The increase in cytosolic levels of protein kinase C precedes the growth factor requirements during the transition stem cells to differentiated progeny in the MCF-7 breast tumor cells.

The expression of regulatory genes within the context of a differentiation program can have profound long-term consequences in tissues with permanent renewing populations. The breast tumor cell line MCF-7 retains in culture some of the characteristics of a unidirectional differentiation pathway. We show that the cytosolic activity of the regulatory enzyme protein kinase C (PKC) precedes and continues the sequence of maturation in pre-differentiated subpopulations derived from a stem cell fraction. However, the activity declines in the most differentiated, postmitotic fraction. These results indicate that PKC may be considered among the regulatory genes in MCF-7 cells that specify maturation of the stem cell progeny.

Adriamycin effects on hydroperoxide metabolism and growth of human breast tumor cells.

Human breast tumor cells MCF-7 were grown during 5 days in the presence of Adriamycin and the IC50 was 50 nM with the highest sublethal concentration 0.1 microM. At this latter concentration Adriamycin produced a complete inhibition of cell division and a partial reversion to a normal breast epithelial appearance. Similar effects of Adriamycin were observed in cells cultured in the presence of 10% FBS and in a chemically defined medium, with Se-glutathione peroxidase activities of 3.8 and 1.3 U/mg of protein, respectively. Cell size and cell oxygen uptake were increased by 41% and by 50%, respectively, in Adriamycin-treated cells. The spontaneous chemiluminescence of monolayers of intact MCF-7 cells (81 +/- 9 cps/mg protein) was increased by 48% in the Adriamycin-treated cultures (120 +/- 11 cps/mg of protein) in agreement with a 91% higher concentration of malondialdehyde in the same cultures. Adriamycin treatment produced a 71% increase in the steady state concentration of H2O2, which was estimated assuming diffusion equilibrium with the external medium, from 1.38 microM in the control cells to 2.38 microM in the treated cells. Cyanide-insensitive respiration was also higher in the cells exposed to the drug than in the control cells. Adriamycin did not affect the activity of the antioxidant enzymes, Cu-Zn and Mn-superoxide dismutase, Se and non-Se-glutathione peroxidase, and catalase. These results contribute to the current hypothesis that oxygen free radicals produced by Adriamycin redox cycling are responsible for at least part of the cytotoxic effects due to this drug.

Increased secretory activity and estradiol receptor expression are among other relevant aspects of MCF-7 human breast tumor cell growth which are expressed only in the absence of serum.

We compared the morphology, clonogenic ability, Percoll gradient distribution, estrogen receptor proteins, and interactions with mesenchymal cells in MCF-7 breast tumor cells grown in medium containing fetal calf serum and insulin (FCS-I) or in a defined medium with insulin (ID) as the only growth factor. In the absence of serum and at densities below 5000-8000 cells/cm2, MCF-7 cells required epidermal growth factor, insulin, and thrombin. When cells reached a density of 23,000-26,000 cells/cm2, only insulin was necessary for optimal growth. In ID medium cells showed an enlarged Golgi apparatus and marked plasma membrane modifications, suggesting increased secretory activity. Moreover there was an increase in the release of protein products to the culture medium and a time-dependent ability of these cells to form macrocolonies in soft agar. On the contrary, cells in FCS-I showed no Golgi complex and few plasma membrane modifications. In both culture media tight junctions, desmosomes, and tonofilaments were present. We investigated the effect of conditioned media from MCF-7 cells growing in FCS-I or ID on the growth of primary rat vaginal fibroblasts. The growth of these mesenchymal cells was stimulated by FCS-I medium and inhibited by ID medium. By contrast, the embryonic fibroblast (preadipocyte) line CHEF/18 was also stimulated by FCS-I for the first 48 h, but thereafter ceased growth and acquired lipid droplets and a differentiated morphology. With ID medium, CHEF/18 cells were only partially inhibited with no changes in morphology. The Percoll gradient profiles of ID cells showed the same six fractions of increasing density as recently described. However, there was a progressive increase in subpopulations with higher growth rates and a decrease in the relative amount of the most differentiated cells. A unique feature of the growth analysis of MCF-7 cells in the absence of serum is the increased expression of the estradiol receptor gene. These studies show that the growth and differentiated properties of tumor cells can depend upon the cellular environment and offer a model system in which to further study this modulation.

Characterization of IIB-MEL-J: a new and highly heterogenous human melanoma cell line.

