Effects of diabetes and insulin on phosphoinositide metabolism in R3230AC mammary tumors.

The effects of diabetes and insulin administration on certain aspects of phosphoinositide metabolism in R3230AC mammary tumors were studied in vivo. Three weeks after diabetes was induced by streptozotocin, [3H]myoinositol incorporation into PI, PIP and PIP2 was increased in R3230AC tumors, whereas the formation of [3H]IP, [3H]IP2 and [3H]IP3 was decreased. Administration of protamine zinc insulin (3IU, twice daily, for 3 days) to diabetic rats decreased [3H]myoinositol incorporation into phosphoinositides and inositol phosphates in these mammary tumors. The R3230AC tumor from insulin-treated diabetic hosts had lower levels of unmetabolized [3H]-myoinositol compared to tumors from diabetic animals. Enzymatically-dissociated tumor cells from insulin-treated animals displayed decreased myoinositol transport in vitro. These findings suggest that the insulin-induced decrease in the turnover of inositol lipids in vivo in R3230AC mammary tumors could have resulted from the decreased level of [3H]myoinositol in these cells.     

Dual secretory and myoepithelial differentiation in the transplantable R3230AC rat mammary carcinoma

The hormone-responsive R3230AC mammary carcinoma, serially transplantable in Fisher rats, shows striking functional and morphological similarities to the normal mammary gland. We have studied its cellular composition by both light and electron microscopy, employing markers of myoepithelial and epithelial cells. We identified two cell types: the major cellular component corresponded to epithelial milk-protein secreting cells, while a second component showed immunocytochemical and ultrastructural characteristics of the myoepithelial cells. These cells were positive with a monoclonal antibody detecting alpha smooth muscle actin. The dual differentiation which normally occurs in breast ducts is therefore reproduced in a malignant experimental tumor. The coexistence of neoplastic cell populations, divergent in morphology and function, that persist in a tumor despite many transplant generations, leads to reconsideration of the relationship between cellular differentiation and malignant transformation.     

Acyl specificity in triacylglycerol synthesis by mammary adenocarcinoma R3230AC in Fischer rats

Activities and acyl specificities of both sn-glycero-3-phosphate and diacylglycerol acyltransferases in microsomal fractions isolated frorn homogenates of the mammary adenocardinoma R3230AC carried by Fischer rats were compared to those from normal mammary glands of lactating Fischer rats. Although the neoplasm exhibited lower activities for these two enzyme reactions, the specificities for acyl-CoAs as donors were quite similar to those found in the normal tissue counterpart. Long-chain acyl-CoAs were preferred substrates for the sn-glycero-3-phosphate acyltransferase reaction while acyltransferase with diacylglycerol as acceptor showed much less preference. With both normal and neoplastic tissues, the products of each reaction were the same i.e., phosphatides with sn-glycero-3-phosphate and triacylglycerol with diacylglycerol as acyl acceptors, respectively. All results support the concept of a non-random distribution of fatty acids in the triacylglycerol of this mammary adenocarcinoma in virgin rats which is the same as that from the normal tissue in lactating animals.     

Insulin binding and glucose transport in the R3230AC mammary adenocarcinoma

Cells dissociated from the R3230AC mammary adenocarcinoma from intact and diabetic rats were examined for insulin binding and glucose transport. The K_d for insulin binding, ～ 10^?10 M, was similar in all tumors studied. However, the apparent number of receptor sites per cell increased in cells from diabetic rats. Kinetic analysis of 3-0-methyl glucose (3-OMG) entry showed both diffusional and passive carrier characteristics. Insulin (4 × 10^?9 M) in vitro did not affect diffusional entry, whereas the hormone altered the passive carrier system, as reflected by an increase in K_m and V_max. Insulin decreased initial velocity of glucose transport at 4–6 mM glucose levels but increased initial velocity of glucose transport at 20 mM glucose. An explanation of the role of insulin on tumor growth in vivo from effects on glucose transport in vitro is proposed.     

