Intracellular distribution of selenium and the growth of mammary cells in culture

Retention of Se in CMT-13 cells increased with an increase in the concentration of selenite in the incubation medium, the duration of exposure, and the density of the culture. The enhanced toxicity of selenite coincided with a proportional increase in Se in both the cytoplasm and nucleus. About 90% of the accumulated Se was isolated with cytoplasmic macromolecules. Increased nuclear Se retention correlated with increased cytoplasmic Se retention. Greater quantities of cytosolic Se-containing proteins (74, 55, 41, 34, and 28 kDa) and a nuclear Se-containing protein (56 kDa) were detected as the quantity of Se within CMT-13 cells increased. These findings suggest that cellular retention and distribution of Se are determinanants of the degree of cellular growth inhibition caused by this trace element.     

Effect of selenium compounds and thiols on human mammary tumor cells

The effect on cell viability and growth rate of sodium selenite, selenocystine, sodium selenate, and selenomethionine at selenium concentrations of 6.25 and 12.5 uM was studied in vitro on cells of the human mammary tumor cell line HTB123/DU4475. Selenite and selenocystine affected both cell viability and growth rate of the tumor cells at these selenium concentrations. Selenite and selenocystine decreased intracellular glutathione concentrations, but did not affect tumor cell glutathione peroxidase activity. After six days of exposure to either selenate or selenomethionine, the viability of tumor cells remained stable, but cell growth, as measured by numbers of cells, was retarded. Neither selenate nor selenomethionine produced changes in concentrations of intracellular glutathione. The toxic effect of selenite on tumor cells was enhanced by addition of 0.25 mM glutathione to the growth medium. Preincubation of the tumor cells with 62.5 uM buthionine sulfoximine decreased cellular glutathione to 15% of controls at 24 h and enhanced the toxicity of selenite toward the tumor cells. Glutathione, 2-mercaptoethanol, and L-cysteine were all toxic to the tumor cells in a dose-dependent manner.     

Immunotherapy with SLPI over-expressing mammary tumor cells decreases tumor growth

We have demonstrated previously that the inoculation of murine mammary tumor cells genetically modified to express high levels of secretory leukocyte protease inhibitor (2C1) do not develop tumors in immunocompetent mice and these cells are more prone to apoptosis than control cells. The aim of the present study was to evaluate the role of the adaptive immune response in the lack of tumor growth of 2C1 cells and the possibility of using these cells for immunotherapy. The s.c. administration of mock transfected F3II cells induces tumor in BALB/c and Nude mice. However, the inoculation of 2C1 cells develops tumor in Nude but not in BALB/c mice. The inoculation of mock transfected F3II cells to 2C1 immunized BALB/c mice by repeated administration of 2C1 cells (once a week for 3 weeks) developed significantly smaller tumors than those observed in non-immunized mice. Remarkably, survival of tumor-bearing immunized mice was higher than non-immunized animals. Herein, we demonstrate that an immunotherapy with SLPI over-expressing non-irradiated tumor cells which do not develop tumor in immunocompetent mice, partially restrain the tumor growth induced by F3II cells and increase the survival of the mice.     

Identification of estrogen-inducible growth factors (estromedins) for rat and human mammary tumor cells in culture

