Human Mammary Tumor Cell Line Research Articles

Effect of 5‐AmJnolevulinic Acid on Protoporphyrin IX Accumulation in Tumor Cells Transfected with Plasmids Containing Porphobilinogen Deaminase DNA

Recently, we reported that the delta-aminolevulinic acid (delta-ALA)-induced increase in porphobilinogen deaminase (PBGD) activity was closely correlated with an increase in the accumulation of protoporphyrin IX (PPIX), resulting in augmented phototoxicity. In this report, we asked whether increasing the cellular expression of PBGD by use of gene transfection techniques in vitro would further enhance delta-ALA-induced PPIX accumulation and hence, phototoxicity. For these experiments we constructed plasmid vectors containing the PBGD-DNA, using a reverse transcription-polymerase chain reaction-generated cDNA fragment encoded from its published sequence. Subsequently, transfection of the human mammary tumor cell line, MCF-7, and the human mesothelioma cell line, H-MESO-1, with the PBGD-DNA-containing plasmids was shown to produce a 2.5-2.7-fold increase in enzyme activity. Twenty-four hours after completion of the transfection procedure, transfectants were exposed for 3 h to 0.5 mM delta-ALA. Exposure of either wild type or transfectants to delta-ALA led to measurable levels of PPIX. Although this produced a modest but significant increase in intracellular PPIX content in H-MESO-1 cells compared to wild-type cells incubated with delta-ALA alone, the increase above the transfection control did not reach statistical significance. Likewise, a significant increase in PPIX was not observed in transfected MCF-7 cells subsequently exposed to delta-ALA. These data demonstrate that transient transfection of cells with the cDNA of PBGD was successful in elevating enzyme activity in both tumor cell lines, but this did not result in a comparable difference in the levels of PPIX. Such an approach for the study of other enzymes in the heme pathway should provide a model to better define rate-limiting steps in the delta-ALA induction of PPIX, and ultimately, to enhance the effectiveness of photodynamic therapy.

Inhibition of tenascin-C expression in mammary epithelial cells by thyroid hormone.

Multiple data suggest a relationship between thyroid hormone (triiodothyronine (T3)) and carcinogenesis. Studies on breast cancer have been inconclusive, suggesting contradictory effects of thyroid status and diseases. Recently, we reported that expression of the extracellular matrix glycoprotein tenascin-C is modulated by T3 during rat brain development. Because tenascin-C has been reported to have growth-, motility-, and angiogenic-promoting activities and to become upregulated during tumorigenesis in breast carcinoma and stromal cells, we analyzed the effects of T3 on tenascin-C expression in mammary epithelial cells. In this study, we showed that tenascin-C RNA expression was inhibited by T3 in normal un-transformed EpH4 mouse mammary epithelial cells expressing appropriate receptors. T3's action appeared to be due to a decreased half-life of the tenascin-C mRNA, with a maximum effect (85% at 100 nM) 48 h after addition. T3 also downregulated tenascin-C in the human mammary tumor cell line SKBR-3, which expresses endogenous thyroid receptors. Immunoprecipitation experiments confirmed that tenascin-C protein content was also decreased by T3 in EpH4 cells (70% reduction at 100 nM). Dexamethasone had a similar inhibitory effect (70% at 100 nM), whereas estradiol, the antiestrogen ICI 164,384, progesterone, and all-trans retinoic acid did not alter tenascin-C expression. Our data demonstrate an inhibitory action of T3 on tenascin-C expression in mammary epithelial cells that may play a role in the physiological regulation of this gene and in neoplastic processes.

Response of primary rabbit kidney proximal tubule cells to estrogens.

