Amphotericin-B and monensin potentiation of murine erythropoiesis in vitro: a possible role for sodium ions.

To study the role of monovalent cation flux in erythropoiesis we cultured mouse bone marrow cells with amphotericin B (AmB), monensin, valinomycin, or Etruscomycin. At low doses the polyene antibiotic AmB has been shown to increase cell permeability to Na+ and K+ and we found that it potentiated erythropoietin (epo)-stimulated erythroid-colony (CFU-E) and burst (BFU-E) growth at concentrations ranging from 0.5-1.0 micrograms/ml. Monensin, a sodium-specific ionophore, potentiated epo-stimulated erythroid growth at concentrations of 1-30 nM. On the other hand, a potassium-specific ionophore, valinomycin, did not cause potentiation, but rather suppressed epo-dependent colony formation. Etruscomycin, another polyene, but one which in mammalian cells increases ion permeability only at toxic concentrations, was also suppressive. Potentiating concentrations of AmB and monensin increased the sensitivity of CFU-E and BFU-E to epo and at saturating epo levels increased the numbers of erythroid colonies and bursts by about 40%. Neither AmB nor monensin stimulated erythroid growth in the absence of epo. We found a 20-fold difference in the AmB concentrations comprising the maximally potentiating dose in C57BL/6 and AKR marrow cultures. This is consistent with observed differences between these two mouse strains with regard to other effects of AmB on them, including the immunoadjuvant properties of AmB. Our results showing potentiation due to sodium ion flux may be related to previous work showing potentiation of erythroid differentiation caused by calcium ion flux, since sodium ion movement may directly affect the intracellular calcium ion concentration.

Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells.

We have systematically studied the effect on hemopoietic colony-forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30-60 min incubation with a 5 microM concentration of stain. Flow-cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony-forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU-M, a megakaryocyte colony-forming cell; CFU-E, a relatively differentiated erythroid precursor; BFU-E, a primitive erythroid precursor; CFU-GM, a granulocyte-macrophage precursor; and CFU-S, the spleen colony-forming cell or hemopoietic stem cell. A staining procedure involving a 30-min exposure to 5 microM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU-M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.

Most cystosarcoma phyllodes and fibroadenomas have progesterone receptor but lack estrogen receptor: stromal localization of progesterone receptor.

Biochemical study of fibroepithelial tumors of the female breast showed presence of progesterone receptor (PgR) in all five cystosarcoma phyllodes (two malignant, three benign), and in 11 of 13 fibroadenomas tested. Estrogen receptor (ER) was detected in only one of five cystosarcomas and 2 of 13 fibroadenomas. The relative volumes occupied by epithelium and stroma in each tumor were measured from histologic sections. The results were consistent with presence of PgR in the stroma and ER in the epithelium. Different types of cystosarcoma (benign and malignant) and different types of fibroadenomas (intracanalicular, pericanalicular, and mixed) did not differ significantly in content of PgR, and mean levels of PgR in cystosarcoma were comparable with those in fibroadenomas. The presence of PgR in cystosarcomas suggests that progestational therapy, and possibly other forms of hormonal therapy, should be tested in the treatment of advanced, malignant cystosarcoma phyllodes.

A fluorescent probe for rapid detection of estrogen receptors.

Fluorescein conjugates to estradiol-17 beta by the sixth carbon (6-FE) and to estrone by the 17th carbon (17-FE) were used to detect estrogen receptors (ERs) in breast cancer tissue sections and in cultured cell lines (human mammary carcinoma MCF-7 and Copenhagen rat prostatic tumor R3327-AT). 17-FE was found to interact with ERs better than 6-FE by biochemical and histochemical techniques. Thin layer chromatography analysis of ethanolic extracts of 17-FE incorporated in tissues and cultured cells showed that over 95% of 17-FE was not metabolized. We concluded that the fluorescence observed in tissue sections and cultured cells was due to 17-FE and not to fluorescein dissociated from the conjugate. Analysis of 65 human breast cancer showed that 74% of the cases were positive for specific 17-FE uptake and 26% were negative. The fluorescence was consistently brighter in the ductal and glandular epithelial cells than in the stroma. specific 17-FE uptake in the nucleoli was observed in MCF-7 and in R3327-AT tumor cells in in vitro cultures, suggesting that these nucleolar estrogen receptors may play a key role in the mechanism of estrogen action. Problems of fluorescence quantitation in tissue sections and cells are discussed.