Diphtheria toxin effects on human cells in tissue culture.
HeLa cells exposed to a single sublethal concentration of diphtheria toxin were found to have diminished sensitivity when subsequently reexposed to the toxin. Three cells strains exhibiting toxin resistance were developed. In the cells that had previously been exposed to toxin at 0.015 mug/ml, 50% inhibition of protein synthesis required a toxin concentration of 0.3 mug/ml, which is more than 10 times that required in normal HeLa cells. There appears to be a threshold level of diphtheria toxin action. Concentrations of toxin greater than that required for 50% inhibition of protein synthesis (0.01 mug/ml) are associated with cytotoxicity, whereas those below this concentration may not be lethal. Several established human cell lines of both normal and neoplastic origin were tested for their sensitivity to the effects of the toxin. No special sensitivity was observed with the cells of tumor origin. Fifty % inhibition of protein synthesis of HeLa cells was achieved with diphtheria toxin (0.01 mug/ml) as compared to the normal human cell lines tested (0.03 and 0.5 mug/ml) and a cell line derived from a human pancreatic adenocarcinoma (0.2 mug/ml). A human breast carcinoma cell line showed a maximum of 45% inhibition of protein synthesis. This required a diphtheria toxin concentration of 5 mug/ml. These results suggest that different human cell lines show wide variation in their sensitivity to the toxin. (+info)
The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture.
We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites. (+info)
The effects of glucocorticoids and progesterone on hormone-responsive human breast cancer in long-term tissue culture.
Glucocorticoids, at physiological concentration, inhibit cell division and thymidine incorporation in three lines of human breast cancer maintained in long-term tissue culture. At steroid concentrations sufficient to inhibit thymidine incorporation 50%, little or no effect is seen on protein synthesis 48 hr after hormone addition. All three of these lines are shown to have glucocorticoid receptors demonstrable by competitive protein binding assays. Receptors are extensively characterized in one line by sucrose density gradient analysis and binding specificity studies. Good correlation between receptor-binding specificity and biological activity is found except for progesterone, which binds to glucocorticoid receptor but is noninhibitory. Cross-competition and quantification studies demonstrate a separate receptor for progesterone. This receptor has limited binding specificities restricted largely to progestational agents, whereas the glucocorticoid receptor bound both glucocorticoids and progesterone. Two other human breast cancer lines neither contain glucocorticoid receptor nor are inhibited by glucocorticoids. It is concluded that in some cases glucocorticoids can directly limit growth in human breast cancer in vitro without requiring alterations in other trophic hormones. (+info)
The effects of androgens and antiandrogens on hormone-responsive human breast cancer in long-term tissue culture.
We have examined five human breast cancer cell lines in continuous tissue culture for androgen responsiveness. One of these cell lines shows a 2- to 4-fold stimulation of thymidine incorporation into DNA, apparent as early as 10 hr following androgen addition to cells incubated in serum-free medium. This stimulation is accompanied by an acceleration in cell replication. Antiandrogens [cyproterone acetate (6-chloro-17alpha-acetate-1,2alpha-methylene-4,6-pregnadiene-3,20-dione) and R2956 (17beta-hydroxy-2,2,17alpha-trimethoxyestra-4,9,11-triene-1-one)] inhibit both protein and DNA synthesis below control levels and block androgen-mediated stimulation. Prolonged incubation (greater than 72 hr) in antiandrogen is lethal. The MCF- cell line contains high-affinity receptors for androgenic steroids demonstrable by sucrose density gradients and competitive protein binding analysis. By cross-competition studies, androgen receptors are distinguishable from estrogen receptors also found in this cell line. Concentrations of steroid that saturate androgen receptor sites in vitro are about 1000 times lower than concentrations that maximally stimulate the cells. Changes in quantity and affinity of androgen binding to intact cells at 37 degrees as compared with usual binding techniques using cytosol preparation at 0 degrees do not explain this difference between dissociation of binding and effect. However, this difference can be explained by conversion of [3H]-5alpha-dihydrotestosterone to 5alpha-androstanediol and more polar metabolites at 37 degrees. An examination of incubation media, cytoplasmic extracts and crude nuclear pellets reveals probable conversion of [3H]testosterone to [3H]-5alpha-dihydrotestosterone. Our data provide compelling evidence that some human breast cancer, at least in vitro, may be androgen dependent. (+info)
Activation of Src in human breast tumor cell lines: elevated levels of phosphotyrosine phosphatase activity that preferentially recognizes the Src carboxy terminal negative regulatory tyrosine 530.