The effects of estrogens on the growth and function of primary rabbit kidney proximal tubule (RPT) cells have been examined in hormonally defined phenol red-free medium. 17beta-estradiol was observed to stimulate growth at dosages as low as 10(-10) M. The growth stimulatory effects of 17beta-estradiol were mitigated in the presence of hydrocortisone, suggesting that these two steroid hormones acted at least in part by common mechanisms. The effects of other steroids known to interact with the estrogen receptor were examined. Alpha estradiol was found to be growth stimulatory over a concentration range of 10(-9) to 10(-8) M, albeit to a lower extent than beta estradiol. In addition, the anti-estrogen tamoxifen was also growth stimulatory (unlike the case with the human mammary tumor cell line MCF-7). The effects of several metabolic precursors of 17beta-estradiol were examined, including testosterone, which was growth stimulatory, and progesterone, which was growth inhibitory. The growth stimulatory effects of 17beta-estradiol, alpha estradiol, and tamoxifen could possibly be explained by their interaction with an estrogen receptor. Indeed, metabolic labelling and immunoprecipitation studies indicated the presence of such an estrogen receptor in the primary cultures. The rate of biosynthesis of the estrogen receptor was found to be affected by the presence of exogenously added 17beta-estradiol. 17beta-estradiol was also observed to increase the activity of two brush border enzymes, alkaline phosphatase and gamma glutamyl transpeptidase, during the growth phase of the primary cultures.

Expression of Peroxisome Proliferator Activated Receptor mRNA in Normal and Tumorigenic Rodent Mammary Glands

The peroxisome proliferator activated receptors (PPARs) α, β/δ, and γ are novel nuclear hormone receptors activated by long chain fatty acids and synthetic ligands and which regulate lipid metabolism. Recent studies have detected PPARγ mRNA in human mammary tumor cell lines. The current study examined the expression profile of PPAR mRNAs in normal and malignant rodent mammary tissues. Virgin murine mammary glands contained PPAR α, β/δ, and γ mRNAs based on northern blot analysis. The PPARγ isoform was predominantly γ2 based on quantitative PCR analysis. During pregnancy and lactation, the PPARα and γ mRNAs decreased while the PPAR β/δ mRNA remained relatively unchanged. NMuMG cells, an epithelial line derived from normal murine mammary gland, expressed PPAR α, β/δ, and γ mRNAs, independent of the presence or absence of compounds modifying PPAR activity. In rats, the physiologic expression pattern of PPARγ mRNA paralleled the murine model; levels were detected in virgin but not lactating mammary glands. In addition, the PPARγ mRNA was not detected in several histologically distinct 7,12-dimethylbenz(a)anthracene induced mammary tumors. These findings suggest that PPARs may regulate mammary epithelial and stromal cell function in response to physiologic or pathologic stimuli that profoundly alter lipid metabolism.

The oncogene heregulin induces apoptosis in breast epithelial cells and tumors

The products of a growing number of genes have been shown to display seemingly contradictory functions; namely, the induction of tumorigenesis and the induction of apoptosis. Heregulin's involvement in oncogenesis occurs through its interactions with members of the EGF receptor tyrosine kinase family. Recently one isoform of heregulin, beta2b, was isolated in an in vitro screen for dominant, apoptosis-inducing genes in kidney epithelial cells. Here we show that heregulin is also capable of mediating apoptosis in human and murine mammary tumor cell lines and murine tumors. Furthermore, through transfection of the human breast cancer cell line MCF-7 with the truncated transmembrane/cytoplasmic segment of the heregulin gene, we show that the intracellular region of the heregulin precursor is sufficient for induction of apoptosis. Through the use of DNA fragmentation assays we also show that apoptosis occurs in cell lines established from heregulin-induced mammary gland tumors. TdT addition of digoxigenin labeled nucleotides to 3' OH ends of DNA breaks recapitulated these results in the actual tumors. Finally, over-expression of heregulin is shown to lead to the down-regulation of Bcl-2, an inhibitor of apoptosis. Conversely, the transfection of Bcl-2 into MCF-7 cells inhibits heregulin-mediated programmed cell death.

