- to 15-day fetuses of the same strain. Rats previously unsensitized to syngeneic embryonic tissue were inoculated with fetal material sc three times during exposure to 1, 2-dimethylhydrazine dihydrochloride (DMH), a gastrointestinal (GI) carcinogen in rodents. Properly timed immunization inhibited the development, growth, and metastasis of primary GI tumors. This effects was observed in both sexes; however, it was more prounced in male rats. Nine WF rats with DMH-induced carcinoma of the GI tract were inoculated sc with syngeneic fetal tissue. Of 9 rats, 7 rejected the embryonal tissue implants, which thus demonstrated the presence of a concomitant immune response to embryonic antigen(s). Two rats in which fetal tissue grew out to palpable nodules had multiple GI tumors with metastasis and extra-GI tumors, i.e., a massive tumor load. Ten other rats with DMH-induced GI tumors were treated with unblocking serum. The unblocking serum was inoculated to counteract serum-blocking factors in vivo. These rats were inoculated intradermally with syngeneic fetal tissue. In all 10 rats, inflammation and necrosis were noted at the inoculation site after 24-72 hours, which thus demonstrated a delayed hypersensitivity reaction to embryonic antigens. Our experiments suggest that embryonic antigens common to fetal and tumor cells can induce immunity in an autochthonous host and can act as rejection antigens.

Restriction to the Cell Nucleus of Virus-Induced Host RNA's in a Rat Kidney Tumor Induced by Polyoma Virus 2

RNA transcripts of repetitive DNA of WF rat kidney, which were derepressed during transformation by polyoma virus, were not transported to the cytoplasm in the tumor cells. Transcripts of repetitive DNA found in the normal cell nucleus were all transported to the cytoplasm in the tumor cells, including those restricted to the nuclei in the normal cells.

Separation of rat leukocytes by countercurrent distribution in aqueous two-phase systems: II. Subpopulations which mediate selective and nonselective lysis of normal and colon carcinoma target cells in vitro

Spleen cells from WF rats immunized to allogeneic lymphoid cells or to syngeneic colon carcinomas and from unimmunized controls were separated by countercurrent distribution in aqueous two-phase systems. The cells were assayed for cytotoxicity to allogeneic fibroblasts or syngeneic colon carcinoma cells and to syngeneic fibroblasts in a 24-hr 51Cr-release assay. Cells from immunized rats which selectively lysed the specific target cells were repeatedly found in one area of the distribution separate from the majority of cells, which nonselectively lysed syngeneic fibroblasts as well. A similar subpopulation which nonselectively lysed all target cells assayed was recovered from the spleens of unimmunized rats. The cells were also assayed for the ability to lyse antibody-coated thymocytes in a 4-hr 51Cr-release assay. The peak of K cells was found to overlap partially that of cells with nonselective cytotoxicity.

Measurement of mammary tumor blood flow in unanesthetized rats.

Blood flow to mammary tumor MT-W9B in minimally disturbed, unanesthetized inbred WF rats was measured with radioactively labeled 25-mu (diameter) microspheres. The distribution of the blood flow estimates was not described by a normal probability density function but could be normalized with a natural log transformation of the data. The relative blood flow to the tumor tissue was always greater than that to the surrounding normal tissue and was dependent on the tumor size, transplantation site (axillary vs. inguinal), and physiologic state of the animal. The growth of this tumor did not alter the blood flow to the adjacent normal tissues. However, the cardiac output and coronary blood flow increased with increasing tumor size. Two blood flow estimates could be performed on the same animal within a time-span of 15 minutes.

Trend in blood pressure among employees: Gurunanjappa S. Bale, Metropolitan Life Insurance Company, New York, New York; Frederic Seltzer; John B. Cromie

Study of blood group A- and B-active glycosphingolipid content of the epithelium of the large intestine of 16 strains of inbred rats led to the discovery of two related strains, SHR and WKY, devoid of A-active glycolipids, whereas all strains expressed B-active glycolipids. This finding evidenced a new A/non-A genetic polymorphism in the rat. Blood group A-active glycolipids were isolated from the large intestine of F344 rats and purified by affinity chromatography on immobilized Helix pomatia lectin. Three glycolipid fractions were separated by preparative thin-layer chromatography and characterized by electron-impact mass spectrometry of their permethylated and permethylated-LiAlH4-reduced derivatives. They were identified as a tetraglycosylceramide (A-4), a hexaglycosylceramide (A-6), and a difucosylated heptaglycosylceramide (A-7) with small amounts of monofucosylated octaglycosylceramide (A-8). Methylation analysis and fragmentation indicated that A6 and A-8 had a lacto- and A-7 a neolactotetraosylceramide core, respectively, identical to the core structures of B-6 and B-7 previously characterized in the large intestine of WF rats (Ångström et al. (1987) Biochim. Biophys. Acta 926, 79–86). Upon methylation analysis, B-6 and B-7 purified from SHR (A-deficient) and F344 (A-expressing) were found identical to those of WF rats. This result indicated that precursor substrates for the synthesis of A-active glycolipids were available in SHR rats and thus the genetic deficiency of A-active glycolipid expression probably originated in a defect of the termination of the blood group A determinant by the α-3-N-acetylgalactosaminyltransferase.

Transplantable Mammary Tumors in Wistar/Furth Rats: Development, Antigenicity, and Effect of Hormone Manipulations2

Transplantable tumor lines were developed from 7,12-dimethylbenz[a]anthracene-induced mammary tumors in inbred WF rats. Although the primary tumors regressed following oophorectomy, the growth of late generations of the transplantable lines was not affected by castration or by treatment with estrogens, androgens, and progesterone. This result coincided with a change in the morphology of the tumors from well-differentiated to poorly differentiated anaplastic tumors. The transplantable mammary tumors were antigenic in vitro as evidenced by stimulation of syngeneic lymphocytes in mixed lymphocyte-tumor cell cultures. However, prior sensitization by excision of a first tumor graft failed to protect the animals against a second challenge with cells from the same tumor line.

Secondary cell-mediated cytotoxic response to challenge of rats with syngeneic Gross virus-induced lymphoma.

Secondary cell-mediated cytotoxicity generated in vivo against a syngeneic Gross virus-induced lymphoma [(C58NT)D] in WF rats was detected by the 4-hour 51Cr release assay. At 30 days or more following primary tumor cell inoculation, after the tumors had regressed, lymphoid cells had little or no detectable direct cytotoxic reactivity. At rechallenge with tumor cells, high levels of cytotoxicity were detected in the peritoneal exudate, peripheral blood, mesenteric lymph node, and spleen cells. The secondary cellular immune response after challenge developed earlier, reached higher levels, and lasted longer than the primary immune response. The secondary cytotoxic reactivity was shown to be immunologically specific by the use of various tumor cells both as target and inhibitor cells. Treatment of immune spleen cells with specific antiserum to rat T-cells and complement abolished their cytotoxic reactivity, whereas removal of complement receptor-bearing cells or phagocytic cells did not reduct the cytotoxicity. These data demonstrated that specific-memory T-cells persisted for long periods in the lymphoid organs of immune rats and could rapidly become cytotoxic from rechallenge with the tumor.