Rat Mammary Adenocarcinoma Research Articles

Purification and comparison of several catalytic parameters of the γ-glutamyltranspeptidase of rat mammary adenocarcinoma (13762) and of normal rat mammary gland

A method for the purification of a membrane-bound glycoprotein, γ-glutamyltranspeptidase ((γ-glutamyl)-peptide:amino-acid γ-glutamyltransferase, EC 2.3.2.2), from a transplantable rat mammary tumor (13762 MT) is described. The properties of the tumor enzyme were compared with those of γ-glutamyltranspeptidase similarly isolated from mammary tissue of non-pregnant multiparous rats. Evidence has been presented elsewhere that the mammary and tumor enzymes exist as groups of species differing in isoelectric point and that the tumor enzyme contains more of those species with lower isoelectric points. In this study the normal and tumor enzyme preparations are found to be identical or very similar in regards to the effect of papain on molecular size, the ratios of the enzymatic activities as measured with various amino acids, the K m for · γ- glutamyl-p- nitroanilide , and the K rmi for inhibition by glutathione. Neuraminidase treatment had no effect on these catalytic properties. The properties observed were generally similar to those previously reported for highly purified rat kidney preparations.

The binding of tamoxifen to oestrogen receptor proteins under equilibrium and non-equilibrium conditions

Data have been presented from sucrose density gradient analysis, protamine sulphate precipitation and specificity studies to show that oestradiol-17β and tamoxifen share a common binding protein. In addition, the binding of [ 3H ]-tamoxifen to cytosol preparations from human and rat mammary adenocarcinomas and rat uteri was measured under equilibrium conditions using Scatchard analysis. The relative affinity values obtained were, however, greater than those calculated from competitive binding studies. In addition the competition between tamoxifen and oestradiol- 17β for oestrogen binding sites at 4°C decreased with increasing incubation time. Based on competitive binding experiments, the principal metabolite of tamoxifen, metabolite B, associated with oestrogen receptor proteins with a higher affinity than tamoxifen. Kinetic studies indicate that these variations probably result from the relative dissociation rates of metabolite B and tamoxifen from the oestrogen receptor, tamoxifen dissociating much more rapidly than metabolite B. It is also proposed that kinetic studies may have important implications with respect to the mechanism of action of tamoxifen.

Glucose-6-Phosphate Dehydrogenase from Lactating Rat Mammary Gland and R3230AC Adenocarcinoma

A number of properties of glucose-6-phosphate dehydrogenase from lactating rat mammary gland and R3230AC rat mammary adenocarcinoma are compared. The main electrophoretic forms of the enzyme from these sources are indistinguishable with respect to charge and molecular weight whereas the minor forms show differences in these properties. The subunit molecular weight and steroid inhibition of the enzymes from the lactating gland and tumor are not significantly different. These results are contrasted with similar studies in mice.

Differences in Kinetic Parameters of Lysis by Syngeneic and Allogeneic T Lymphocytes Immune to the Same Tumor

Abstract Kinetic parameters of cell-mediated cytotoxicity (CMC) to the 13762A rat mammary adenocarcinoma were studied by using immune syngeneic (Fischer) and allogeneic (Wistar Furth) spleen cells. Treating the spleen cells with rabbit anti-T cell serum and complement eliminated cytotoxicity. Both types of cytolytic cells bound to tumor cells in Ca2+-free medium but required Ca2+ to lyse them. Cytochalasin A did not interfere with the lysis of target cells already bound to cytolytic cells but it did prevent attachment of cytolytic cells to new targets. Cytolysis of the tumor occurred in three sequential steps: 1) binding of the lymphocytes to the target; 2) production of a lytic lesion in the target by the lymphocyte; and 3) lysis of the target. Allogeneic and in vitro augmented syngeneic lymphocytes required about 30 min for maximum binding to targets compared to 4 hr for syngeneic cells sensitized in vivo. Induction of the lytic lesion required 1 hr for the allogeneic cells and up to 8 hr for both types of sensitized syngeneic cells. Target cell death was slightly faster after incubation with allogeneic (8 hr) than with syngeneic (12 hr) cells. Pretreatment of the tumor cells with mitomycin C or actinomycin D did not enhance target lysis. Despite the many similarities between allogeneic and syngeneic effector cells, the increased time required for induction of the lytic lesion by syngeneic effector cells might indicate employment of a different lytic mechanism or a lower lytic efficiency compared to allogeneic cells.