1-β-d-Arabinofuranosylcytosine-5′-alkylphosphonophosphates and diphosphates: new orally active derivatives of ara-C

ara-Cytidine-5′-alkylphosphonophosphates and the corresponding -diphosphates were found to be cytostatically active in vitro against the human mammary epithelial cell line H184 A1N4 and the human mammary tumor cell line MaTu. Our results indicate that the replacement of the diphosphate by the phosphonophosphate group has no influence on antiproliferative activity in this case. The compounds were more active than the corresponding cytidine phospholipid conjugates and related compounds lacking a cytostatically active nucleoside, the ara-C prodrug Cytoros, and were slightly less active than ara-C. The cytostatic effect was prevented by 2′-deoxycytidine indicating their action as prodrugs of ara-C. In contrast to ara-C, they increase [Ca2+]1 in H184 A1N4 cells, pointing to a different mechanism of action in addition to their prodrug effect. In combination with phospholipid analogs, synergistic effects could be observed. Further studies within the disease-oriented in vitro Anticancer Screening Program of the National Cancer Institute show selectivity for certain cancer cell lines. The hexadecyl derivatives revealed a significant antitumor activity in vivo against the murine lymphatic leukemia P 388 cells being equally potent or even superior to ara-C. In contrast to ara-C, they were found to be orally active. Side effects measured as leukopenia and body weight reduction were less pronounced than with the parent drug.—Brachwitz, H., J. Bergmann, I. Fichtner, Y. Thomas, C. Vollgraf, P. Langen, and W. E. Berdel. 1-β-d-Arabinofuranosylcytosine-5′-alkylphosphonophosphates and diphosphates: new orally active derivatives of ara-C. J. Lipid Res. 1998. 39: 162–172.

Human endogenous retrovirus expression and reverse transcriptase activity in the T47D mammary carcinoma cell line.

We have examined the human mammary tumor cell line T47D and have found that these cells produce virus-like particles which band at the typical density for retroviral particles on a sucrose gradient, possess reverse transcriptase activity, and package HERV-K10-like sequences. Using this information and a bacterial expression system to identify long open reading frames, we have identified individual clones which have full-length open reading frames for reverse transcriptase and RNase H and which could encode the reverse transcriptase activity detected in these cells.

Antiproliferative effect and interaction of fucans with cells

Fucans were extracted from the brown seaweed Ascophyllum nodosum, degraded by acidic hydrolysis, fractionated by gel nitration and characterized by high performance steric exclusion chromatography. Different fucan fractions of average molecular weight 6000–150000 g mol −1 were thus obtained. The effects of these low molecular weight (LMW) fucans on cells were examined. The results show that these fucans can inhibit the growth of some cell lines. The Chinese hamster fibroblasts (CCL 39) are strongly inhibited, without any cytotoxicity, showing an inhibition percentage of 80% at a fucan concentration of 1000 μg ml −1, while inhibition does not exceed 50% on human colon adenocarcinoma cells (COLO 320DM) at the same fucan concentration. Other tumor cell lines examined here are not sensitive to these fucans (human mammary tumor cell line MCF7, murine leukemic cells P388 and human fibroblasts McCoy). This fucan inhibitory effect seems to be dependent on the sulfate group content. We show that no fucan molecular weight effect was observed for fractions in the range 10000–150000 g mol −1. However, the fucan fractions with a molecular weight under 10000 g mol −1 are less active. The analysis of the cells by flow cytometry shows that fucans did not induce any perturbations on the cell distribution in the various phases of the cell cycle, but they did influence the synthesis and secretion of thrombospondin. On human fibroblasts (McCoy), no antiproliferative effect was observed, but it seems that fucans led to substantial alterations of cell adhesion without any cytotoxicity. More information concerning the fucan mechanism is necessary to understand how they interact with the extracellular matrix.

Demonstration of ligand-dependent signaling by the erbB-3 tyrosine kinase and its constitutive activation in human breast tumor cells.

The predicted human erbB-3 gene product is closely related to epidermal growth factor receptor (EGFR) and erbB-2, which have been implicated as oncogenes in model systems and human neoplasia. We expressed the erbB-3 coding sequence in NIH 3T3 fibroblasts and identified its product as a 180-kDa glycoprotein, gp180erbB-3. Tunicamycin and pulse-chase experiments revealed that the mature protein was processed by N-linked glycosylation of a 145-kDa erbB-3 core polypeptide. The intrinsic catalytic function of gp180erbB-3 was shown by its ability to autophosphorylate in vitro. Ligand-dependent signaling of its cytoplasmic domain was established employing transfectants that express a chimeric EGFR/erbB-3 protein, gp180EGFR/erbB-3. EGF induced tyrosine phosphorylation of the chimera and promoted soft agar colony formation of such transfectants. These findings combined with the detection of constitutive tyrosine phosphorylation of gp180erbB-3 in 4 of 12 human mammary tumor cell lines implicate the activated erbB-3 product in the pathogenesis of some human malignancies.