Summary The role of polypeptide growth factors (estromedins) as mediators of estrogen-responsive mammary tumor growth is studied in this report. Three possible new mechanisms were investigated that include endocrine, autocrine, and paracrine related growth factors. The first hypothesis being tested is whether estrogens interact with target tissues and cause the biosynthesis and secretion of polypeptide growth factors, which then act as mitogens for normal and neoplastic mammary tissues. Data presented suggest that this mechanism involves estrogen interaction with uterus, kidney, and pituitary gland causing production of growth factors, which then enter the general circulation and promote growth of distant target tissues. This is an endocrine type mechanism. Another type of estromedin control (autocrine control) may be exerted in an autostimulatory way in which the target tissue produces the polypeptide factors for its own growth in response to estrogen stimulation. A variation of the autocrine mechanism may be a paracrine mechanism in which some cells of an estrogen-responsive normal or neoplastic tissue produce growth factors that act on adjacent or neighboring cells. From the data available, all three possible types of growth factors could be functioning synergistically to yield the final result of continuous estrogen responsive tumor growth in vivo. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by American Cancer Society Grant BC-255; D. A. S. is the recipient of an American Cancer Society Faculty Research Award, FRA-212. D. D. is supported by a Rosalie B. Hite predoctoral fellowship from the Rosalie B. Hite Foundation, Houston, Texas. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Effects of phosphoethanolamine and ethanolamine on growth of mammary carcinoma cells in culture

Phosphoethanolamine (PEtn) was previously shown to be a potent mitogen for a rat mammary carcinoma cell line, 64-24. The objective of this study was to elucidate the role of phosphoethanolamine as a mitogen. The growth stimulatory effect of various compounds related to PEtn was examined, and ethanolamine (Etn) and compounds whose structures resembled Etn were also found to be active. Among several neoplastic mammary epithelial cell lines of human and rat tested, a human line, T47-D, gave a positive growth response to phosphoethanolamine. The results suggest that a positive growth response to PEtn is correlated with growth response of a cell line to prolactin in culture. PEtn and Etn (10^?6 M) added to the medium incorporated efficiently into cellular phosphatidylethanolamine. After 3–4 days in culture more than 50% of the cellular phosphatidylethanolamine appeared to be derived from exogenous PEtn or Etn. Moreover, phosphatidylethanolamine content in phospholipid became three times higher than that without Etn in the medium.     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture.     

Activation of the estrogen receptor from N-nitrosomethylurea-induced rat mammary tumors

The activation of the estrogen receptor (ER) from N-nitrosomethylurea (NMU)-induced rat mammary tumors was studied in vitro. The activation of the receptor induced by heating of the cytosol containing occupied ER was measured by a 3-4-fold increase of receptor binding to nuclei in comparison with the nuclear binding of the nonactivated ER. The activation of the ER was further shown by alteration of the elution profile from DEAE-cellulose. A shift of the receptor peak from 234 mM (Peak II, nonactivated ER) to 70 mM (Peak I, activated ER) phosphate buffer could be obtained. The overall recoveries of activated ER following chromatography on DEAE-cellulose were significantly lower than the recoveries of the nonactivated ER, 71 and 85%, respectively. Binding of the activated ER to nuclei and chromatography of the supernatant which is not able to bind to nuclei on DEAE-cellulose resulted in a decrease of Peak I and in an increase of the overall recovery. These findings suggest that the nuclear bound ER consists of two parts. One is represented partially by Peak I of the elution profile and the other one by that part of the receptor which can not be eluted from the column under the conditions used. Furthermore, the dissociation of tritiated estradiol (E3H) from the nonactivated ER followed a two component exponential function whereas after activation a monophasic dissociation curve could be observed. The mean half times for the dissociation of E3H from the activated and nonactivated ER were 101 and 7.2 min, respectively. Finally, the nonactivated molybdate stabilized ER sedimented in 5-20% sucrose density gradients as two peaks, one at 9.5 S and the other at 4 S. After activation of the ER only the smaller 4 S peak was evident. Molybdate inhibited the activation of the ER measured by nuclear binding assays, sucrose density gradient analysis, dissociation kinetics or ion exchange chromatography but not completely in every case.     

