Effect of serum on cells cultured from human mammary tumors

Summary Clusters of cells derived from biopsy specimens of human mammary ductal carcinomas form two morphologically distinct epithelial colonies in culture, designated as E and E′. The proportion of E′ cell clusters that attached and formed colonies ranged from 0.3 to 13.0% with different tumors. Attachment was independent of tumor grade. Microscopic observations revealed that the survival of E′ cell colonies was limited to approximately 10 days with rapid cell degeneration commencing about 7 days. A comparison of sera showed that colony formation by cells from malignant tumors during the 1st week of culture was maximum in the presence of fetal bovine serum. Human serum alone was 70 to 100% less effective in promoting E′ colonies. The most significant finding was that human serum from normal donors inhibited E′ colony development in the presence of FBS. Although human serum was less effective than FBS in promoting colony formation by clusters of E cells, an inhibition was not observed. Inhibitory activity could not be attributed to either antagonistic hormones or the source of human serum. THese results demonstrate that normal human serum contains a factor(s) that exhibits an inhibitory activity specific for human epithelial cells (E′) derived from malignant tumors. Supported by NCI Contract CB-33898 and a Fellowship from the Imperial Cancer Research Fund, London, England.     

Prolactin binding to dissociated cells from rat mammary tumors and mammary gland.

Prolactin binding activity was studied in suspensions of cells which had been enzymatically dissociated from R3230AC mammary tumors, 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors and lactating rat mammary glands. Prolactin bound specifically with high affinity (apparent binding affinity = 4.0 X 10(9) M-1) to R3230AC tumor cells. Hormone binding at room temperature was proportional to cell number and increased with time of incubation up to 120-180 min. Prolactin binding to R3230AC tumor cells from diabetic animals was reduced by about 50%. Specific prolactin binding activity was also demonstrated in preparations of cells from DMBA-induced tumors and lactating mammary gland. The levels of hormone binding in both dissociated cells and subcellular particles prepared from these tissues varied as follows: DMBA-induced tumors > lactating mammary gland > R3230AC mammary adenocarcinoma.     

Effect of serum on phenylalanine hydroxylase levels in cultured hepatoma cells.

Continued high levels of phenylalanine hydroxylase in cultured H4-II-E-C3 rat hepatoma cells require either serum or glucocorticoids in the culture medium. Upon withdrawal of serum, cellular phenylalanine hydroxylase levels decay exponentially with a half-life of 22 hours for about 60 hours, after which time a low, constant enzyme content persists for at least 96 hours. This decline of phenylalanine hydroxylase is fully reversible; normal enzyme levels are restored in a time- and dosage-dependent fashion upon addition of serum to basal cultures. The serum factor is nondialyzable and moderately heat-stable. The stimulation by serum of the phenylalanine hydroxylas content of basal cultures is blocked by 3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide and requires ongoing cellular protein synthesis. When added to the enzyme-assay mixture in vitro, serum does not alter the phenylalanine hydroxylase activity of extracts from basal cultures. Three lines of evidence suggest that serum contains a nonsteroidal phenylalanine hydroxylase stimulatory components(s): (a) glucocorticoid antagonists inhibit less than one-half of the biological activity of serum; (b) exhaustive extraction of endogenous serum glucocorticoids with charcoal reduces the activity of serum to about one-half of control values; and (c) the stimulatory effects of charcoal reduces the values; and (c) the stimulatory effects of charcoal-extracted serum and hydrocortisone are additive. The phenylalanine hydroxylase stimulatory activities of the charcoal-extracted sera from four mammalian species and from three stages in development in one mammalian species are comparable. A survey of partially purified preparations of a number of known hormones failed to reveal any one capable of elevating the phenylalanine hydroxylas levels of basal cultures in a manner comparable to that of charcoal-extracted serum.     

Regulation of vimentin expression in cultured human mammary epithelial cells

Using five different monoclonal antibodies to vimentin, we have examined the expression of vimentin in cryostat sections and serum-free cultures of normal human breast tissue. In cryostat sections, myoepithelial cells as well as stromal cells showed immunoreactivity to vimentin, irrespective of the antibody used. In contrast, luminal epithelial cells were negative for vimentin, but positive for keratin K18. In culture, myoepithelial cells showed immunoreactivity to vimentin from their first appearance in monolayer. Moreover, a fraction of luminal epithelial cells expressed vimentin in addition to keratin K18. We found a clear, reversible correlation between proliferation, determined by incorporation of [3H]-TdR, and induction of vimentin in the luminal epithelial cells. Thus, in growth-stimulated cultures on a medium containing cholera toxin (CT), epidermal growth factor (EGF), transferrin (Tf), hydrocortisone (H) and insulin (I), the fraction of vimentin-positive luminal epithelial cells increased, while it decreased within 14 days from approximately 36% to 3% on a medium containing CT and EGF, only. We therefore conclude: (1) vimentin is constantly expressed in myoepithelial cells in situ and in vitro, and (2) expression of vimentin in luminal epithelial cells in vitro is not a result of monolayer cultivation as such, but rather associated with the increased growth rate seen in culture.     