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract Collagen-binding proteins ( CBPs) of rat mammary tumors are identical to Ca 2+-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I. In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.

Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family.

This report describes the isolation and recombinant expression of a cDNA clone encoding HER4, the fourth member of the human epidermal growth factor receptor (EGFR) family. The HER4/erbB4 gene encodes a 180-kDa transmembrane tyrosine kinase (HER4/p180erbB4) whose extracellular domain is most similar to the orphan receptor HER3/p160erbB3, whereas its cytoplasmic kinase domain exhibits 79% and 77% identity with EGFR and HER2/p185erbB2, respectively. HER4 is most predominantly expressed in several breast carcinoma cell lines, and in normal skeletal muscle, heart, pituitary, brain, and cerebellum. In addition, we describe the partial purification of a heparin-binding HER4-stimulatory factor from HepG2 cells. This protein was found to specifically stimulate the intrinsic tyrosine kinase activity of HER4/p180erbB4 while having no direct effect on the phosphorylation of EGFR, HER2, or HER3. Furthermore, this heparin-binding protein induces phenotypic differentiation, and tyrosine phosphorylation, of a human mammary tumor cell line that overexpresses both HER4 and HER2. These findings suggest that this ligand-receptor interaction may play a role in the growth and differentiation of some normal and transformed cells.

Generation of reactive oxygen species from the reaction of selenium compounds with thiols and mammary tumor cells

Sodium selenite, sodium selenate, selenocystine and selenomethionine were tested for their abilities to generate Superoxide by the oxidation of glutathione and other thiols in the absence and presence of cells of the human mammary tumor cell line HTB123/DU4475. Free radical generation was measured by lucigenin- or luminol-amplified chemiluminescence. In the absence of tumor cells, lucigenin-dependent chemiluminescence was observed from the reaction of selenite with the thiols glutathione, 2-mercaptoethanol and l-cysteine, but not with oxidized glutathione. Superoxide dismutase, catalase, and glutathione peroxidase all suppressed the observed chemiluminescence; but when these enzymes were heat inactivated they had little suppressive inhibition on chemiluminescence. Luminoldependent chemiluminescence from the reaction of selenite with glutathione was much less than that observed by lucigenin-amplified chemiluminescence. In the presence of the HTB123/DU4475 mammary tumor cells, lucigenin-dependent chemiluminescence was observed from the reactions of selenite and selenocystine with glutathione which were 5 and 23 times greater than their respective reactions with glutathione in the absence of tumor cells. The enhanced chemiluminescence generated by selenite and selenocystine in the presence of the tumor cells was also suppressed by Superoxide dismutase, catalase and glutathione peroxidase. These data suggest that a free radical, the Superoxide anion (O 2 −, and H 2O 2; are produced from the reaction of selenite and selenocystine with glutathione. These free radical reactions may account for the toxicity of selenite and selenocystine in vitro in comparison to a near absence of acute tumor cell toxicity and Superoxide generation by selenate and selenomethionine with thiols. Enhanced chemiluminescence in the presence of tumor cells may be an expression of cellular selenium metabolism and the capability of cells to form selenium metabolites that more easily oxidize glutathione and other thiols producing reactive free radicals and peroxides.

Epidermal growth factor reduces HER-2 protein level in human ovarian carcinoma cells.

Over-expression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian, endometrial, and mammary carcinoma is an indicator of poor prognosis. Interactions between the epidermal growth factor (EGF) receptor and the HER-2 protein have been described. The aim of this study was to elucidate the effects of EGF on HER-2 expression. In the human ovarian carcinoma cell lines HTB-77, OVCAR-3, 2780, SKOV-6, SKOV-8 and 2774, and the human mammary tumor cell line SKBR-3, total cellular p185HER-2 was determined by an ELISA, whereas the surface p185HER-2 was measured with a living-cell RIA. Stimulation of these cell lines with either EGF (0.1-30 nM) or TGF-alpha (0.1-30 nM) led to a significant reduction in p185HER-2 expression. The effect was more pronounced in cells with normal HER-2 expression. A reduction of mRNA levels for p185HER-2 by EGF was observed in OVCAR-3 cells but not in the over-expressing lines HTB-77 and SKBR-3. Interestingly, the EGF-induced effect was not always associated with growth stimulation and was not correlated with the number of EGF binding sites detected by a radioligand assay. Our data indicate that EGF treatment results in a down-regulation of p185HER-2.

Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumor cell lines.

Subtractive hybridization, selecting for mRNAs expressed in normal human mammary epithelial cells (NMECs) but not in mammary tumor cell lines (TMECs), led to the cloning of the human gap junction gene connexin 26 (Cx26), identified by its sequence similarity to the rat gene. Two Cx26 transcripts derived from a single gene are expressed in NMECs but neither is expressed in a series of TMECs. Northern analysis using rat Cx probes showed that Cx43 mRNA is also expressed in the normal cells, but not in the tumor lines examined. Connexin genes Cx31.1, Cx32, Cx33, Cx37, and Cx40 are not expressed in either normal cells or the tumor lines examined. In cell-cell communication studies, the normal cells transferred Lucifer yellow, while tumor cells failed to show dye transfer. Both Cx26 and Cx43 proteins were immunolocalized to membrane sites in normal cells but were not found in tumor cells. Further analysis demonstrated that Cx26 is a cell-cycle regulated gene expressed at a moderate level during G1 and S, and strongly up-regulated in late S and G2, as shown with lovastatin-synchronized NMECs. Cx43, on the contrary is constitutively expressed at a uniform low level throughout the cell cycle. Treatment of normal and tumor cells with a series of drugs: 5dB-cAMP, retinoic acid, okadaic acid, estradiol, or TGFb had no connexin-inducing effect in tumor cells. However, PMA induced re-expression of the two Cx26 transcripts but not of Cx43 in several TMECs. Thus Cx26 and Cx43 are both downregulated in tumor cells but respond differentially to some signals. Modulation of gap-junctional activity by drug therapy may have useful clinical applications in cancer.

In vitro antitumour activities of a novel 2:3 condensation product of salicylaldoxime with di‐n‐butyltin(IV) oxide

AbstractThe antitumor activities of three novel condensation products of salicylaldoxime with di‐n‐butyltin(IV) oxide (compound 1), di‐t‐butyltin oxide (compound 2) and diphenyltin oxide (compound 3) are presented. Against MCF‐7, a human mammary tumor cell line, compounds 1 and 2 are characterized by inhibition doses ID50 in vitro of 67 and 49 ng cm−3 respectively, whereas cis‐platin, an antitumor drug used clinically, has an ID50 of 850 ng cm−3. Against WiDr, a colon carcinoma, they also score better than cis‐platin: 215 and 121 ng cm−3 versus 624 ng cm−3. In contrast, the diphenyltin compound, 3, is inactive against both tumor cell lines.The results of a pre‐screening performed on compound 1 by the National Cancer Institute (NCI) on a panel of 60 human tumor cell lines show that two of the selectivity parameters calculated by the NCI for that compound are statistically significant, namely DG150 (51.9>50) and MGDH (80.1>75). One is almost satisfactory (DH = 72.4 = ca75). The other two, DTGI (40.0<50) and DLC50 (16.7<50) are not. (Abbreviations are explained in the text and in Gielen, M. and Willem, R. Anticancer Res., 1992, in press).

Direct effects of luteinizing hormone-releasing hormone agonists and antagonists on MCF-7 mammary cancer cells.