Characteristics of proline transport into R3230AC mammary tumor cells

Cells separated by enzyme treatment of the R3230AC mammary carcinoma were used to characterize the entry of proline. These cells showed minimal changes in cell viability and intracellular volume and were found to be suitable for transport studies, since the vi of proline was maintained for at least 4 h when cells were stored at 37 or 4 degrees C, or when transport was measured in the presence or absence of Na+. Proline was acitvely transported by these tumor cells, reaching a distribution ratio ([proline] intracellular/[proline] extracellular) of 20 after 2 h. Proline entry consisted of two processes, one saturable (carrier mediated) and the other, non-saturable. The carrier-mediated entry, Km - 0.83 mM and V = 151.10(-5) mumol/min per 5.10(6) cells, was Na+-dependent, sensitive to pH and metabolic inhibitors, and completely inhibited by alpha-(methylamino)-isobutyric acid (Ki = 0.34 mM). Proline entry in the absence of Na+ was 20% that in the presence of Na+ and was found to be due to a non-saturable process, since (a) vi of proline uptake in the absence of Na+ increases linearly with increasing proline concentration and (b) was not suppressed by either 20 mM alpha-(methyl-amino)-isobutyric acid, 50 mM glycine +20 mM phenylalanine, or 50 mM serine +20 mM phenylalanine when proline uptake was measured in the presence or absence of Na+. Therefore, under the conditions studied, we conclude that proline transport appears to be restricted to the A (alanine-preferring) system. Furthermore, these cells should provide a suitable model to study the effect of hormonal manipulations on the amino acid transport process.     

Influence of estrogen on glutathione levels and glutathione-metabolizing enzymes in uteri and R3230AC mammary tumors of rats

The glutathione content and the activities of several enzymes in its metabolism, glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase, were assayed in uteri obtained from estrogen-treated rats and in R3230AC mammary adenocarcinomas obtained from ovariectomized, intact and estrogen-treated hosts. Normal mammary glands, obtained 10–12 days post-partum, were also examined for these parameters.A daily pharmacological dose of 0.4 μg of estradiol-17β induced a maximal increase in uterine weight and in reduced glutathione (GSH); higher doses of estrogen did not significantly increase either of these parameters. Levels of oxidized glutathione (GSSG) were comparable in both estrogen-treated and untreated rats. The time course of the estrogen-induced uterotrophic response was associated with increases in glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase activities with the increased GSH level preceding the increase in uterine weight. Compared to neoplasms from intact or ovariectomized animals, tumors from estrogen-treated hosts exhibited significant decreases in levels of GSSG and GSH, as well as in glutathione reductase and glutathione peroxidase activities, but demonstrated a significant elevation of γ-glutamyl transpeptidase activity. Normal glands from lactating rats had decreased GSH levels, lower activities of glutathione reductase and glutathione peroxidase, but elevated γ-glutamyl transpeptidase activity versus tumors from intact rats. Tumors from estrogen-treated rats more closely resembled mammary glands during lactation. The divergent growth responses elicited by estrogen in the uterus and mammary tumor are correlated with the observed changes in GSH levels and enzymes involved in glutathione metabolism.     

Effect of estradiol-17β on glucose and proline uptake in plasma membrane vesicles from the R3230AC rat mammary carcinoma

Purified plasma membrane vesicles isolated from R3230AC rat mammary tumors displayed carrier-mediated and stereospecific uptake. Uptake was shown to be proportional to protein concentration, sensitive to increasing osmolarity, and inhibited only by substrates entering by the same carrier. Carrier-mediated glucose uptake was inhibited rapidly by estradiol-17β and phloretin in a dose-dependent manner, whereas proline uptake was not affected by estradiol-17β. The data suggest that the inhibition of glucose by estradiol and phloretin, originally observed in whole cells, occurs by an interaction of the steroid with a component on the plasma membrane. In contrast, the lack of effects of estradiol on proline transport into vesicles implies that intracellular components may have mediated the estrogen-induced effects observed in whole cells.     