Role of serum and hormones during the growth and development of rat mammary tumor epithelial cells in collagen gel culture

Summary The characteristics of hormone-dependent rat mammary tumors in response to serum and hormones were determined in collagen gel matrix culture. Epithelial cells from 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas were embedded in collagen gel and the effect of estrogen, progesterone, prolactin, insulin, and serum was tested. The total cell number and [³H]thymidine incorporation were used to determine the growth pattern of the cells in culture. It was found that in medium containing 20% porcine serum and supplemented with insulin, estrogen, progesterone and prolactin, both the cell number and [³H]thymidine labeling index increased with time, after an initial lag. Serum seemed to be essential to maintain growth of the tumor cells, because hormones alone, in the absence of serum, were unable to sustain growth of the cells. When estrogen, progesterone, prolactin, and insulin were tested individually in the presence of 20% porcine serum, only estrogen demonstrated a significant stimulatory effect.     

Eicosanoids and linoleate-enhanced growth of mouse mammary tumor cells.

Metastatic mouse mammary tumor cell line 4526 was used to determine whether linoleate (LN)-derived cyclooxygenase metabolites were involved in the mechanism of LN-enhanced 4526 tumor growth. Unstimulated line 4526 cells converted LN to both PGE1 and PGE2 in serum free medium (SFM). However, neither prostaglandin (PG) influenced growth, while db-cGMP, but not db-cAMP, stimulated growth to the same extent as LN. Cyclooxygenase inhibitors stimulated growth while suppressing PG synthesis. Lipoxygenase inhibitors decreased growth in a dose dependent manner. Supplemental LN had no effect on cyclooxygenase inhibition while the IC50s for lipoxygenase inhibition were increased several fold. These results indicate that lipoxygenase products rather than cyclooxygenase metabolites play a major role in LN-stimulated growth of line 4526 cells.     

Inhibition of the growth of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced mammary gland neoplasms by methionine in rats

The diets supplemented with 0.5% methionine inhibited the development of mammary adenocarcinomas induced in female rats with 7,12-dimethylbenz(a)anthracene and N-nitrosomethylurea by 3.8 and 2.3 times, respectively. Methionine produced no effect on the incidence of other new-growths. It is suggested that the mechanism by which methionine exerts an antineoplastic effect on mammary carcinomas is a consequence of its hypolipidemic and neurotropic activity.     

Influence of estradiol on mammary tumor collagen solubility in DMBA-induced rat mammary tumors

Estradiol plays a vital role in the growth and development of mammary glands. It is a potent stimulator of metabolic processes in normal and carcinoma breast. A critical factor in determining mammary glandular morphology is the stroma. Collagen is a predominant component of the extracellular matrix and cell-collagen interactions are essential carcinogenesis. The present investigation explored the influence of estradiol on collagen solubility and metabolism in mammary tumors during tumor progression and regression. A single injection of 20 mg of 9,10-dimethyl-1,2-benzanthracene was given to rats at 7 weeks of age. With the appearance of the first palpable mammary tumor, the rats were treated with 0.5 microg estradiol or 50 microg tamoxifen daily for 30 days. The rats were sacrificed 24 h after 30 days of treatment. Estradiol appears to stimulate the synthesis of new collagens and thus contributes to the enlargement of the mammary tumors. This might have created a potential microenvironment by increasing the synthesis of suitable matrix that sustains the growth of the mammary tumors. In short, the present findings emphasize a definite mediatory role for collagen in estradiol promoted mammary tumor growth.     

Testosterone-induced growth of S115 mouse mammary tumor cells is dependent on heparan sulfate

The androgen-induced proliferation of S115 mouse mammary tumor cells has been suggested to involve autocrinic fibroblast growth factor signaling. Heparan sulfate proteoglycans are required for fibroblast growth factor signaling, presumably due to their ability to alter binding of fibroblast growth factors to their receptors. We have investigated the role of heparan sulfate proteoglycans in the testosterone-induced proliferation of S115 cells. We demonstrate that when the cells are treated with sodium chlorate, which inhibits the sulfation of endogenous heparan sulfate proteoglycans, cell growth becomes dependent on exogenous heparin. The shortest heparin oligosaccharides supporting cell growth were octasaccharides, whereas dodecasaccharides were almost as effective as native heparin. The N-, 2-O-, and 6-O-sulfate groups of heparin were all required for full testosterone response. Treatment of S115 cells with chlorate or testosterone did not alter the expression of fibroblast growth factor receptors 1 or 3, whereas the expression of fibroblast growth factor receptor 2 was down-regulated. We have previously shown that overexpression of syndecan-1 heparan sulfate proteoglycan renders S115 cells insensitive to testosterone and now demonstrate that this effect can be overcome by sodium chlorate treatment in combination with exogenous heparin. Our results suggest that heparin-like molecules are intimately involved in the androgen-mediated proliferation of S115 cells.     