Expression of RNA of an Endogenous Replication-Defective Retrovirus in Rat Mammary Adenocarcinomas Induced by 7,12-Dimethylbenz[a]anthracene2

An investigation into the possible relationship between chemical carcinogen induction of rat mammary tumors and the expression of an endogenous retroviral genome was initiated. Mammary tumors were induced in female SD rats with 7,12-dimethylbenz[a]anthracene (DMBA). Tumors, identified histologically as mammary adenocarcinomas, were analyzed for RNA of a replication-defective endogenous retrovirus or RNA of a helper-independent endogenous type C virus. Expression of RNA of the replication-defective virus was detected in mammary tumors weighing 0.2--2.0 g. Larger tumors, for which histologic examination revealed proportionally more fibroblastic tissue than epithelial cells, did not contain comparable concentrations of this viral RNA. RNA homologous to a helper-independent rat type C retrovirus was not detected in tumors of any size. A cell line was established from a primary DMBA-induced mammary adenocarcinoma and appeared similar to the small mammary tumors with respect to endogenous type C viral RNA expression. We discuss possible implications of the expression of endogenous replication-defective viruses for use as markers for the effects of chemical carcinogens.

Target cell - substratum interaction. I. Effect of primed lymphocytes on a rat mammary adenocarcinoma tumor cell line.

The differential effects of normal and immunologically primed lymphocytes on the adherence of trypsinized rat mammary adenocarcinoma tumor cells (DMBA"8) to their substratum are described, utilizing both allogeneically and syngeneically primed lymphoid cells as effectors. This observation may illustrate an important immunological phenomenon, and may result in a simplified assay procedure for the detection of cell-mediated immunity.

Poly(U) binding to ascites cells of the 13762A rat mammary adenocarcinoma: Evidence for a protein receptor on the cell surface

Summary Ascites cells of the 13762 rat mammary adenocarcinoma bind poly(U) in a reaction that is complete within 5 min at 0°C. Poly(U) binding is saturable; the capacity of these cells is 5×10 7 UMP residues/cell (approx. 2×10 5 chains/cell). Most [ 3 H]poly(U) bound in the rapid reaction can be recovered in an undergraded state. However, it is rapidly degraded by low concentrations of exogenous pancreatic ribonuclease. The magnitude of binding is independent of temperature and ionic conditions, and is unaffected by metabolic inhibitors or concanavalin A (ConA). Radioactivity presented as [ 3 H]poly(U) tends to co-fractionate with 5′-nucleotidase after homogenization of cells in the media of low ionic strength, but is efficiently released from cells exposed to protein denaturants that effectively fix cellular RNA in situ. Cells pretreated with proteolytic enzymes have sharply reduced capacities to bind poly(U). Autoradiography of cells bearing [ 3 H]poly(U) demonstrates a uniform distribution of radioactivity through the cell population and is consistent with binding to the plasma membrane. These and other results imply that binding of poly(U) to 13762 ascites cells is mediated by protein receptors on the cell surface.

Ca2+-stimulated ribonucleases from rat mammary gland and R3230AC mammary adenocarcinoma nuclei.

Ca2+-stimulated ribonucleases from rat mammary gland and R3230AC mammary adenocarcinoma nuclei.

