Insulin stimulation of fatty acid synthesis in human breast cancer cells.

Four human breast cancer cell lines were tested for their response to hormones with respect to the lactational function, fatty acid synthesis. Physiologic concentrations of insulin enhanced the incorporation of [14C]acetate into fatty acids in two of four cell lines tested. All cell lines had specific, high-affinity insulin receptors; therefore, the failure of two lines to respond could not be attributed to the absence of receptor. The effect of insulin involved an increase in the maximun velocity of incorporation rather than a decrease in the Michaelis constant.

Vasopressin: action on WRK-1 rat mammary tumor cells.

WRK-1, a cell line in long-term culture derived from a 7, 12-dimethylbenz[a]anthracene-induced rat mammary tumor, responds to physiologic concentrations of vasopressin with increased precursor incorporation into phospholipids and with increased protein accumulation. Because vasopressin has been reported to be a potent mitogen for Hela cells and 3T3 cells, a study was conducted to determine whether it could act as a mitogen for WRK-1 cells. Under no conditions was a clear-cut mitogen response to vasopressin demonstrated.

Effects of estrone, estradiol, and estriol on hormone-responsive human breast cancer in long-term tissue culture.

The effects of estrone, estradiol, and estriol on MCF-7 human breast cancer are compared. In this estrogen-responsive cell line, all three estrogens are capable of inducing equivalent stimulation of amino acid and nucleoside incorporation. Estriol is capable of partially overcoming antiestrogen inhibition with Tamoxifen (lCl 46474), even when antiestrogen is present in 1000-fold excess. Antiestrogen effects are completely overcome by 100-fold less estriol. Studies of metabolism of estrogens by MCF-7 cells revealed no conversion of estriol to either estrone or estradiol. All three steroids bind to a high-affinity estrogen receptor found in these cells. The apparent dissociation constant is lower for estradiol than for estrone and estriol, but all three bind to an equal number of sites when saturating concentrations are used. Tritiated estrogens used in binding studies were shown to be radiochemically pure. We conclude that estriol can bind to estrogen receptor and stimulate human breast cancer in tissue culture. Our data do not support an antiestrogenic role for estriol in human breast cancer.

Unsaturated fatty acid requirements for growth and survival of a rat mammary tumor cell line.

A cell line, the growth and survival of which is markedly affected by linoleic acid, has been established from a carcinogen-induced rat mammary tumor. The cells have been continuously passaged in 5% rat serum plus 10% fetal calf serum-supplemented medium. The rat serum component was found to be indispensalbe, for when it was omitted the growth rate rapidly declined and the cells died by 5 to 7 days. Removal of the rat serum from the growth medium also resulted in a dramatic loss of Oil Red O-positive droplets in the cells, suggesting that the lipid component of rat serum might be a major growth-promoting principle in rat serum. This is likely since the total lipid fraction, but not the delipidized protein fraction, could largely supplant requirement of the cells for rat serum. Pure linoleic acid was found to be effective in maintaining the cell growth in delipidized serum or in whole fetal calf serum-supplemented medium. Fatty acid analysis revealed a 19-fold higher amount of linoleic acid in rat serum than in fetal calf serum.

Vasopressin stimulation of acetate incorporation into lipids in a dimethylbenz(a)anthracene-induced rat mammary tumor cell line.

In a preliminary report we described the effects of rat prolactin on the incorporation of [14C]acetate into lipids by a cell line from a dimethylbenz(a)anthracene-induced rat mammary tumor. The characteristics of the response to prolactin were very similar to those described for the normal rat mammary gland; namely, insulin was required for full expression of the response, maximal activity was not seen until 36 hr after the addition of the hormones, and growth hormone was able to elicit the same response. However, we were unable to detect binding of 125I-labeled prolactin to these cells, and furthermore, other more purified prolactin preparations were inactive. Upon further investigation we discovered that the activity resided in a low-molecular-weight fraction of the rat prolactin B-1 preparation and was probably either vasopressin or oxytocin or both. These data suggest the possibility that vasopressin may play a role in rodent mammary tumorigenesis.

Direct inhibition of growth and antagonism of insulin action by glucocorticoids in human breast cancer cells in culture.

