Rat Mammary Carcinoma Cell Line Research Articles

Glutathione depletion in human and in rat multi-drug resistant breast cancer cell lines

The effects of GSH depletion in a human breast cancer cell line and a multi-drug resistant subline (ADRr) were determined in a number of experimental conditions. The ADRr cells contained lower GSH concentration which cannot be explained solely on the basis of differences in cell kinetics, and yet the rate-limiting synthetic enzyme gamma-glutamylcysteine synthetase was increased 2-fold. Inhibition of GSH synthesis by BSO resulted in more rapid and more pronounced GSH depletion in ADRr compared to the wild-type cells, suggesting that enhanced GSH utilization and efflux in the resistant cells account for the lowered basal concentration. In addition, the gamma-glutamyl moiety salvage enzyme gamma-glutamyltranspeptidase was reduced markedly in the ADRr cell line. Since these cells have overexpression of the efflux pump protein P-glycoprotein, we examined the effects on cellular GSH of inhibition of the pump's function by verapamil. We found that verapamil significantly depleted cellular GSH. In a rat mammary carcinoma cell line selected in Adriamycin for multi-drug resistance, a similar molecular phenotype has been described including diminished cellular GSH concentration. Verapamil treatment of these cells also resulted in significant depletion of cellular GSH. These results are consistent with the recent report that combined treatment of BSO and verapamil has an additive effect on cytotoxicity. It is likely that decreased basal GSH concentration is due to oxidation and conjugation of it in reactions catalyzed by the enhanced peroxidase and GST found in these cells.

Identification, partial purification and characterization of high-molecular-weight gelatin-degrading metalloproteinases produced by a rat mammary carcinoma cell line

BC1 rat mammary carcinoma cells were found to secrete a unique profile of metalloproteinases, distinguished by two gelatin-degrading metalloproteinases of M r > 220 · 10 3and M r ≫ 220 · 10 3. These enzymes were each partially purified by gel-filtration chromatography, and inhibitor studies showed them to be metalloproteinases. Under conditions where denatured collagen types I, II, and V were completely degraded, native collagen types I, II, IV and V, fibronectin, fibrinogen, C1q, casein, and denatured transferrin were not degraded significantly by these enzymes. The relationship of these enzymes to other extracellular matrix-degrading metalloproteinases and their possible significance in tumour invasion and metastasisis is discussed.

Identification of a metalloproteinase co-purifying with rat tumour collagenase and the characteristics of fragments of both enzymes

A metalloproteinase similar or identical to stromelysin was shown to co-purify with interstitial collagenase from the rat mammary carcinoma cell line, BC1. The mixture of BC1 metalloproteinase and collagenase degraded casein, gelatin, fibronectin, fibrinogen, laminin, proteoglycan and type IV collagen, in addition to types I and II collagen. Using SDS-PAGE and zymography, the M 4 of both enzymes was 51·10 3. During storage, the 51·10 3 protein converted to fragments of M r 34·10 3 and 24·10 3, and isoelectric points of 4.6–5.3 and 5.7–6.0, respectively. The fragments were separated from the intact ( M r 51·10 3) enzymes by DEAE-Sepharose chromatography, but intact metalloproteinase and collagenase activities resisted separation by a range of chromatographic methods. The M r 34·10 3 fragment retained the proteinolytic activities of the intact enzymes, excepting collagenase cleavage of collagen types I and II. The M r 24·10 3 fragment had no proteinolytic activity, showed an increase in M r of 6·10 3 upon reduction, in common with the intact enzymes, and also had similar chromatographic properties to the intact enzymes. The data presented are consistent with a pattern of breakdown which is common to both collagenase and the metalloproteinase, and suggest that both enzymes are comprised of two protein domains.

Cellular interactions determining the production of collagenase by a rat mammary carcinoma cell line

The cellular interactions regulating the production of collagenase by a cell line derived from a spontaneously arising rat mammary carcinoma have been studied. The cell line, BC1, was grown permanently under defined serum-free conditions, so that the poorly characterized and variable effects of serum on collagenase expression were avoided. Two stable subpopulations of cells present in BC1 cultures were defined as epithelioid cells ("E-cells") and myoepithelioid cells ("M-cells"). These subpopulations differed in their morphology, pattern of growth and susceptibility to detachment from culture vessels by trypsin. Seven clones of M-cells and 7 clones of E-cells, obtained by the limiting dilution technique, were used to determine the cellular source of collagenase and the interactions which led to its expression. M-cells displayed an absolute dependence on a soluble factor produced by E-cells for their survival in vitro. The presence of both cellular types in culture was necessary for collagenase secretion to occur, E-cells being the major source of enzyme in mixed cultures. A soluble factor produced by M-cells was largely, if not completely, responsible for the induction of collagenase secretion by E-cells. Clones representative of both subpopulations were tumorigenic in syngeneic host animals. These results suggest that the phenotypic diversity which occurs within populations of neoplastic cells may give rise to subpopulations of cells which display a more aggressive phenotype in coexistence than in isolation.