Enhancement of R3230AC Rat Mammary Tumor Growth and Cellular Differentiation by Dibutyryl Cyclic Adenosine Monophosphate23

Daily sc injections of 8 mg N6, O2'-dibutyryl 3',5'-cyclic AMP (DBcAMP) beginning 1 day after tumor implantation significantly increased the growth rate of R32230AC rat mammary adenocarcinomas, which nearly doubled in in situ volume by day 40 compared to similarly implanted tumors in saline-injected controls. Weights of excised tumors, intact, drained, and dried all increased approximately 80%, which suggested that the increase in tumor size was not due to accumulation of secreted fluid or tissue water. Injections of 17beta-estradiol valerate (0.1 mg/wk) from day 1 or of DBcAMP from day 22 resulted in insignificant changes in growth--28% and 35% increases in tumor volume and a 5% decrease and an 18% increase, respectively, in drained wet weight. Electron microscopic examination revealed that estrogen and DBcAMP caused differentiation of the tumor cells into two different states: Estrogen-treated tumors resembled lactating mammary glands; they contained large lipid droplets, organized rough endoplasmic reticulum, and vesicles containing electron dense granules resembling protein. DBcAMP-treated tumor cells were marked by a proliferation of the Golgi complex and numerous vesicles containing fine granular material.

Tumour angiogenesis factor (TAF) in human and animal tumours

Extracts were made from Walker 256 carcinoma, spontaneous rat mammary adenocarcinoma, Wilms' tumour, human neuroblastoma and human haemangioma. Chromatography of the extracts on Sephadex G-100 yielded four fractions, A, B, C and D. Injection of fractions B and C resulted in the growth of new capillaries in the subcutaneous fascia or rats. Controls, e.g. similar extracts of rat liver or human kidney, did not induce neovascularisation. The endothelium of newly-formed blood vessels contained many mitotic figures. A limitation of this method is that it is qualitative only. In order to develop a quantitative in vitro assay for a tumour angiogenesis factor (TAF), short-term primary cultures were initiated from adult rat brain white matter, as cells from such cultures were shown to be vascular in origin. Addition of fractions containing TAF (B and C) which were active in vivo failed to stimulate thymidine uptake by the cells. The possible reasons for this failure and the therapeutic potential of TAF in cancer control are discussed.

Suitability of Rat Mammary Adenocarcinoma 13762 as a Model for BCG Immunotherapy2

We determined the optimal conditions for conducting experiments with the solid and ascites sublines of the 13762 rat mammary adenocarcinoma and examined the response of the tumor growth rate to BCG administered in admixture with tumor cells or separately at a remote site. Versene dissociation of the 13762 solid tumor produced better growth rates than did pronase-DNase, but the former decreased cell viability and yields. A dose of 10(6) or 10(5) tumor cells produced 100% growth by the sc and iv routes. Both sublines grew slower but produced metastases slightly sooner in the intradermal than in the sc site. The frequency of axillary lymph node metastases from the sc site increased as a function of the duration of the time interval between tumor implantation and surgical excision. Both solid and ascites tumors were weakly immunogenic. Administration of BCG in a split adjuvant protocol did not improve tumor immunity. Admixture of tumor cells with BCG suppressed tumor growth but when given at a remote site, BCG was ineffective. We concluded that the 13762 rat mammary adenocarcinoma is a useful system for BCG immunotherapy.

Anti-tumour efficacy of calusterone against DMBA-induced rat mammary adenocarcinoma in vivo and in organ culture.

The effect of calusterone (7beta,17alpha-dimethyltestosterone) on rat mammary DMBA-induced adenocarcinoma was studied both in vivo and in organ culture. In vivo all 8 tumours with a diameter of less than 30 mm regressed following calusterone injection (10 mg/day for 2-3 weeks). In organ culture calusterone (20 mug/ml medium) inhibited the synthesis of DNA and RNA in all 7 cases examined. Testosterone also inhibited the synthesis of DNA and RNA in organ culture in 12 out of 14 and 10 out of 14 tumours respectively. Oestradiol-17beta on the other hand had no effect on DNA and RNA synthesis in organ culture although 70% of the tumours examined were ovarian dependent, i.e. regressed following castration. This could be explained by the direct effect of calusterone on rat adenocarcinoma compared with the indirect effect of oestradiol-17beta which probably exerts its action by activating the secretion of prolactin which acts on the tumour.

Cytoplasmic actomyosin fibrils in tissue culture cells: direct proof of contractility by visualization of ATP-induced contraction in fibrils isolated by laser micro-beam dissection.

