Enhanced rates of fluid pinocytosis during exponential growth and monolayer regeneration by cultured arterial endothelial cells

Rates of fluid pinocytosis by bovine aortic endothelial cells were measured during various manipulations of growth status in vitro. Sparsely seeded cultures grew exponentially until a confluent monolayer was formed, at which time growth slowed. This change in growth rate coincided with a decline in the rate of pinocytosis to about one-third that in the growing cultures. During the subsequent attainment of maximal cell density in the confluent monolayer, the pinocytic rate remained constant. There was close correlation between ³H-thymidine labelling indices, as measured by autoradiography, and the rates of pinocytosis. Mechanical “wounding” of the confluent monolayer resulted in cell migration and proliferation. Twenty-four hours after “wounding,” rates of pinocytosis per mg. cell protein were significantly enhanced. When regeneration of the monolayer was blocked by cytochalasin B, pinocytosis remained at the same rate as in the uninjured, confluent monolayer. These experiments support, and extend to endothelium, earlier observations that in growing cells pinocytosis proceeds at a higher rate than in non-growing, quiescent cells. Furthermore, they raise the possibility that the transendothelial transport of macromolecules such as lipoproteins by receptor-in-dependent fluid pinocytosis in vivo may be altered by the growth status of the endothelium.     

Differential Estrogenic Responsiveness of MCF-7 Cells Relationship to the Presence of two Different Estrogen Receptors

Abstract

Estradiol stimulation of thymidine incorporation and progesterone receptor synthesis is at a maximum in exponentially growing cells. These activities are found to disappear in confluent MCF-7 cells. Since no significant differences in the binding of estradiol to its receptor site (Kd = 10^?10 M, Bmax = 150 fm/mg protein) are observed in these two conditions, receptor structure was analyzed in both cell populations. Various methods demonstrated that receptor size is related to the state of confluence. The hydrodynamic properties of estradiol receptors complexed with ³H-estradiol from cells in the two different growth phases are similar in low ionic strength but different in high ionic strength media. Moreover, when the cell extracts are analyzed in denaturing     

Synergistic cytotoxic effects of tamoxifen and black cohosh on MCF-7 and MDA-MB-231 human breast cancer cells: an in vitro study

Breast cancer cell cultures were exposed to different concentrations of black cohosh, estradiol (E2), and tamoxifen to examine the effect on cell proliferation; cytotoxicity was assessed by using sulforhodamine B (SRB) dye solution. E2 (10(-10) - 10(-8) mol/L) markedly stimulated the proliferation of MCF-7 cells (p < 0.01). Tamoxifen stimulated MCF-7 cell proliferation at 10(-6) mol/L and 10(-5) mol/L (p < 0.005) but inhibited in a dose-dependent fashion the proliferative effect of E2 (p < 0.001). Black cohosh alone did not show any stimulatory effect, but exhibited a cytotoxic effect, which was significant at 10(3) microg/mL (p < 0.001). Adding black cohosh at 10(0)-10(3) microg/mL to E2 at 10(-9) mol/L also resulted in a dose-dependent inhibition of E2 proliferative effect. Interestingly, the combination of black cohosh (10(0)-10(3) microg/mL) with increasing tamoxifen concentrations further inhibited MCF-7 cell growth. On MDA-MB-231 cells, neither E2 nor tamoxifen displayed any detectable effect. However, black cohosh inhibited MDA-MB-231 cell proliferation at 10(3) microg/mL (p < 0.05), and this inhibitory effect was enhanced by increasing tamoxifen concentrations. This study reveals a cytotoxic effect of black cohosh on both estrogen-sensitive and estrogen-insensitive breast cancer cells and a synergism with tamoxifen for inhibition of cancerous cell growth.     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Cell surface-associated growth inhibitory proteins : Evidence for conservation between mouse and human cell lines

The ability of the normal human fibroblast line (IMR91) to exhibit density-dependent regulation of growth has been examined. The line exhibits density-dependent regulation of growth; saturation density in 15% fetal bovine serum is 2 × 10⁵ cells/cm². Membranes prepared from confluent monolayers of these cells contained growth inhibitory factors to both exponentially growing IMR91 and Swiss 3T3 cells. This factor(s) appears to be similar to a previously described factor found on the surface of Swiss 3T3 cells [14]. The inhibition of DNA synthesis in growing IMR91 cultures by membranes was both time- and concentration-dependent. The effect was reversible by high serum. Specificity experiments utilizing membranes prepared from Swiss 3T3 cells indicated some species specificity for inhibition by membranes, but this specificity was no longer exhibited by solubilized membrane preparations. These results are compatible with the suggestion that both the growth inhibitory factors and their receptors are conserved through evolution.     

