MATERIALS AND METHODS HUVEC. HUVECs were obtained from umbilical cords from cesarean sections. The cells were cultured by standard methods in the presence of heparin and endothelial-cell growth supplement (7). They were characterized by their homogeneous and typical cobblestone morphology, factor VIII antigen positivity, and the presence of Weibel-Palade bodies on electron microscopy. HUVECs were used for these studies after three to five passages.For clonal culture to exclude contamination with hematopoietic stem cells, HUVECs (1-5 x 105 cells per ml) were plated in methylcellulose cultures as described (8), with the modification that half of the fetal bovine serum was replaced with human umbilical cord blood serum. Hematopoietic growth factors were added as follows: rhEpo, 2 units/ml; stem-cell factor, 10 pg/ml; granulocyte/macrophage-colonystimulating factor, 200 units/ml; interleukin 3, 200 units/ml; endothelial-cell growth factor, 20 ktg/ml.Preparation of mRNA. After 1-5 x 107 cells were harvested and washed twice with phosphate-buffered saline, RNA was extracted with guanidinium thiocyanate and lauryl sarcosinate (9). mRNA was adsorbed onto oligo(dT)-cellulose columns (Pharmacia) and, after the columns were washed with high-and low-salt solutions, was eluted with 10 mM Tris'HCl buffer containing 1 mM EDTA (pH 7.4) at 650C. Total amount and concentration of mRNA were determined spectrophotometrically and confirmed by agarose gel electrophoresis.

Erythropoietin is known to be a hematopoietic growth factor with a sinulrly specific action on the proliferation and differentiation of erythroid progenitor cells. We have observed a dose-dependent proliferative action of human recombinant erythropoletin on human umbilical vein endothelial cells and bovine adrenal capillary endothelial cells. Binding studies with radioiodinated recombinant human erythropoietin revealed a large number (s27,000) of an apparent single class of receptors with an affinity in the 10-9 M range.Linkage of the radiolabeled ligand to its receptor via a bifunctional crosslinking agent allowed us to identify an endothelial cell protein of 45 kDa as the principal receptor associated with this mitogenic effect of erythropoietin. Recombinant human erythropoietin also enhanced the migration ofendothelial cells.Human umbilical vein endothelial cells (HUVECs) have been reported to produce a variety of colony-stimulating factors and lymphokines, although not erythropoietin (Epo) (1-5). Epo has been considered unique among the other hematopoietic stimulators because its only generally accepted action has been the proliferation and differentiation of cells of erythroid lineage (6). Colony-stimulating factors, on the other hand, are known to affect a variety of hematopoietic cells, and even cells of nonhematopoietic origin (6)(7)(8). Recombinant human Epo (rHuEpo) has been used therapeutically in thousands of uremic, anemic patients, with the only side effects arising from the vascular system (hypertension and thrombosis) (9).In experiments designed originally to study additional mechanisms of the vascular manifestations (thrombosis and hypertension) of Epo treatment (aside from the ones ascribed to increased blood viscosity), we serendipitously noticed significant cell proliferation when rHuEpo from Integrated Genetics (Framingham, MA) was added to HUVEC cultures. We pursued this observation with a series of new experiments, verifying that rHuEpo from various manufacturers has indeed a mitogenic effect on both HUVECs and bovine adrenal capillary endothelial cells (BACECs). We further analyzed the number and molecular weight of the putative receptor through which this action may be exerted. Since many agents with a mitogenic effect on endothelial cells have also an effect on endothelial cell migration, we investigated this aspect, finding again that the rHuEpo preparations enhanced the chemotaxis of both HUVECs and BACECs. MATERIALS AND METHODSCell Culture. Cesarian section-derived HUVECs were cultured at 37°C and in 5% C02/95% air by standard methodologies (10) in T 25-cm2, 50 ml capacity, tissue culture flasks (Falcon, Becton Dickinson Labware) in the presence of heparin and endothelial cell growth supplement (Sigma). They were characterized by the homogeneous and typical cobblestone morphology, factor VIII antigen positivity, and the presence of Weibel-Palade bodies on electron microscopy. In general, HUVECs were used for our experiments after two to four passages. rHuEpo, initially from I...

Vitamin E, best known as a potent antioxidant, has been shown to have other functions that are not mediated by this activity. Recent reports have suggested that vitamin E may inhibit smooth muscle cell and also cancer cell growth. We have studied the effect of dl-alpha-tocopherol (vitamin E) on a series of well-established cancer cell lines that included two erythroleukemia cell lines and a hormone-responsive breast and prostate cancer cell line. Cell proliferation was examined in these cell lines, which were maintained at optimal growth conditions. A dose-dependent inhibition of cell growth was found in all cell lines examined, with the MCF-7 breast and CRL-1740 prostate cancer cell lines showing potent suppression of growth at 0.1 mM vitamin E, whereas the erythroleukemia cell lines, HEL and OCIM-1, responded only at > 0.25 mM vitamin E with inhibition of proliferation. Studies of [3H]thymidine incorporation showed that vitamin E supplementation reduced DNA synthesis in all cell lines. Analysis of high-molecular-weight DNA revealed extensive fragmentation, indicating apoptosis of all cell lines supplemented with vitamin E. Our studies thus give evidence of a general inhibition of cell proliferation by dl-alpha-tocopherol, with breast and prostate cancer cells distinctly more sensitive than erythroleukemia cells.

Organosulfur compounds are the biologically active components of allium vegetables. Many health benefits have been ascribed to them, including inhibition of carcinogenesis. Inasmuch as several of these thioallyl compounds are quite unstable and others are rapidly inactivated in the body, we have investigated one of the stable components present in aged garlic extract, S-allylmercaptocysteine (SAMC), in an effort to determine whether it can inhibit proliferation of cancer cells. Proliferation and viability of two erythroleukemia cell lines, HEL and OCIM-1, two hormone-responsive breast and prostate cancer cell lines, MCF-7 and CRL-1740, respectively, and normal human umbilical vein endothelial cells in response to different concentrations of SAMC were studied for up to two weeks. There were variations in sensitivity to this organosulfur compound in the different cell lines examined, but the two hormone-responsive cancer cell lines of breast and prostate clearly were far more susceptible to the growth-inhibitory influence of the thioallyl compound. The antiproliferative effect of SAMC was limited to actively growing cells. Human umbilical vein endothelial cells that had reached confluence escaped the reduction in viability so noticeable in the cancer cell lines tested. Our studies thus give evidence of a direct effect of SAMC on established cancer cells.

The antiproliferative potential of S-allylmercaptocysteine (SAMC), a stable organosulfur compound of aged garlic extract, has been investigated using two erythroleukemia cell lines, HEL and OCIM-1. It induces a dose-dependent inhibition of cell growth with a 50% lethal dose of 0.046 mM for OCIM-1 cells and 0.093 mM for HEL cells. [3H]thymidine incorporation was reduced in cells treated with this thioallyl compound, and analysis of high-molecular-weight DNA showed fragmentation compatible with apoptosis. Flow cytometric analyses of DNA revealed an abnormal cell cycle progression in both types of erythroleukemia cells, with the major portion of the unsynchronized cells in the G2/M phase. Measurement of acid-soluble free sulfhydryl groups showed an initial increase in response to SAMC followed by a progressive dose-dependent decrease with extended incubation of cells. We conclude from these studies that SAMC is an effective antiproliferative agent against erythroleukemia cells that induces cell death by apoptosis.