Phosphatidylethanolamine biosynthesis in rat mammary carcinoma cells that require and do not require ethanolamine for proliferation.

Epithelial cells and some of their transformed derivatives require ethanolamine to grow normally in defined culture medium. When these cells are cultured without ethanolamine, the amount of cellular phosphatidylethanolamine is considerably reduced. Using a set of rat mammary carcinoma cell lines whose growth is responsive (64-24 cells) and not responsive (22-1 cells) to ethanolamine, the biochemical mechanism of ethanolamine responsiveness was investigated. The biosynthesis and metabolism of phospholipid, particularly of those involving phosphatidylethanolamine, were thus compared between the two types of cells. The incorporation of [3H]serine into phosphatidylserine and phosphatidylethanolamine in 64-24 cells was 60 and 37%, respectively, of those in 22-1 cells. However, the activity of phosphatidylserine decarboxylase was virtually the same in these cell lines. When these cells were cultured in the presence of [32P]phosphatidylcholine and [32P]phosphatidylethanolamine, the rate of accumulation of 32P-labeled phosphatidylserine from the radioactive phosphatidylethanolamine was considerably reduced in 64-24 cells compared to that in 22-1 cells, although the rate of synthesis of phosphatidylserine and phosphatidylethanolamine from the radioactive phosphatidylcholine was similar between the two cell lines. The rate of labeling phosphatidylcholine from the radioactive phosphatidylethanolamine was also reduced in 64-24 cells, although the difference was not as great as that of phosphatidylserine. Incorporation of 32P into phosphatidylethanolamine was correlated with the concentration of ethanolamine in the culture medium in 64-24 cells, whereas in 22-1 cells the incorporation was not influenced by ethanolamine. Enzyme activities of the CDP-ethanolamine pathway were not significantly different between the two cell lines. The rate of degradation of phosphatidylethanolamine was also similar in these cell lines. These results show that ethanolamine responsiveness of 64-24 cells, and probably other epithelial cells, is due to a limited ability to synthesize phosphatidylserine resulting from a limited base-exchange activity utilizing phosphatidylethanolamine.

Inverse correlation between the metastatic capacity of cell clones derived from a rat mammary carcinoma and their intercellular communication with normal fibroblasts.

We used three highly and two weakly metastatic clones obtained from a rat mammary carcinoma cell line (c-SST-2) to examine the relationship between the metastatic capacity of tumor cells and their intercellular communication with normal fibroblasts. By employing the dye-transfer method, we found that the frequency of intercellular communication between weakly metastatic clone cells and fibroblasts was significantly higher than that between highly metastatic clone cells and fibroblasts. These results suggest that normal fibroblasts may regulate the metastatic capacity of tumor cells by intercellular communication.

A comparative analysis of drug-induced DNA effects in a nitrogen mustard resistant cell line expressing sensitivity to nitrosoureas

In the Walker 256 rat mammary carcinoma cell line, WR, resistance to nitrogen mustards (NM) is accompanied by collateral sensitivity to chloroethylnitrosoureas (CENUs). DNA-interstrand cross-links, DNA-protein cross-links, and sister chromatid exchange (SCE) induction were assayed in WR and the parent cell line (WS) after treatment with nitrogen mustard (HN2), phosphoramide mustard (PM), chlorozotocin (CLZ) and l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU). Treatment of cells with HN2 caused extensive levels of cross-links, approximately 50% of which were DNA-interstrand, equal in both WR and WS, whereas PM caused no detectable cross-links in either cell line. CLZ induced low levels of DNA-interstrand cross-links, similar in WR and WS, but no DNA-interstrand cross-links could be detected in either cell line after treatment with CCNU. Both CLZ and CCNU induced low levels of DNA-protein cross-links in both cell lines, though higher in WR than WS. There was no difference in the rate of removal of HN2-induced DNA-interstrand or DNA-protein cross-links or total CLZ-induced cross-links by the two cell lines, suggesting that differential repair was not relevant to the expression of resistance. Both HN2 and PM caused more SCEs in WS than in WR, whereas CLZ and CCNU induced more SCEs in WR. Thus, NM-induced SCEs were related to cell killing but not cross-linking, whilst CENU-induced SCEs were related to cell killing and DNA-protein but not DNA-interstrand cross-links. Furthermore, the collateral sensitivity of WR cells to CENUs was not due to the differential induction of DNA-interstrand cross-links or repair of total cross-links, although higher levels of DNA-protein cross-links occurred in WR, and these may be either a cause or a consequence of increased susceptibility of these cells to CENUs. Presumably NMs and CENUs have several distinct and separate macromolecular targets which result in differential cell killing. It is concluded that a range of lesions occurred after treatment of WR and WS cells with either NMs or CENUs and that, in these cell lines, there is no simple correlation between drug-induced cross-linking, SCE induction and cytotoxicity.