We have examined the interaction of dexamethasone with the ZR75-1 human breast cancer cell line to determine if glucocorticoids might directly inhibit growth of breast cancer cells. Growth of these cells in serum-free medium was stimulated significantly by physiological concentrations of insulin (0.1 to 1.0 nM). Pharmacological concentrations of dexamethasone (10 nM) reduced cell number below that found in controls and nearly abolished the effect of insulin after several days in culture. Thymidine and uridine, but not leucine, incorporation into macromolecules or acetate incorporation into fatty acids were similarly inhibited by dexamethasone in the presence of absence of insulin. Dexamethasone did not inhibit insulin effects by altering insulin receptor affinity or concentration, as determined by Scatchard analyses of insulin binding. Net thymidine uptake into the trichloroacetic acid-soluble fraction of the cell was stimulated by insulin and inhibited by dexamethasone also inhibited thymidine kinase activity multiple potential sites of glucocorticoid action that directly oppose the effects of insulin. They also suggest that glucocorticoids have a direct inhibitory effect on proliferation of human breast cancer cells, which may help explain breast tumor regression following pharmacological glucocorticoid therapy.

Purification and properties of estrogen-responsive cytoplasmic thymidine kinase from human breast cancer.

The effect of 17 beta-estradiol on cytoplasmic thymidine kinase activity was studied in MCF-7, a human breast cancer cell line in culture which responds to estrogens with an increase in the rate of growth. Levels of 17 beta-estradiol which maximally stimulate [3H]thymidine incorporation into DNA also maximally stimulate thymidine kinase activity. The Vmax for thymidine increased while the Km was not affected by estrogen stimulation when performed on nonpurified enzyme. Tamoxifen, an antiestrogen, decreased the specific activity of the enzyme. To further study its hormonal regulation, cytoplasmic thymidine kinase was purified greater than 2000-fold by affinity column chromatography. The purified preparation migrated in one band to a pI of 8.5 on an isoelectric focusing gel. The purified thymidine kinase was further characterized by examining its molecular weight, pH optimum, heat stability, utilization of phosphate donors, inhibition by nucleotides, and the effect of pyrimidine nucleoside analogs.

Casein production by human breast cancer.

Casein was measured in the sera of breast cancer patients, in breast cancer tumors, and in breast cancer cells in long-term tissue culture using a sensitive and specific radioimmunoassay. Levels present in breast cancer sera were not elevated above control values. Eight of forty-seven (17%) of the tumor samples tested were positive for casein, the highest level representing 0.003% of the soluble protein. When seven human breast cancer cell lines were assayed for casein, the results were uniformly negative even under conditions of stimulation by lactogenic hormones. In addition, direct immunoprecipitation of labeled cellular protein supported the negative result of the radioimmunoassay. Thus it appears that casein production is not a common characteristic of most human breast cancers.

Characterization of the vasopressin receptor on WRK-1 cells.

Specific vasopressin binding to WRK-1 rat mammary tumor cells was assessed and compared with vasopressin-induced alterations in phosphatidylinositol metabolism. Scatchard analysis revealed the presence of two binding sites: a saturable, high affinity site with a dissociation constant of 1 X 10(-9) M and an n of 2700 sites per cell, and a nonsaturable, apparent lower affinity site. The higher affinity site appeared to have V1a specificity and to correlate with vasopressin's ability to stimulate phosphatidylinositol turnover in the cells.

Insulin stimulation of fatty acid synthesis in human breast cancer in long term tissue culture.

The effect of various steroid and peptide hormones on the rate of incorporation of (14C)acetate into fatty acids was studied in MCF-7 cells, a human breast cancer cell line in continuous tissue culture. Physiologic concentrations of insulin elicited an increase in the rate of precursor incorporation. Addition of human placental lactogen and dexamethasone with insulin caused no further increment. Other hormones, such as 17beta-estradiol and 15a-dihydrotestosterone, had no effect on the rate of net labeled-acetate incorporation, although they stimulated the rate of general macromolecular synthesis. The stimulation by insulin appeared to be independent of glucose concentration, since the effect was seen in the absence of glucose. The insulin-induced increase was observed as early as 1 h following hormone addition and appeared to be maximal by 4 h. Inhibitors of RNA and protein synthesis had no effect on the insulin-mediated stimulation of precursor incorporation into fatty acids. Thus, these human cell lines in long term tissue culture may be of value as a model system in studying the action of physiologic concentrations of insulin.

Mitogenic activation of human prostate-derived fibromuscular stromal cells by bradykinin.