Temperature-sensitive mutants for steroid-sensitive growth of S115 mouse mammary tumor cells

We have generated temperature-sensitive (ts) mutants for steroid-regulated anchorage-independent cell growth. Androgen-responsive S115+A mouse mammary tumor cells were mutagenized with ethyl methane sulfonate and the variants which were growth-arrested in suspension at the nonpermissive temperature of 41 degrees C were selected by killing dividing wild-type cells with the DNA synthesis inhibitors 5-fluoro-2'-deoxyuridine or cytosine arabinoside. Fifteen clones were isolated and characterized for morphology and growth properties. Three (ts21, ts27, ts33) of the phenotypic variants were ts for androgen-maintained anchorage-independent growth, two of them (ts27 and ts33) also for growth in monolayer. Growth arrest at 41 degrees C was not due to a defect in androgen receptor function in any of the mutant cell lines as shown by steroid binding assays and by the androgen-stimulated expression of both endogenous MMTV RNA and the transiently transfected LTR-CAT gene at the nonpermissive temperature. It remains to be determined for clone ts33 whether the defect is in postreceptor events of steroid action or in genes affecting general mechanisms of cell growth. However, since in clones ts21 and ts27 general cell growth remains functional at 41 degrees C under serum stimulation, defects may be in postreceptor steroid-related pathways. It is hoped that these mutants will provide a useful tool for study of steroid regulation of cell growth and in particular of the property of anchorage-independent growth.     

Effect of selenium on malignant tumor cells of brain

Some reports have demonstrated that selenium can inhibit tumorigenesis in some tissues of animal. However, little is known about the inhibitory effect on malignant tumor cells of brain. The purpose of our study was to determine the biological effect of selenium on growth of rat glioma and human glioblastoma cell lines. Cell lines C6 and A172 were obtained from Japanese Cancer Research Resources Bank, Tokyo, Japan (JCRB). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere of air and 5% CO₂. Antiproliferative effects of selenium were evaluated using growth rate assay quantifying cell number by MTT assay. An antiproliferative effect of selenium was found in two cell lines, which was more effective on human A172 glioblastoma and less effective on rat C6 glioma.     

Hormonal regulation of mouse mammary tumor growth

In view of reports that human breast cancer cells secrete growth factors that can replace estradiol in sustaining tumor growth [1], we have investigated whether hormone independent (HI) GR mouse mammary tumors can sustain growth of estrogen-depleted hormone dependent (HD) tumors. HD GR mammary tumor TSl 106 was grafted subcutaneously in the right flank of estrone plus progesterone treated castrated (020 X GR)F1 mice. After 2 weeks the estrone treatment was stopped and the mice received 50, 100 or 150 mg HI GR mammary tumor TSl 104 in the left flank. However, the regression of the HD tumor due to estrone depletion was not prevented or retarded by the HI grafts. In other experiments we investigated integrations of mouse mammary tumor virus (MMTV) proviral DNA in the DNA of GR mammary tumors. We could demonstrate the presence of two cell populations in tumor TSl 96, both HD but differing in MMTV DNA integration events. Our data indicate that exogenous integrations of MMTV proviruses can take place in mouse mammary tumor DNA without loss of hormone dependency of the tumors. Like in GR/Mtv-2+ mice, mammary tumor transplants differing in MMTV proviral integrations are also observed in 020/Mtv-2+ mice.