Modulation of growth, prostaglandin synthesis, and prolactin-binding in two cultured rat mammary carcinoma cell lines by flurbiprofen

The effect of treatment with flurbiprofen (FB), a nonsteroidal anti-inflammatory drug, on growth, prostaglandin synthesis, and prolactin receptor levels was examined in two established rat mammary carcinoma cell lines. Growth of NMU cells was suppressed with a concentration of 1 ug FB/mlculture medium (4 × 10 −5 M); RBA cells, in contrast, were less sensitive, being inhibited only by a 100 μg/ml (4 × 10 −4M) concentration of the drug. Prostaglandin (PG) synthesis by both cell lines, as indicated by decreased release of PGE 2 and PGF 2α into the culture medium, was inhibited by 0.1, 1 and l0μg/ml of FB. Both carcinoma cell lines exhibited high levels of specific prolactin receptors (PRLR) (9–11,000 sites/cell); binding was diminished in cells exposed to 1 μg/ml (4 × 10 −6 M), and abolished completely by 10 μg/ml (4 × 10 −5 M) of FB. In marked contrast to the results at higher concentrations, at 0.1 ug/ml (4× 10 −7 M), the drug caused a significant increase in the prolactin binding capacity of RBA cells and a diminution in PG production, but in the absence of any measurable effect on cell proliferation. A similar, but less pronounced trend was seen in the NMU cell line. When NMU cells were cultured in the presence of 10 μ/ml FB for 4 days, and then in inhibitor-free medium for a further 3 days, recovery of growth was demonstrated, together with the reappearance of prolactin-binding capacity. The effect of FB on RBA cell PRLR expression was also reversible, though concommitant changes in cell growth were less obvious. Hence, the inhibitory effect of FB on PG synthesis, and the associated decrease in prolactin binding capacity, was specific and reversible and not the result of a generalized toxic effect.

Establishment of two nonmetastasizing and one metastasizing rat mammary carcinoma cell lines.

Two continuous rat mammary tumor cell lines have been established in culture from the lymphogenously metastasizing rat mammary carcinoma TMT-081 and one from the nonmetastasizing MT-100 and some of their in vitro and in vivo characteristics studied. Cell line TMT-081-MS was established as a free-floating cell suspension from the metastasis-free spleen of a rat bearing TMT-081 in the ascites form and is characterized by a high level of mammary tissue specific antigen (MTA), an antigen present on lactating or hormonally stimulated rat mammary tissues but not detected on normal mammary tissue. This line metastasizes in the syngeneic host but is rejected by the nude mouse without metastases. Cell line TMT-081-NM is a line derived from the ascites of a rat also bearing TMT-081 ascites. Cell line MT-100-TC is a line derived from the ascites of a rat bearing the ascites form of MT-100. Neither TMT-081-NM nor MT-100-TC has ever shown metastases in the syngeneic host but they are lethal; in the nude mice they grow rapidly, are lethal, and sometimes show hematogenous metastases. Both grow in small clusters and show a low level of MTA. These cell lines have been in continuous culture for a year and have proliferated and maintained their individual in vitro and in vivo growth characteristics during more than 100 consecutive subcultivations.

Phosphatidylethanolamine synthesis in ethanolamine-responsive and -nonresponsive cells in culture.