Biologically active kinin peptides are released from precursor kininogens by kallikreins. Kinins act on kinin receptors to mediate diverse biological functions including smooth muscle contraction, inflammation, pain and mitogenicity. All components of the kallikrein-kinin system exist in human male genital secretions suggesting that these molecules participate in physiological and pathophysiological genitourinary function. The objective of this study was to assess the consequences of kinin action on prostate cells. Primary cultures of prostate secretory epithelial (PE) and prostate fibromuscular stromal (PS) cells were established from human prostate tissue. Transcripts encoding both the human B1 and B2 bradykinin receptor subtypes were detected in human prostate transition-zone tissue and in cultured cells by RT-PCR. In receptor binding assays, the B1 subtype predominated on PE cell membranes and the B2 subtype predominated on PS cell membranes. In PS cells, but not in PE cells, BK induced significant inositol phosphate accumulation and [3H]-thymidine uptake. These responses were mediated through the B2 receptor subtype. The use of signal transduction inhibitors indicated that mitogenic activation by BK occurred through both protein kinase C (PKC) and protein tyrosine kinase dependent mechanisms. PMA (phorbol 12-myristate 13-acetate) produced maximal [3H]-thymidine uptake by PS cells, resulted in cell elongation and caused the alpha-actin fibres present in PS smooth muscle cells to became organized into parallel arrays along the length of the elongated cells. In summary, the prostate contains a functional kallikrein-kinin system, which could be significant in physiological and pathophysiological prostate function.

An antibody-toxin conjugate directed against a human mammary cancer antigen.

A conjugate has been constructed consisting of diphtheria toxin fragment A (DTA) linked to a monoclonal antibody, BLMRL-HMFG-Mc5 (MC5), directed against a mammary cancer antigen. The conjugate retains both binding and DTA enzymatic activity when tested against target MCF-7 cells, although the conjugate binds less well than the unconjugated antibody. The conjugate is toxic to MCF-7 cells. Toxicity is both dose- and time-dependent. Half-maximal toxicity is observed with 2-5 X 10(-8) M conjugate after 5 days of incubation. However, the conjugate need only be in contact with the cells for 1 day in order to effect complete killing by 5 days. The earliest that an effect can be seen on cell growth is 2 days. When tested against WRK-1 rat mammary tumor cells, the conjugate is inactive.

Failure of breast cancer cells in long-term culture to aromatize androstenedione.

The ability of breast cancer cells in long-term tissue culture to aromatize androstenedione to estrone and estradiol was examined. (3H) androstenedione (1.1 x 10(-7)M) was incubated with 9 cell lines of human breast cancer and one line derived from a dimethylbenz(a)anthracene induced rat mammary tumor. No conversion to estrone or estradiol was detected. The findings are discussed in light of previous studies showing aromatization of androgens in breast cancer tissues.

First Report of Colletotrichum gloeosporioides on Strawberry in Northwestern Argentina.

Colletotrichum gloeosporioides was isolated from symptomatic strawberry (Fragaria × ananassa Duch. 'Chandler') growing in Lules (Tucumán, Argentina). Isolates were characterized based on several criteria. Potato dextrose agar (PDA) was used to evaluate cultural and morphological characteristics of the isolates. After 10 days on PDA at 28°C under continuous white light, colonies showed abundant aerial, cottony white to pale beige growth, with orange asexual fruiting bodies in older colonies. Isolates displayed cylindrical conidia, rounded at both ends, averaging 10.4 × 3.9 µm (length by width). A sexual phase (perithecia) was observed in all isolates in 2-month-old cultures on PDA at 28°C under continuous white light. Pathogenicity tests were conducted with healthy plants of cvs. Pájaro and Chandler. Spray inoculation with conidial suspensions (106 conidia per ml) resulted in disease symptoms (petiole and crown lesions with wilting of crown-infected plants) 7 days after inoculation. Infection progressed at a higher rate in Pájaro than in Chandler. Reisolations from infected strawberry lesions yielded isolates with characteristics identical to the isolate used to inoculate the host. Based on morphological and cultural characteristics, isolates were identified as C. gloeosporioides Penz. & Sacc. (teleomorph Glomerella cingulata Spauld & H. Schenk) (1). This is the first report of C. gloeosporioides causing strawberry anthracnose in northwestern Argentina. Reference: (1) P. S. Gunnell and W. D. Gubler. Mycol. 84:157, 1992.

Cell adhesion glycoprotein vitronectin during Xenopus laevis embryogenesis.

Vitronectin (vn) is a cell-adhesive glycoprotein present in blood and extracellular matrix of all vertebrates. In the present study we reported the cDNA cloning of Xenopus laevisvitronectin and its spatial and temporal expression pattern during the embryonic development of this important model organism. The deduced amino acid sequence of Xenopus laevis vn showed 49%, 47% and 43% identity with human, chicken and zebrafish orthologs, respectively, whereas the comparison with Xenopus tropicalis vn presented 85% identity. The structural organization consisting of a somatomedin B domain and two hemopexin-like domains was similar to higher vertebrate vitronectins. The vn transcripts were detected from stage 28 onward. At tadpole stages, vn is expressed in heart, gut derivatives and in the notochord. The protein was detected in heart, liver, foregut, pronephros and notochord at stages 43 and 47 of Xenopus embryos. Our results suggest that vitronectin is developmentally regulated and could participate in embryo organogenesis.