Relationship between cell proliferation, chromatin template activity and accumulation of nuclear proteins

Trypsinization of confluent monolayers of WI-38 cells causes an extensive loss of nuclear proteins. The loss of nuclear proteins is restored only several hours after the cells have been replated at a lower density in 10% serum. When trypsinized fibroblasts are replated at a lower density in 10% serum, there is also a sustained progressive leading to DNA synthesis and cell division. If 0.3% serum is used instead of 10%, there is a modest increase in nuclear template activity, but not accumulation of nuclear proteins and no DNA synthesis or cell division.     

Induction and inhibition of estradiol hydroxylase activities in MCF-7 human breast cancer cells in culture

The effects of estradiol, progesterone, and tamoxifen on the activity of estradiol 2- and 16 alpha-hydroxylases were studied in human breast cancer cell cultures using a radiometric assay. After 5 days' exposure to these compounds, incubations in the presence of either [2-3H]estradiol or [16 alpha-3H]estradiol as substrate were carried out. In MCF-7 cells, estradiol (10(-8) M), progesterone (10(-6) M) and tamoxifen (10(-6) M) significantly increased 16 alpha-hydroxylase activity (estradiol; 21% progesterone 10% to 32%; tamoxifen 21% to 31%; P less than 0.01). Synergistic effects were observed when the cells were successively exposed to tamoxifen and progesterone. Simultaneous treatment with tamoxifen plus estradiol or estradiol plus progesterone showed no change from estradiol alone. On the other hand, although estradiol had no direct effects on 2-hydroxylase activity, tamoxifen decreased this enzymatic activity significantly at 10(-6) M (23% to 37%). Progesterone acted synergistically to further decrease this reaction. Treatment with only progesterone caused an increase in 2-hydroxylation. In contrast, a subline of MCF-7 cells with low estrogen receptor levels showed only minimal enzyme-hormone responses. Likewise, treatment of the estrogen receptor-negative MDA-MB-231 human breast cancer cell line with these compounds showed no effects on either 2- or 16 alpha-hydroxylase activity. In the progesterone receptor-rich T47D cell line, estradiol decreased both activities while progesterone increased both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-cycle-dependent repair of damage in alpha and bulk DNA of monkey cells

Excision repair of bulky chemical adducts in alpha DNA of confluent cultures of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have compared the removal of adducts produced by treatment with aflatoxin B1 (AFB1) and N-acetoxy-2-acetylamino-fluorene (NA-AAF) from alpha DNA sequences in synchronized and exponentially growing cultures of monkey cells. Proficient removal of AFB1 adducts in alpha DNA was observed in exponentially growing cultures. However, as the cultures approached confluence, adduct removal from alpha DNA became deficient. Cells synchronized by subculturing confluent cultures exhibited proficient removal of adducts from both alpha and bulk DNA when treated in early G1 or late S/G2 while those cells treated in early S phase did not remove adducts from either alpha or bulk DNA. We conclude that the accessibility of chemical adducts to repair in alpha chromatin is influenced by the growth state and the cell cycle stage.     

The production and localization of laminin in cultured vascular and corneal endothelial cells