Mammalian cells can be classified into two types based upon whether or not they show growth response to ethanolamine (Etn) in culture. The content of phosphatidylethanolamine (PE) in phospholipid and incorporation of radioactive Etn into the cells were examined in the Etn-responsive and -nonresponsive cells in order to elucidate the mechanisms of growth stimulation by Etn. In all Etn-responsive cells tested, 5 microM Etn significantly altered the composition of cellular phospholipid compared to that grown without Etn, while Etn-nonresponsive cells had a similar phospholipid composition whether the growth medium contained Etn or not. Using two rat mammary carcinoma cell lines, 64-24 (responsive type) and 22-1 (nonresponsive type), further studies were carried out. In 64-24 cells there was a proportional increase in PE content as the dosage of Etn in the medium was increased. The increase in PE content leveled off at 10 microM. Further, the increase in PE content was correlated with increased rate of growth. In contrast, PE content or growth rate did not change at all in 22-1 cells. In 64-24 cells radioactive Etn (0.1-50 microM) was incorporated four- to five-fold more efficiently into phospholipid, and the aqueous pool of precursors of PE was ten times less as compared to 22-1 cells, indicating that Etn-responsive cells utilize Etn supplied in the medium to synthesize PE far more efficiently than Etn-nonresponsive cells. De novo synthesis of PE must not be sufficient to support optimum growth in Etn-responsive cells.

Sera from irradiated rats contain antibodies to a ubiquitous tumour-associated antigen

Female rats of the inbred strains BN/BiRij and WAG/Rij were irradiated with 300 kV X-rays, or 15 MeV or 0.5 MeV fast neutrons. Sera were collected several months after irradiation and found to be negative for antibodies reacting with the murine mammary tumour virus as tested by a solid-phase radioimmunoassay and an immunofluorescence absorption test. We found, however, that in an immunofluorescence assay several sera from irradiated rats reacted with a cytoplasmic antigen in rat mammary, ureter and skin carcinoma cell lines as well as a mouse mammary tumour and a transformed BALB/3T3 mouse fibroblast line. No reaction was found with normal fibroblast cell lines of rat or murine origin. Endpoint titres of the sera on tumour cells ranged from 1:20 to 1:160. Of 48 sera from unirradiated rats 18 also stained tumour cells, but usually at the low dilution of 1:10. Irradiation seems to enhance antibody activity to a ubiquitous tumour-associated antigen.

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.

Phosphoethanolamine as a growth factor of a mammary carcinoma cell line of rat.

We have identified phosphoethanolamine as the pituitary-derived growth-promoting material that specifically stimulates the rat mammary carcinoma cell line 64-24. We have been studying the growth characteristics of the 64-24 cell line, which was isolated from a highly hormone-dependent tumor and which retains in culture many characteristics of the original tumor. Previously, crude bovine pituitary extract was shown to contain a significant amount of growth-stimulating activity for these cells, and a growth factor from this extract was purified to homogeneity. This report describes the identification of the growth factor as phosphoethanolamine. Further, the biological activity of phosphoethanolamine was found to be virtually identical to that of the purified growth factor. A possible role of phosphoethanolamine in the growth of mammary tumor cells as well as of normal mammary epithelial cells and other tissues is discussed.

Gangliosides of cultured cells of a rat mammary carcinoma cell line.

Ganglioside content of rat mammary carcinoma-derived cells grown in layers in vitro was nearly as high as that of apical membrane-derived milk fat globule membrane and nearly four time higher than the content of normal, lactating mammary tissue on a protein basis. The major ganglioside of these carcinoma-derived cells was identified as GDla (sialic acid-Gal-GalNAc-sialic acid)-Gal-Glc-Cer. Relative to carcinoma-derived cells, rat mammary tissue and milk fat globule membrane had more complex ganglioside patterns but appeared to lack substantial quantities of GDla.

Growth stimulating activity in bovine pituitary extract specific for a rat mammary carcinoma cell line.

We report the isolation of a bovine pituitary growth factor (MGF) for a rat mammary carcinoma cell line, 64-24, which was isolated from a highly hormone-dependent mammary tumor. The MGF has been partially purified by a series of Diaflo ultrafiltration membrane sievings, isoelectric focusing and Sephadex columns. The MGF has a molecular weight of approximately 1,000 to 2,000 daltons and has a U.V. absorption spectrum typical for a polypeptide. Its isoelectric point is approximately pH 3.8-4.0. The factor is heat stable. The growth stimulating activity of the MGF does not stimulate other rat cell lines (22-1, RMG or HTC lines) but is specific for the 64-24 mammary tumor cell line. The MGF is not among previously reported pituitary hormones or growth factors.