Identification of multiple proliferative growth factors in breast cyst fluid.

Gross cystic disease is a common benign breast disease that is associated with a twofold to fourfold increase in breast cancer risk. Both diseases are hormonally induced and may share a common biochemical environment conducive to abnormal proliferative responses. A large collection of breast cyst fluid samples was analyzed for growth factors associated with cell proliferation: epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), platelet-derived growth factor (PDGF), transforming growth factor-alpha (TGF-alpha), and transforming growth factor-beta (TGF-beta). The data demonstrate that significant amounts of proliferative growth factors are present in breast cyst fluid of all cyst subtypes. The presence of IGF-II, PDGF, and TGF-beta in breast cyst fluid was confirmed for the first time. EGF, PDGF, and TGF-beta concentrations in breast cyst fluid were several times greater than reported for serum; IGF-I and IGF-II concentrations were several times lower. In the first 100 samples tested, no TGF-alpha was detected. Only EGF and IGF-II levels demonstrated a consistent correlation with apocrine type 1 cysts. These results demonstrated that effective concentrations of proliferative growth factors are in breast cyst fluid and suggest that adjacent breast tissue may be a probable source of synthesis. Growth factor profiles of breast cyst fluid may indicate the presence in breast tissue of a hormonal and proliferative environment permissive to subsequent cancer growth.

Specific growth factor activity identifies and predicts murine mammary tumor.

In milk from substrains of mice with varying incidences of developing mammary tumor, we have isolated a specific mitogenic activity, which is unique in that it identifies mice with mammary tumor and predicts those mice that will eventually develop mammary tumor. None of the milk samples from control mice, who never developed mammary tumor, contained this specific predictive mitogenic activity. Chemical characterization has shown this specific mitogenic activity to be acid- and heat-stable and resistant to reducing agents. Partial purification, by ion-exchange, high-performance liquid chromatography size-exclusion, and isoelectrofocusing techniques, of this specific mitogenic activity from milk of mice that had or eventually developed mammary tumor identifies several peptide growth factor components in a 6-10 kDa molecular weight range. Of known growth factors, radioassay techniques identify an insulin-like growth factor-1-like peptide as a major component. Small amounts of platelet-derived growth factor and transforming growth factor-beta activities also were present. Our results suggest that a subset of growth factors that are diagnostic of the presence of murine mammary tumor and predictive of eventual tumor development may be early indicators of the transition of a competent cell to a progressively malignant state. Similar studies of a secreted body fluid from women at risk for breast cancer may lead to the identification of a specific biologic tumor marker for breast cancer.

Regulation of galactokinase gene expression in Tetrahymena thermophila. I. Intracellular catecholamine control of galactokinase expression.

The addition of glucose to the medium of Tetrahymena thermophila results in a 7-fold repression of galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase). The presence of millimolar amounts of the catecholamines dopa, dopamine, norepinephrine, and epinephrine or the hormone glucagon also results in the repression of galactokinase in the absence of glucose. The addition of millimolar amounts of adrenergic agonists (isoproterenol, tyramine, 2-amino-6,7-dihydroxytetrahydronaphthalene) results in significant repression of galactokinase in the absence of glucose; concentrations of 2-amino-6,7-dihydroxytetrahydronaphthalene less than or equal to 10(-4) M result in a derepression of galactokinase specific activity. Addition of adrenergic antagonists (propranolol, dichloroisoproterenol) have no effect on galactokinase activity at concentrations less than 10(-4) M but do arrest cell growth at greater concentrations. The addition of the cAMP analogs caffeine or theophylline in millimolar amounts results in repression of galactokinase activity; however, cell growth is greatly slowed or completely arrested at these concentrations. Analysis of the repression response of several mutants demonstrates that mutants deficient in catecholamine biosynthesis are altered in their regulation of galactokinase. Measurements of intracellular cAMP levels for 0-24 h following the addition of several of the above compounds to exponentially growing cells did not demonstrate any change over this period. Measurement of intracellular cAMP levels for 24 h following the addition of glucose or galactose to exponentially growing wild-type and mutant cell strains did not demonstrate any difference in cAMP concentrations over this period although a wide range of galactokinase activity was exhibited. Starvation of wild-type cells prior to the addition of glucose in minimal medium without added carbohydrate resulted in a significant increase in cAMP following the addition of glucose. This increase is demonstrated to be dependent upon the ability of the cells to resume division after the arrest of growth and is not correlated with galactokinase regulation. These results support the conclusion that cAMP is not involved in the repression of galactokinase gene expression initiated by glucose or hormone-like effectors and demonstrate the participation of an adrenergic control system in galactokinase regulation which is subordinate to the regulation by glucose. A possible model is discussed.

Regulation of galactokinase gene expression in Tetrahymena thermophila. II. Identification of 3,4-dihydroxyphenylalanine as a primary effector of adrenergic control of galactokinase expression.

Intracellular concentrations of catecholamines were determined in wild-type and mutant Tetrahymena thermophila, using the highly sensitive techniques of high-performance liquid chromatography and electro-chemical detection. Catecholamines were determined in these cell strains grown under various steady-state conditions, including those which initiate and maintain repression of galactokinase gene expression. Wild-type cells grown in defined minimal medium supplemented with 1% glycerol, exhibiting derepressed galactokinase synthesis, were found to contain considerable quantities of dopa (3,4-dihydroxyphenylalanine) and dopamine, but no detectable levels of either norepinephrine or epinephrine. Analyses of wild-type cells revealed a strong positive correlation between the internal concentration of dopa and expression of the galactokinase gene, both of which are regulated by exogenous carbohydrates, catecholamine agonists, or dibutyryl-cAMP; an analogous relationship between intracellular dopamine concentrations and galactokinase activity was not found. In addition, a correlation between intracellular dopa content and the phenotypic expression of galactokinase in various mutants deficient in the catecholamine biosynthetic pathway or in glucokinase further confirms the role of dopa as a primary effector in the regulation of galactokinase gene expression.