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Biomedical subjects

M E Monaco

Publications and source records attributed to M E Monaco.

At least 37 records · Page 2Linked to original sources
PubMed1986

The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell.

We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1-phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone-sensitive PI is likewise hormone-sensitive, while that synthesized from hormone-insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin.

Animals↗
PubMed1986

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.

Antibodies↗
PubMed1983

Characterization of the hormone-sensitive phosphatidylinositol pool in WRK-1 cells.

WRK-1 rat mammary tumor cells respond to vasopressin with an increase in the rate of phosphatidylinositol turnover. Evidence derived from a series of experiments performed under various prelabeling conditions suggests that the hormone-sensitive phosphatidylinositol resides in a distinct pool within the cell, accounting for approximately 17% (8-37%) of the total cellular phosphatidylinositol. The possibility that two distinct cell types might explain this finding is unlikely since neither newly cloned nor thymidine-blocked cells exhibit any alteration in the nature of their response. This hormone-sensitive phosphatidylinositol moiety has the following characteristics. 1) Under equilibrium labeling conditions, it is completely turned over within 5 min of hormone addition. 2) It is both synthesized and degraded even in the absence of hormone, although at a much slower rate. 3) Under the conditions employed, there does not appear to be transfer of phosphatidylinositol from the insensitive to the sensitive pool. A model of these events is outlined.

Animals↗
PubMed1983

Characterization of insulin regulation of lipid synthesis in MCF-7 human breast cancer cells.

Stimulation of lipid synthesis by insulin in MCF-7 human breast cancer cells is characterized by an increase in acetate incorporation into long-chain fatty acids. The effects occurs in the absence of an increase in glucose uptake by the cells, and cannot be explained by a decrease in turnover of cellular fatty acids. Differential substrate experiments as well as direct measurement of enzyme activities indicate that insulin stimulates increases in activity of the first enzyme of the de novo pathway, acetyl CoA carboxylase. [32Pi] incorporation into phospholipids is also stimulated by insulin. Thin layer chromatography reveals five peaks of [32Pi]-labeled phospholipids corresponding in mobility to the following standards: lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. [32Pi] incorporation into each of these peaks is stimulated, although the degree of stimulation varies.

Breast Neoplasms↗
PubMed1982

The phosphatidylinositol cycle in WRK-1 cells. Evidence for a separate, hormone-sensitive phosphatidylinositol pool.

Using the vasopressin-sensitive rat mammary tumor cell line, WRK-1, we examined conditions under which vasopressin was able to stimulate turnover of prelabeled, radioactive phosphatidylinositol. Only cells which had been preincubated with 32Pi in the presence of hormone were able to subsequently respond to the hormone by increased loss of radioactivity from phosphatidylinositol. In addition, in experiments performed with uniformly labeled cells, we estimated that the hormone-sensitive phosphatidylinositol accounted for 17% of the total cellular phosphatidylinositol. Implications of these findings are discussed.

Animals↗
PubMed1982

A new model system for studying the phosphatidylinositol cycle.

An early manifestation of the response of WRK-1 rat mammary tumor cells to vasopressin is an increase in incorporation of (32P)Pi into phospholipids. Incorporation into all classes of phospholipids is stimulated; however, incorporation into phosphatidylinositol (PI) is increased to the greatest degree (3- to 10-fold as compared with 1.3- to 2-fold for the other phospholipids). Furthermore, increased incorporation into PI is accompanied by an increased rate of PI turnover; turnover rates of the other phospholipids are unaffected by vasopressin.

Acetates↗
PubMed1982

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.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1981

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.

Breast Neoplasms↗
PubMed1980

Neurohypophysial-hormone-responsive cell line derived from a dimethylbenzanthracene-induced rat mammary tumour.

WRK-1, a cloned cell line derived from a rat mammary tumour, responds to physiological concentrations of vasopressin and pharmacological concentrations of oxytocin with increased incorporation of [14C]acetate into lipids and increased protein accumulation. The presence of pharmacological concentrations of insulin, which itself is active on the WRK-1 cells, further enhances the effects of the neurohypophysial hormones. Unlike the action of vasopressin on other responsive tissues, the stimulation of acetate incorporation by WRK-1 cells is not observed until 24 h after the addition of the hormone. The lipids synthesized in response to the hormones are predominantly polar lipids, rather than the triclyclycerold characteristic of the differentiated mammary gland. [1-Deaminocysteine, 8-D-arginine] vasopressin, a vasopressin analogue that lacks pressor activity, has no effect on WRK-1 cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1979

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.

Acetates↗
PubMed1978

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.

Androstenedione↗
PubMed1978

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.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1978

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.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1977

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.

Acetates↗
PubMed1977

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.

Acetates↗