A special cell line derived from a rat mammary adenocarcinoma (RMCD cells) displays a distinct pattern of actomyosin fibrils (AM fibrils) visible with phase contrast, Nomarski interference and polarized light optics. It was shown that the cytoplasmic AM fibrils are arranged as bundles of highly parallel F-actin filaments. The chimical nature of the filaments was identified by incubation with heavy meromyosin from rabbit skeletal muscle. These cytoplasmic actomyosin fibrils actively contract under isotonic conditions. This was shown by contraction experiments under polarized light optics, by cinematographic analysis and by direct proof of the contractility of AM fibrils isolated by laser micro-dissection. Thus, cytoplasmic AM fibrils can be assumed to represent structures essential for motive force generation in contraction processes in non-muscle cells.

Effects of Estradiol and Prolactin on Growth of Rat Mammary Adenocarcinoma Cells in Monolayer Cultures

The effects of estradiol and ovine prolactin on the growth of a rat mammary adenocarcinoma cell line were investigated. The action of estradiol was dose-dependent. At less than or equal to 1 mug/ml it did not influence growth, at 5-10 mug/ml it was inhibitory, and at 20 mug/ml it was toxic. Prolactin alone had little effect on growth. However, the inhibitory effect of estradiol could be counteracted by a high concentration of prolactin. It appears, therefore, that the growth of cultured rat mammary adenocarcinoma cells can be regulated by the ratio of prolactin to estradiol: When the ratio is high growth is favored; when it is low, growth is inhibited.

Potent inhibitory activity of a new antiestrogen, RU 16 117, on the development and growth of DMBA-induced rat mammary adenocarcinoma.

When initiated the same day as dimethylbenzanthracene (DMBA) administration, daily treatment with 8 or 24 mug of the new antiestrogen RU 16117 (11alpha-methoxy ethinyl estradiol) completely prevented the appearance of mammary tumors in all animals up to the last time interval studied (130 days after DMBA administration). At daily doses of 0.5 and 2.0 mug of RU 16117, the tumor incidence was reduced to 78.6% and 40.0%, respectively. The levels of receptors for estradiol, progesterone, and prolactin in tumor tissue were reduced after treatment with 2.0 mug RU 16117 while the binding of growth hormone and insulin was not affected. While plasma LH levels were decreased after treatment with 8 or 24 mug RU 16117, plasma prolactin levels were slightly increased in animals receiving the highest dose of the antiestrogen. When RU 16117 was given at the daily dose of 24 mug for a period of 4 weeks, RU 16117 led to 65% reduction of the number of already established DMBA-induced mammary tumors. Not only the tumor number but also the tumor size was reduced by RU 16117 in a manner similar to that following ovariectomy. That the inhibitory effect of RU 16117 was not due to its low estrogenic activity is indicated by the absence of inhibitory effect of similar treatment with a range of doses (0.1-12.5 mug per day) of estradiol-17beta which cover the low estrogenic activity of the doses of RU 16117 used. Decreased levels of receptors for estradiol-17beta, progesterone, and prolactin were found in the tumors remaining after ovariectomy while treatment with the dose (24 mug) of RU 16117, efficient to inhibit tumor growth, has a similar inhibitory effect on the levels of estradiol-17beta and prolactin receptors. The present data indicate that the potent inhibitory effect of RU 16117 on the development and growth of DMBA-induced mammary tumors results from actions at both the hypothalamic-pituitary and tumor levels. The action at the peripheral level would be possibly secondary to a reduced sensitivity of the tissue to circulating hormones through lowering of hormone receptor concentrations.

Immune Response to a Syngeneic Mammary Adenocarcinoma

Abstract The rat mammary adenocarcinoma 13762A is weakly immunogenic in syngeneic hosts. Transplantation immunity is not developed after injection of irradiated tumor cells. Two cell types were isolated from the tumor: one grew slowly forming solid implants; the second grew rapidly in ascites form, even in allogeneic hosts. Spleen cells cytotoxic for a tissue culture derivative of the tumor were produced in animals after injection of either cell type indicating a common antigen. The kinetics of tumor growth and production of cytotoxic spleen cells were compared in syngeneic and allogeneic animals. In vivo rejection of the tumor in allogeneic hosts did not correlate with the in vitro assay of spleen lymphocyte cytotoxicity. Furthermore, the cytotoxic response of spleen cells from syngeneic tumor-bearing hosts parallels that found in allogeneic hosts.