Effect of hormones on primary cell cultures from pregnancy-dependent mouse mammary tumors.

No hormone combination was successful in maintaining long term primary cultures of pregnancy-dependent mammary tumors. Insulin provided the most consistent dose dependent stimulatory effect on short term proliferation as measured by 3H-TdR incorporation into DNA. Insulin in combination with corticosterone and prolactin produced the greatest stimulatory effect in most tumors. Both insulin at 5 microgram/ml and insulin, prolactin and hydrocortisone induced a partially synchronous wave of DNA synthesis in restimulated cultures. The time course of the wave of DNA synthesis was different for different hormone treatments. Insulin as 5 microgram/ml caused an earlier wave of DNA synthesis than insulin, prolactin plus corticosterone.     

Biological characteristics of cultured cells derived from various types of human brain tumors

We placed in culture brain tumors from 45 cases (7 cases of astrocytoma, 2 from oligodendrogliomas, 2 glioblastomas, 2 ependymomas, 13 meningiomas, 6 pituitary adenomas, 5 neurinomas, a malignant lymphoma, a choroid plexus papilloma, and 6 metastatic tumors) and succeeded in making a primary culture from 33, and maintained 17 in vitro over a considerable period of time (greater than three months). In the early period of the primary cultures, the astrocytoma cells had cytoplasmic processes which contacted each other, the oligodendroglioma cells were small and spindle-shaped, the glioblastoma cells were neoplastic with pleopmorphic features and possessed cytoplasmic processes, the ependymoma cells formed a rosette-like cell arrangement, the meningioma cells were spindle- or round-shaped cells and characterized as forming psammoma bodies, the pituitary adenoma cells were round- or oval-shaped cells and produced growth hormone (GH), adenocorticoid tropic hormone (ACTH), prolactin, or other hypophyseal hormones, the choroid plexus papilloma cells were round-or polygonal and showed a papillary cell arrangement, the neurinoma cells were spindle- or fibrous-shaped cells, and the malignant lymphoma cells were round and formed cell aggregates floating in the culture medium.     

Origin,concentration and structural features of human mammary gland cells cultured from breast secretions

Summary This study traced the origin of cells observed in human breast secretion samples obtained during lactation and describes the appearance of these cells following prolonged maintenance in vitro. Human milk contains a large number of single vacuolated foam cells and a small proportion of non-vacuolated epithelial cells in clusters. Foam cells are identified by their large size, the polarity of their cytoplasmic organelles, the variation in number and size of lipid vacuoles and the condensed chromatin of their eccentrically located nucleus. Both cell types originate by exfoliation from the mammary gland. This was established by comparing the structural characteristics of cells isolated from milk with those of the cuboidal cell linings of ducts and alveoli in lactating mammary tissue. Relatively pure populations of foam cells could be established from early lactation samples (3–7 days post/partum) while non-vacuolated epithelial cell clusters were more frequently cultured from late lactation specimens (1–10 days postweaning). Foam cells did not divide and lost cytoplasmic organization during prolonged culture. In contrast, non-vacuolated epithelium in clusters proliferated to form colonies of polygonal cells. These results, which imply that foam cells are an active form of the non-vacuolated mammary cells in clusters, call attention to one system for the study of the complex hormonal interactions necessary to induce and maintain lactation.Supported in part by NCI contract NO 1-CB-33898     

Response of cultured normal human mammary epithelial cells to X rays

The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique.     

BIOCOCKTAIL对乳腺癌细胞MDA-MB-231凋亡的促进作用

目的从细胞凋亡方面观察BIOCOCKTAIL（益生菌微生物制剂）对乳腺癌细胞MDA-MB-231的抑制作用。方法采用FITC和PI双染色法及TUNEL法检测凋亡。结果通过FITC和PI双染色法,观察到BIOCOCKTAIL可显著增加乳腺癌细胞MDA-MB一231凋亡细胞。I组乳腺癌细胞系（8．1％绑38．7％,P〈0．05）,Ⅱ组乳腺癌细胞系（6．9％掷19．4％,P〈0．05）。TUNEL法检测MDA-MB-231细胞凋亡现象明显增多。结论BIOCOCKTAIL能够有效诱导乳腺癌细胞MDA-MB-231凋亡,有可能成为乳腺癌治疗的补充方案。     

Effect of hormones on cyclic AMP levels in cultured human cells.

Cultured cells derived from human adipose tissue grew more slowly and had significantly higher basal levels of cyclic AMP than cultured fibroblasts. Cyclic AMP levels in cultured adipose tissue cells were unaffected by epinephrine and were elevated 15-fold by prostaglandin E₁ while fibroblast cyclic AMP levels were elevated 27-fold by epinephrine and 95-fold by prostaglandin E₁. These results support the postulate that the cultured adipose tissue cell is a distinct cell type which may represent an adipocyte or preadipocyte in culture.     