Elevated levels of Src kinase activity have been reported in a number of human cancers, including colon and breast cancer. We have analysed four human breast tumor cell lines that exhibit high levels of Src kinase activity, and have determined that these cell lines also exhibit a high level of a phosphotyrosine phosphatase activity that recognizes the Src carboxy-terminal P-Tyr530 negative regulatory site. Total Src kinase activity in these cell lines is elevated as much as 30-fold over activity in normal control cells and specific activity is elevated as much as 5.6-fold. When the breast tumor cells were grown in the presence of the tyrosine phosphatase inhibitor vanadate, Src kinase activity was reduced in all four breast tumor cell lines, suggesting that Src was being activated by a phosphatase which could recognize the Tyr530 negative regulatory site. In fractionated cell extracts from the breast tumor cells, we found elevated levels of a membrane associated tyrosine phosphatase activity that preferentially dephosphorylated a Src family carboxy-terminal phosphopeptide containing the regulatory tyrosine 530 site. Src was hypophosphorylated in vivo at tyrosine 530 in at least two of the tumor cell lines, further suggesting that Src was being activated by a phosphatase in these cells. In preliminary immunoprecipitation and antibody depletion experiments, we were unable to correlate the major portion of this phosphatase activity with several known phosphatases. (+info)
Growth inhibition of breast cancer cells by Grb2 downregulation is correlated with inactivation of mitogen-activated protein kinase in EGFR, but not in ErbB2, cells.
Increased breast cancer growth has been associated with increased expression of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases (RTKs). Upon activation, RTKs may transmit their oncogenic signals by binding to the growth factor receptor bound protein-2 (Grb2), which in turn binds to SOS and activates the Ras/Raf/MEK/mitogen-activated protein (MAP) kinase pathway. Grb2 is important for the transformation of fibroblasts by EGFR and ErbB2; however, whether Grb2 is also important for the proliferation of breast cancer cells expressing these RTKs is unclear. We have used liposomes to deliver nuclease-resistant antisense oligodeoxynucleotides (oligos) specific for the GRB2 mRNA to breast cancer cells. Grb2 protein downregulation could inhibit breast cancer cell growth; the degree of growth inhibition was dependent upon the activation and/or endogenous levels of the RTKs. Grb2 inhibition led to MAP kinase inactivation in EGFR, but not in ErbB2, breast cancer cells, suggesting that different pathways might be used by EGFR and ErbB2 to regulate breast cancer growth. (+info)
Increased expression of fibroblast growth factor 8 in human breast cancer.
Fibroblast growth factor 8 (FGF8) is an important developmental protein which is oncogenic and able to cooperate with wnt-1 to produce mouse mammary carcinoma. The level of expression of FGF8 mRNA was measured in 68 breast cancers and 24 non-malignant breast tissues. Elevated levels of FGF8 mRNA were found in malignant compared to non-malignant breast tissues with significantly more malignant tissues expressing FGF8 (P=0.019) at significantly higher levels (P=0.031). In situ hybridization of breast cancer tissues and analysis of purified populations of normal epithelial cells and breast cancer cell lines showed that malignant epithelial cells expressed FGF8 mRNA at high levels compared to non-malignant epithelial and myoepithelial cells and fibroblasts. Although two of the receptors which FGF8 binds to (FGFR2-IIIc, FGFR3-IIIc) are not expressed in breast cancer cells, an autocrine activation loop is possible since expression of fibroblast growth factor receptor (FGFR) 4 and FGFR1 are retained in malignant epithelial cells. This is the first member of the FGF family to have increased expression in breast cancer and a potential autocrine role in its progression. (+info)
Estrogen-dependent and independent activation of the P1 promoter of the p53 gene in transiently transfected breast cancer cells.
Loss of p53 function by mutational inactivation is the most common marker of the cancerous phenotype. Previous studies from our laboratory have demonstrated 17 beta estradiol (E2) induction of p53 protein expression in breast cancer cells. Although direct effects of E2 on the expression of p53 gene are not known, the steroid is a potent regulator of c-Myc transcription. In the present studies, we have examined the ability of E2 and antiestrogens to regulate the P1 promoter of the p53 gene which contains a c-Myc responsive element. Estrogen receptor (ER)-positive T47D and MCF-7 cells were transiently transfected with the P1CAT reporter plasmid and levels of CAT activity in response to serum, E2 and antiestrogens were monitored. Factors in serum were noted to be the dominant inducers of chloramphenicol acetyltransferase (CAT) expression in MCF-7 cells. The levels of CAT were drastically reduced when cells were maintained in serum free medium (SFM). However, a subtle ER-mediated induction of CAT expression was detectable when MCF-7 cells, cultured in SFM, were treated with E2. In serum-stimulated T47D cells, the CAT expression was minimal. The full ER antagonist, ICI 182 780 (ICI) had no effect. Treatment with E2 or 4-hydroxy tamoxifen (OHT) resulted in P1CAT induction; OHT was more effective than E2. Consistent with c-Myc regulation of the P1 promoter, E2 stimulated endogenous c-Myc in both cell lines. Two forms of c-Myc were expressed independent of E2 stimuli. The expression of a third more rapidly migrating form was E2-dependent and ER-mediated since it was blocked by the full ER antagonist, ICI, but not by the ER agonist/antagonist OHT. These data demonstrate both ER-mediated and ER-independent regulation of c-Myc and the P1 promoter of the p53 gene, and show differential effects of the two classes of antiestrogens in their ability to induce the P1 promoter of the p53 gene in breast cancer cells. (+info)