Effect of estradiol-17 beta on glucose and proline uptake in plasma membrane vesicles from the R3230AC rat mammary carcinoma

Purified plasma membrane vesicles isolated from R3230AC rat mammary tumors displayed carrier-mediated and stereospecific uptake. Uptake was shown to be proportional to protein concentration, sensitive to increasing osmolarity, and inhibited only by substrates entering by the same carrier. Carrier-mediated glucose uptake was inhibited rapidly by estradiol-17 beta and phloretin in a dose-dependent manner, whereas proline uptake was not affected by estradiol-17 beta. The data suggest that the inhibition of glucose by estradiol and phloretin, originally observed in whole cells, occurs by an interaction of the steroid with a component on the plasma membrane. In contrast, the lack of effects of estradiol on proline transport into vesicles implies that intracellular components may have mediated the estrogen-induced effects observed in whole cells.     

Effect of insulin to decrease glucose transport in dissociated cells from the R3230AC mammary adenocarcinoma of diabetic rats.

Dissociated cells of the R3230AC mammary tumor were found to take up glucose by diffusion and by a passive carrier system. Using labeled 3-O-methylglucose as the probe, the following properties of the passive carrier were identified: (1) specificity for glucose, (2) competition by galactose and mannose but not by mannitol and fructose, (3) inhibition by phloretin but not by phloridzin, (4) temperature sensitivity, and (5) a Km for transport of 3-4 mM. The effects of insulin in vitro on carrier-mediated glucose transport were investigated in tumor cells from diabetic rats. At 10-9 M insulin, a time-related decrease in v for transport was observed resulting in an increased calculated Km (2- to 3-fold increase after 60-90 min incubation with insulin); only slight effects on V were obtained. This unusual response in v to insulin was observed when glucose was present in the medium at 2 mM and 5 mM, but not at 20 mM glucose. The effect of insulin to decrease the v was dose-related, with the major effects seen between 10-10M and 10-8M. The apparent decrease in glucose entry in vitro may in part explain the ability of insulin to inhibit growth of this tumor in vivo.     

Heterogeneous localization of adenylate and guanylate cyclases in R3230AC rat mammary adenocarcinoma cells

Adenylate and guanylate cyclase activities were demonstrated in R3230AC rat mammary adenocarcinomas by electron microscopic cytochemistry. Adenylate (AC) and guanylate (GC) cyclases were detected on plasma membrane of tumor epithelial cells, but not on fibroblasts and endothelial cells in the perivascular space. Both AC and GC activities were enriched in tumor epithelial cells at the periphery of the tumor lobular parenchyma rather than in cells in central core of the lobular parenchyma. Furthermore, the tumor cell plasma membranes facing the connective tissue stroma were in paucity or devoid of either enzyme activity. These heterogeneous distributions of both AC and GC among tumor epithelia suggest that R3230AC epithelial cells in different parts of the tumor mass may vary significantly in their regulation of cellular physiology.     

Effects of trypsin on binding of insulin and concanavalin A and on glucose and proline transport in the R3230AC mammary adenocarcinoma

Effects of trypsin treatment on insulin and concanavalin A binding to, and glucose and proline transport in, dissociated R3230AC mammary adenocarcinoma cells were examined. Reduction of binding of ¹²⁵I-labelled insulin was dependent on the amount of trypsin used, the temperature and the time of the incubation period. Under conditions that reduced insulin binding by greater than 75%, transport of glucose and proline was reduced by less than 15%. Scatchard analysis of insulin binding after trypsin treatment yielded slopes similar to those from cells not exposed to trypsin, assuming either two classes of receptors or an average affinity, $\text{K}\text{?}{}_{\mathrm{<mtext>e</mtext>}}$. Dissociation of bound insulin from untreated or trypsin-treated cells was enhanced by addition of excess unlabelled ligand. Insulin added in vitro, which decreased glucose transport in untreated cells, produced a decrease in glucose transport in cells treated with trypsin for 5 min (insulin binding was decreased 35%), but not in cells treated for 45 min (insulin binding was decreased 90%). Binding of the plant lectin concanavalin A was also reduced by trypsin treatment, but to a lesser extent and with a different time-course than for insulin. Scatchard analysis of the binding of concanavalin A in untreated and trypsin-treated cells yielded comparable values for K_d. The insulinomimetic actions of concanavalin A on glucose transport were abolished after brief exposure to trypsin. Pre-treatment of cells with concanavalin A reduced insulin binding and partially protected insulin receptors from trypsin digestion, but the inability to remove all of the concanavalin A precluded its use as a method to protect insulin receptors. Thus, in this rat mammary tumor, the number, but not the affinity or functional activity, of insulin receptors can be reduced by trypsin treatment without significant effects on glucose or A system amino acid transport.     