A highly heterogeneous cell line, IIB-MEL-J, was established from a human metastatic melanoma. This cell line contains small cells, dendritic cells, and megacells with multiple nuclei. IIB-MEL-J expresses S 100, cytokeratin intermediate filaments and the gangliosides GD2 and GD3. It requires growth factors (insulin, EGF, and transferrin) and antioxidants for optimal growth. When plated under optimal conditions, IIB-MEL-J grows with a doubling time of 70-80 hours. The cells may be fractionated by Percoll gradient centrifugation into several subpopulations (A, B, and C) with different characteristics. Subpopulation A is the slowest growing, and most of the DNA-synthesizing cells are concentrated in fractions B and C. Every subpopulation expresses S 100 and cytokeratin intermediate filaments, whereas only subpopulation B and C express GD2 and GD3. Pigmented cells are concentrated mainly in subpopulation C. Cytogenetic analysis of IIB-MEL-J revealed extensive chromosomal alterations, including a highly heterogeneous chromosome number and chromosomal rearrangements, gains, losses, isochromosomes, and double minutes. This highly heterogeneous cell line may be helpful to study cellular differentiation and interaction between different subpopulations in human melanoma.

Growth factors and hormones which affect survival, growth, and differentiation of the MCF-7 stem cells and their descendants.

The human breast tumor cell line was separated by Percoll density gradient centrifugation into six different subpopulations, A to F, one of which (E) appears to contain the stem cells on the basis of several criteria (M. Resnicoff et al. 1987, Proc. Natl. Acad. Sci. USA 84, 7295. We now analyzed the response of the isolated subpopulations to insulin, thrombin, PGF2 alpha, estradiol, and 13-cis-retinal. We demonstrate that the first two growth factors stimulate [3H]thymidine incorporation in the more differentiated subpopulations (D and F), while PGF2 alpha has mitogenic activity in subpopulations C and D. In the absence of any added growth factor, estradiol has the extreme and transient capacity of allowing the stem cell to detach from the tissue culture dish and to grow in suspension as multicellular aggregates (MCF-7/SE cells). 13-cis-Retinal acts as a negative modulator of differentiation and protects the cells from the inhibitory and differentiation activity of Na-butyrate.

Effect of estradiol and tamoxifen on the anchorage-independent growth of the subpopulations derived from MCF-7 breast carcinoma cells: cytogenetic analysis of the stem cell subpopulation.

The MCF-7 breast carcinoma cell line can be separated by Percoll density gradient centrifugation into several subpopulations, A to F, one of which (E) has been previously suggested to be highly enriched in stem cells. The anchorage-independent growth of the different fractions and its sensitivity to estradiol (E2) and tamoxifen (TAM) was assayed. The anchorage-independent growth capacity of the different fractions was E greater than A greater than B greater than D greater than C,F. The E fraction had the highest clonogenic index (6.62 +/- 1.18) and was unaffected by E2 or TAM. The karyotypic analysis of the E fraction revealed features similar to those of the unfractionated cell line. It is suggested that the high growth rate of fraction E is due to an enrichment in stem cells and not to the existence of a different clone.

Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells.

The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

Role of thrombin in the proliferative response of T-47D mammary tumor cells. Mitogenic action and pleiotropic modifications induced together with epidermal growth factor and insulin.

The growth of the human metastatic cell line (T-47D) in a chemically defined medium (DM) is shown to be dependent on the presence of three traditional growth factors: epidermal growth factor, insulin, and transferrin. The addition of thrombin further stimulates its growth. The mitogenic action on a human mammary tumor cell line from epithelial origin is a novel action of thrombin. Cells in the DM show striking morphological changes which are dramatically enhanced by the addition of thrombin. These observations are part of a pleiotropic response to the growth factors: the protein content of the cells increases in the defined medium; the 2DG gels of the 35S- and 32P-labeled proteins show important changes in spots, several of which are probably of cytoskeletal origin. It is also shown that cells in a semisolid growth factor-supplemented medium have growth advantages over their counterparts grown with serum. All the phenotypic changes mentioned above reveal the important role of growth factors in the growth and behavior of this mammary cell line. The results obtained with thrombin indicate a new site of action of this enzyme which may be important in the metastatic spread of human mammary tumor cells.

Subpopulations of MCF7 cells separated by Percoll gradient centrifugation: a model to analyze the heterogeneity of human breast cancer.

Exponentially growing MCF7 human breast cancer cells were separated in Percoll gradients into six different fractions of increasing density (A to F). These fractions could be subcultured and were found to contain different cellular subpopulations as defined by the following criteria: ability to generate other cellular subpopulations; growth rate; DNA synthesis; and expression of estrogen receptors, ras oncogene-encoded protein p21, and carcinoembryonic antigen. One of the minor fractions (E), which contained about 5% of the total cell number, appeared to contain the stem cells, on the basis of the following criteria: (i) its ability to reproduce the other cellular subpopulations, (ii) its high rate of growth and DNA synthesis, and (iii) the inability of the other subpopulations to generate it. The most differentiated subpopulation appeared to be the densest one (F), since it was the slowest growing and appeared to be the end point of the other subpopulations.