The binding of luteinizing hormone-releasing hormone (LH-RH) analogues to the human mammary tumor cell line MCF-7 and their effect on the cell proliferation was studied to elucidate their direct action on estrogen-dependent mammary tumors. The growth rate of these cells was doubled by the addition of 1 nM estradiol to cells maintained in an estrogen-deficient medium. Although the basal growth rate was only slightly inhibited by the LH-RH antagonist [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH (SB-75), the estrogen-stimulated growth was completely abolished by the antagonist. In contrast, the LH-RH agonist buserelin stimulated cell growth in estrogen-deficient medium, whereas it had no effect in the presence of estrogen. 125I-labeled buserelin was used for the measurement of LH-RH receptors on MCF-7 cells. A Scatchard plot analysis of buserelin-specific binding revealed a nonlinear plot, which suggested the presence of one high-affinity binding site with a Kd of 1.4 +/- 1.0 nM and the remaining sites with low affinity (Kd = 1.3 +/- 1.0 microM). The binding of 125I-labeled buserelin was displaced equally well by unlabeled buserelin and by the LH-RH antagonist SB-75, suggesting that both analogues are bound to the same receptor. When parallel experiments were performed with 125I-labeled SB-75, the binding was displaced by unlabeled SB-75 and other antagonists, but only partially displaced by unlabeled buserelin. The results suggest that in these mammary tumor cells there is a LH-RH antagonist binding site that is not recognizable by LH-RH agonists. This hypothesis was tested by measuring cell growth in the presence of both agonists and antagonists. It was found that SB-75 inhibited the stimulation of growth by buserelin, but buserelin did not prevent the inhibition by the antagonist of the estrogen-dependent growth. These results suggest that antagonists directly inhibit mammary tumor growth, not only by competing with LH-RH high-affinity receptors, but also by other mechanisms mediated by low-affinity antagonist binding sites.

New cell lines of human breast cancer origin

Established human mammary tumor cell lines constitute an important tool in the study of breast cancer. The aim of this work was to isolate and characterize two new mammary tumor cell lines, JCK and GCS, which were obtained from the pleural effusion and ascitic fluid, respectively, from two breast cancer patients. Both cell lines had some properties of transformed cells, namely immortalization and growth in soft agar. The carcinoma cells presented epithelial morphology shown by light and electron microscopy, and antigenic properties shown by different tumor markers such as a cytokeratin cocktail, carcinoembryonic antigen, epithelial membrane antigen, and human milk fat globule membrane antigen. A significant increase was also found (P greater than 0.05) in cell growth and 3H-thymidine incorporation into DNA in the JCK and GCS cell lines in the presence of 17 beta estradiol at concentrations of 10(-9) and 10(-7) M, respectively, after 5 days in culture. These cells presented estradiol receptor levels which were similar in the biopsies and the resulting cell lines. The aromatase activity was also similar in the JCK cell line and the original patient biopsy. However, there was a considerably higher aromatase activity in the GCS cell line than in the biopsy specimen. Southern hybridizations with the neu oncogene showed an additional 12 kb fragment in both cell lines, as also seen in patients with breast cancer. We conclude from these studies that this in vitro system may provide us with a way to study metastatic cells and improve clinical management of breast cancer patients.

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.

Effect of dietary selenium and magnesium on human mammary tumor growth in athymic Nude Mice

The effect of dietary selenium (Se) supplementation and low dietary magnesium (Mg) on growth of cells of the human mammary tumor cell line (HTB123/DU4475) and the tissue glutathione (GSH) content in female athymic nude mice was studied. Sixty three- to four-week-old female athymic nude mice were randomly divided into six dietary groups of 10 animals. The mice were fed a modified AIN-76A diet with two levels of Mg (100 and 665 mg/kg) and three levels of Se (0.04, 0.2, and 4.0 mg/kg). At the fourth week of dietary treatment, mice were subcutaneously inoculated with 2.5 x 10(6) viable tumor cells on the dorsal lumbar region and then fed their respective diets for another four weeks. Dietary Se supplementation had no significant effect on tumor growth or tissue GSH content. Low dietary Mg limited both tumor growth and tissue GSH synthesis but raised Mg and GSH levels in tumor tissues. The growth of mice fed the diet containing 100 mg/kg Mg and 4.0 mg/kg Se was significantly retarded. This study demonstrated that neither Se deficiency nor Se supplementation had any effect on mammary tumor growth or tissue GSH content in athymic nude mice. Low dietary Mg did retard tumor growth and inhibited GSH synthesis. Low dietary Mg also resulted in an apparent increase in Se toxicity in these animals.

Hormonal induction of transfected genes depends on DNA topology.

Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.