The production and localization of laminin, as a function of cell density (sparse versus confluent cultures) and growth stage (actively growing versus resting cultures), has been compared on the cell surfaces of cultured vascular and corneal endothelial cells. Comparison of the abilities of the two types of cells to secrete laminin and fibronectin into their incubation medium reveals that vascular endothelial cells can secrete 20-fold as much laminin as can corneal endothelial cells. In contrast, both cell types produce comparable amounts of fibronectin. Furthermore, if one compares the secretion of laminin and fibronectin as a function of cell growth, it appears that the laminin released into the medium by either vascular or corneal endothelial cells, is a function of cell density and cell growth, since this release is most pronounced when the cells are sparse and actively growing, and decreases by 10- and 30-fold, respectively, when either vascular or corneal endothelial cell cultures become confluent. With regard to fibronectin secretion, no such variation can be seen with vascular endothelial cell cultures, regardless of whether they are sparse and actively growing or confluent and resting. Corneal endothelial cell cultures, demonstrated a twofold increase in fibronectin production when they were confluent and resting as compared to when they were sparse and actively growing. When the distribution of laminin versus fibronectin within the apical and basal cell surfaces of cultured corneal and vascular endothelial cells is compared, one can observe that unlike fibronectin, which in sparse and subconfluent cultures can be seen to be associated with both the apical cell surface. In confluent cultures, laminin can be found associated primarily with the extracellular matrix beneath the cell monolayer, where it codistributes with type IV collagen.     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

Influence of long-term limitation of cell proliferation on the cell cycle duration

The hypothesis was tested that constant, long-term inhibition of cell proliferation is the primary cause of cellular senescence. Mouse L-929 fibroblasts were maintained in confluent cultures for periods of 6 and 12 months with cell viability maintained by regular replacement of medium and serum. The mitotic activity of the cell population under these conditions was one mitosis per 10⁴ cells. No changes in the duration of the cell cycle were observed when, following long-term quiescence, the cell were replated and grown at low cell density. The results do not support the hypothesis that prolonged suppression of cell proliferation induces cell senescence.     

Tissue polypeptide antigen liberation and proliferation of MCF-7 cells after synchronization with hydroxytamoxifen and rescue with estradiol

The kinetic of release of the tissular polypeptide antigen (TPA) by MCF-7 cells synchronised by sequential treatment with hydroxytamoxifen (OH-TAM) and 17 beta-estradiol has been studied. The present findings confirm the proliferative effect of estradiol on MCF-7 cells, with a shortening of the doubling time (TD) (22.2 h versus 24.8 h) and an increase in the growing fraction (Fc) (94% versus 81%) when compared with the same parameters measured in cells rescued from OH-TAM but not treated with estradiol. In addition, the action of estradiol was followed by a simultaneous increase in the amount of TPA in the culture medium related with the phases G2/M and G1 of the mitotic cycle. This phenomenon seems to be the reason for the steplike shape of the TPA released curves. The experimental results suggest that in MCF-7 cells the sequential combination of antiestrogenic agents and estrogens leads to a cellular synchronization. Furthermore this synchronization is maintained for at least 3 cycles of cell division.     

N-Alkoxypyrazoles as biomimetics for the alkoxyphenyl group in tamoxifen

The preparation of a series of novel analogues of the selective antiestrogen tamoxifen is reported. 1Z-alkoxyphenyl group in tamoxifen has been replaced by a N-alkoxypyrazole, while functionalised phenyl groups or heteroaromatics were introduced at the 2Z-position using sequential Suzuki cross coupling of 1,2-(bis)borylpinacol 1-phenylbutene with 4- or 5-iodo-1-N,N-dimethylaminoethyl or propyl-pyrazoles. Approximately 50 tamoxifen analogues were obtained and tested in an estrogen receptor (ERalpha) affinity assay. Several compounds exhibited binding affinities 2-5-fold lower than tamoxifen. Dose-response experiments with six selected compounds were carried out using two different human breast cancer cell lines, MCF-7 and the tamoxifen resistant cell line MCF-/TAM(R)-1. Both cell lines exhibited growth inhibition upon treatment with the tamoxifen analogues. Co-treatment of the cells, with estradiol and the individual compounds, were also performed. The results indicated that the observed growth inhibitory effect was mediated by the ERalpha. Analogues of the potent antiestrogen 4-hydroxytamoxifen (4-OHT) were synthesised where the 1E-4-hydroxyphenyl was replaced by a 1-hydroxypyrazol-4-yl group. However, modest growth inhibition of MCF-7 cells was observed upon treatment with these analogues. In contrast, 1Z-, 2Z-ringclosed tamoxifen analogue (59) was shown to possess antiproliferative effects on MCF-7 and MCF-/TAM(R)-1 cells in lower doses than tamoxifen.     