Glucose formation by the R3230AC rat mammary adenocarcinoma

1. 1. The presence of d-fructose-1,6-diphosphate 1-phosphohydrolase (E.C.3.1.3.11) activity has been established in the mammary glands of late pregnant Fisher 344 rats and in the R3230AC rat mammary adenocarcinoma. 2. 2. Homogenates of the R3230AC tumour are capable of synthesizing glucose from fructose-1,6-diphosphate a and other gluconeogenic precursors. The rate of glucose synthesis from fructose-1,6-diphosphate is decreased by high substrate concentrations, estrogen administration to the tumour bearing rats and by the addition of 5′AMP or ATP to the incubation mixture. 3. 3. Starvation increases glucose formation from fructose-1,6-diphosphate in the estrogen-treated rats but has no effect in the non-treated animals. 4. 4. Tumour homogenates produced α-glycerophosphate from glucose and other glycolytic precursors. The latter finding points to the presence of the α-glycerophosphate shunt in the R3230AC adenocarcinoma.

Sialyltransferase acceptor activity of “antifreeze” glycoproteins from an antarctic fish

Summary The “antifreeze” glycoproteins from the Antarctic fish T. borchgrevinki function as effective acceptors for N-acetylneuraminic acid transfered from cytidine 5′-monophosphate N-acetylneuraminic acid by solublized enzymes from rat liver and a rat mammary adenocarcinoma. The properties of the two preparations are sufficiently similar to suggest that the same enzyme(s) are involved. The similarity between the glycoside structure that must result from this reaction and the known glycoside structures of some mucins and blood group substances invites the suggestion that the antifreeze glycoproteins were evolved during adaptation to a freezing environment by loss of the capability of transferring sialic acid to the antifreeze glycoprotein structure.

Partial fractionation of oestradiol-binding chromatin from dimethylbenzanthracene-induced rat mammary tumour.

SUMMARY The distribution of [6,7-3H]oestradiol-17β binding in chromatin from dimethylbenzanthracene-induced rat mammary adenocarcinoma was studied by differential centrifugation of sonicated nuclei. The evidence suggests that more oestradiol-17β is associated with the 'light' chromatin sedimenting at a higher centrifugal force than with the 'heavy' chromatin. Electron microscopy showed that heterochromatin was present in intact tumour nuclei, but it dispersed during isolation of the nuclei, and probably did not account for the fractionation of chromatin that occurred. No heterochromatin was observed in the centrifuged fractions. The template activity appeared to be higher in the fractions that had bound oestradiol. These results are compatible with the view that oestradiol binds to restricted regions of the chromatin and stimulates local increases in transcription.

Role of ovarian hormones in the growth of transplantable mammary carcinoma.

The ovarian hormone requirement for the growth of a transplantable prolactin-dependent rat mammary adenocarcinoma MTW9 (MT) has been studied. Prolactin stimulation of mammary tumor growth was supplied by coimplantation with the mammosomatotropic tumor MtTW5 (MtT). When serum prolactin measured by radioimmunoassay reached 100 ng/ml intact female rats, MT began measurable growth. Ovariectomy at the time of inoculation of both MtT and MT resulted in slight reduction in growth of MtT and complete inhibition of growth of MT, although serum prolactin rose as high as 600 ng/ml during growth of MtT in the spayed rat. Daily administration of estrogen plus progesterone started 2 months after implantation of MT and MtT in spayed rats failed to stimulate growth of MT. A second transplant of MT to such steroid-treated animals, 3 months after the first transplant failure, resulted in prompt tumor growth. Ovarian hormones are required for survival as well as growth of implants of this prolactin-requiring mammary tumor. ...