Alkaline ribonuclease and ribonuclease inhibitor in mammary gland during the lactation cycle and in the R3230AC mammary tumour.

Alkaline RNAase (ribonuclease) and RNAase inhibitor were assayed to determine the potential role of the degradative process in regulating the amount of RNA in the mammary gland and mammary tumour. Very little free alkaline RNAase activity was found in the cytosol fraction of the mammary gland of virgin, pregnant, lactating or involuting Fischer rats. However, addition of p-chloromercuribenzoate to the assay medium revealed latent RNAase which, when expressed on a DNA basis, decreased during pregnancy and lactation. The cytosol latent RNAase is stable in 0.125 M-H2SO4. The non-cytosol RNAase activity also decreased during pregnancy and lactation. Addition of Triton X-100 produced slightly higher activity at all stages tested. The inhibitor activity in rat mammary gland was very low before pregnancy, increased gradually during pregnancy and more dramatically at parturition, continued to increase throughout lactation and returned to resting-gland values by the sixth day of involution. The increase during pregnancy may be due to the increased cellularity of the gland, whereas the gain during lactation was more than could be accounted for by increases in cell number. The R3230AC transplantable mammary tumour resembles the normal gland in early lactation with respect to both its cytosol and non-cytosol alkaline RNAase activities and its moderately high content of RNAase inhibitor. The relatively high inhibitor and low RNAase activities in both the gland of the lactating rat and in the tumour are of potential significance in maintaining high amounts of RNA and increased rates of protein synthesis in these tissues.     

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.     

Gene expression of PCNA/cyclin in adult tissues and the R3230AC mammary tumor of rat

We have investigated the gene expression of PCNA (Proliferating Cell Nuclear Antigen)/cyclin in rat tissues and the R3230AC mammary tumor. The steady-state mRNA level of PCNA/cyclin in a tissue is related to the proliferation of the tissue. The observation was confirmed with the results from the studies of the immunoblotting analyses and the DNA polymerase activity measurements. Furthermore, an overexpression of PCNA/cyclin was found in the R3230AC mammary tumor, which is accompanied by an altered PCNA/cyclin gene structure detected with the Southern blot analysis.     

Effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase in R3230AC mammary tumors and uteri.

The effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in the R3230AC mammary adenocarcinoma of ovariectomized Fischer rats was investigated. Enzyme synthesis was estimated by techniques using immunochemica precipitation and isolation of enzyme protein from tissues of rats that had been given radioactive leucine prior to sacrifice. The antibody-enzyme complex was dissociated and glucose-6-phosphate dehydrogenase was isolated after electrophoresis on sodium dodecyl sulfate-acrylamide gels. Administration of estradiol-17beta produced a two-fold increase in glucose-6-phosphate dehydrogenase activity, which was preceded by a five-fold increase in specific synthesis of glucose-6-phosphate dehydrogenase in R3230AC tumors. At least a 15-fold increase in enzyme synthesis was observed in the uterus. The rate of enzyme degradation (t 1/2) in the tumor was estimated at 17 h. These data indicate that the estrogen-induced increase in glucose-6-phosphate dehydrogenase activity was due to a de novo increase in enzyme synthesis.     

Intranuclear dynamics of DNA polymerase alpha differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase alpha activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase alpha activity, with the majority (60-75%) localized in soluble nucleoplasm and a significant amount (13-20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase alpha was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60-80%) of DNA polymerase alpha activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60-80%) of DNA polymerase beta activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase alpha molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase alpha which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase beta in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.