In situ lymphoid cells of mouse mammary tumors. I. Development and evaluation of a method for the separation of lymphoid cells from mouse mammary tumors

A method of separating lymphoid cells from solid mouse mammary tumors was developed and evaluated. In this method the tumors are digested with 0.01% collagenase, 0.01% DNAase, and 0.025% trypsin in Dulbecco's PBS into suspensions of cells with a viability of 90%. The suspensions are fractionated on a continuous gradient of Ficoll in tissue culture medium. In model experiments this gradient was found to separate, cleanly, admixed cells of an established mammary tumor cell line and dissociated thymus glands. Recovery rates were 50% for the tumor cells and 80% for the thymocytes. The preparation of the cell suspensions and the gradient separation procedure are not harmful to the cells as indicated by trypan blue exclusion and the ability to grow in cell culture.     

Effect of melanins from black yeast fungi on cultured human cells. II. Differentiation of keratinocytes in vitro

Data on the influence of the black yeast melanin (3 samples) on the in vitro differentiation of human keratinocytes are presented. The effect of melanins was estimated by the morphological state of keratinocytes using electron microscopy. The obtained differences in the state of the formed multilayer keratinocyte sheets depended on the melanin sample.     

Effect of melanins from black yeast fungi on cultured human cells. I. Proliferation of keratinocytes and fibroblasts

Results of screening of the influence exerted by yeast black melanin on the proliferation of human skin keratinocytes and embryonic fibroblasts are presented. The optimal concentration of the investigated melanins was found to be within 0.005 and 0.0001 mg/ml. 17 samples of DHN-melanin from black yeast and 2 commercial samples of [symbol: see text]OPA-melanin (natural and synthetic) were investigated. It was established that keratinocyte proliferation was inhibited by 3 black yeast melanin samples; the influence of other 14 samples was the same as in the control. Keratinocyte proliferation was stimulated only by a commercial sample of natural [symbol: see text]OPA-melanin at concentration 0.005 mg/ml. The synthetic melanin at concentrations 0.005 and 0.001 mg/ml inhibited keratinocyte proliferation. Of the 17 investigated black yeast melanin samples, only one sample stimulated fibroblast proliferation at concentration 0.005 mg/ml. Three other samples inhibited the proliferation; of these one sample did it at all used concentrations, and two samples at concentration 0.0001 mg/ml. The rest 13 samples of black yeast DHN-melanins and the synthetic [symbol: see text]OPA-melanin did not differ in either action from the control.     

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.     

Purification of a mammary-derived growth factor from human milk and human mammary tumors

A growth factor, mammary-derived growth factor 1 (MDGF1), has been purified to apparent homogeneity from human milk. The factor is a pepsin-sensitive, reducing agent-insensitive protein with a molecular mass of 62 kDa and a pI of 4.8. An apparently identical factor has been isolated from human mammary tumors, suggesting that MDGF1 might be made by and act as an autocrine growth factor for mammary cells. High affinity receptors for MDGF1 have been detected on mouse mammary cells, normal rat kidney cells, and A431 epidermoid cells (KD = 2 X 10(-10) M). MDGF1 at picomolar levels stimulates the growth of mammary cells and greatly amplifies their production of collagen, apparently via elevating collagen mRNA levels, an effect that is demonstrated for normal rat kidney cells. The responsiveness of mammary cells to MDGF1 is attenuated when the cells are grown on a basement membrane collagen substratum, a component of the extracellular matrix upon which these cells normally rest in vivo. MDGF1 thus may regulate the production of new basement membrane as mammary epithelium invades the stroma during proliferation.     

Immunogenicity of human B cell antigens solubilized from cultured human lymphoid cells.

Antigens solubilized from culured human lymphoid cells WI-L2 and RPMI 1788 were partially purified by ultracentrifugation on a KBr gradient. These antigens injected into rabbits produced xenoantibodies which after absorption with melanoma cells became specific to B cell antigens. Three such xenoantisera were submitted to the Second Histocompatibility Workshop of the Americas and reacted much like alloantisera to B cell antigens against a large panel of B peripheral lymphocytes and cells from patients with chronic lymphocytic leukemia. Xenoantisera to B cell antigens inhibited the mixed lymphocyte reaction, but did not affect the mitogenic activity of phytohemagglutinin or the functional properties of C3 receptors, monkey red blood cell receptors, or T cell receptors.     

Non-random association of mitotic cells from cultured human lymphocyte suspensions.

By using chromosome markers in two separate cell lines of a human dispermic chimera, it was shown that 4.9% of metaphases in suspensions of PHA-cultured lymphocytes were paired because of non-random factors. A similar amount of metaphase pairing occurred in cultured lymphocytes of normal donors, and evidence from the relative mitotic cycles of the paired cells indicated that some of this pairing was non-random. Such non-random pairing could be a source of bias in cell kinetic and other studies involving metaphase cells in lymphocyte cultures.     

Protein extracts from cultured cells contain nonspecific serum albumin

Serum is an important component of cell culture media. The present study demonstrates contamination of intracellular protein extract by bovine serum albumin from the culture media and illustrates how this contamination can cause the misinterpretation of western blot results. Preliminary experiments can prevent the misinterpretation of some experimental results, and optimization of the washing process may enable specific protein detection.