Response of primary rabbit kidney proximal tubule cells to estrogens

The effects of estrogens on the growth and function of primary rabbit kidney proximal tubule (RPT) cells have been examined in hormonally defined phenol red–free medium. 17β-estradiol was observed to stimulate growth at dosages as low as 10⁻¹⁰ M. The growth stimulatory effects of 17β-estradiol were mitigated in the presence of hydrocortisone, suggesting that these two steroid hormones acted at least in part by common mechanisms. The effects of other steroids known to interact with the estrogen receptor were examined. Alpha estradiol was found to be growth stimulatory over a concentration range of 10⁻⁹ to 10⁻⁸ M, albeit to a lower extent than beta estradiol. In addition, the anti-estrogen tamoxifen was also growth stimulatory (unlike the case with the human mammary tumor cell line MCF-7). The effects of several metabolic precursors of 17β-estradiol were examined, including testosterone, which was growth stimulatory, and progesterone, which was growth inhibitory. The growth stimulatory effects of 17β-estradiol, alpha estradiol, and tamoxifen could possibly be explained by their interaction with an estrogen receptor. Indeed, metabolic labelling and immunoprecipitation studies indicated the presence of such an estrogen receptor in the primary cultures. The rate of biosynthesis of the estrogen receptor was found to be affected by the presence of exogenously added 17β-estradiol. 17β-estradiol was also observed to increase the activity of two brush border enzymes, alkaline phosphatase and gamma glutamyl transpeptidase, during the growth phase of the primary cultures. J Cell Physiol 178:35–43, 1999. © 1999 Wiley-Liss, Inc.     

Histone mediated changes in morphology and confluent density of cultured cells

Calf thymus histones and histone subfractions were added to media overlying subconfluent mouse fibroblast cells in culture. The histones caused significantly higher cell densities at confluence than control cultures and disruption of the normal ordered arrangement of cells. These changes were seen on application of histones to growing cells but not confluent cells and were reversed when the histones were removed and the cells replated with more growth area. The slightly lysine rich histone fraction had the greatest effect and the lysine rich fraction had the least effect on cell morphology and cell number at confluence. These effects could not be duplicated with other highly charged basic proteins.     

Involvement of plasma membrane glycoproteins in the contact-dependent inhibition of growth of human fibroblasts

The human embryonal lung fibroblasts used in this study showed a pronounced inhibition of growth when reaching a critical cell density. This effect has been mimicked by the addition of glutaraldehyde-fixed human fibroblasts to sparsely seeded growing cells. Inhibition of growth was not observed when glutaraldehyde-fixed cells were pretreated with galactosidase or with galactose-specific lectins, or when glutaraldehyde-fixed human or rabbit erythrocytes were added to the proliferating fibroblasts. In addition, glutaraldehyde-fixed mitotic cells were without effect on the proliferation, while cells prepared from sparse culture had lesser potency than cells prepared from confluent cultures. Plasma membranes, isolated from cells of confluent cultures, when added to growing cultures of human fibroblasts inhibited DNA synthesis in a concentration-dependent manner. On the other hand, plasma membranes isolated from sparsely seeded cells had only minor inhibitory potency. When the plasma membranes were isolated from cells treated previously with tunicamycin, an antibiotic which inhibits the synthesis of the oligosaccharide portion of asparagine-linked glycoproteins, the inhibitory effect was abolished. The same effect was observed when plasma membranes were pretreated with galactosidase. These data indicate that the growth of cells in vitro is regulated by specific cell-cell contacts. They also show that one of the molecular reactants in this process are membrane glycoproteins with asparagine-linked oligosaccharides.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Cell-cell contact and growth regulation of pinocytosis in 3T3 cells

In sub-confluent cultures of Balb/c-3T3 cells, pinocytosis rates were increased after exposure to specific growth factors (serum; platelet-derived growth factor, PDGF; epidermal growth factor, EGF). Conversely, as cells became growth-inhibited with increasing culture density, there was a corresponding decline in pinocytosis rate per cell. In order to test whether density-inhibition of pinocytosis was influenced either by the growth cycle or by cell contact independently of growth, cells were induced into a quiescent state at a range of subconfluent and confluent densities. Under such conditions, cell density did not significantly inhibit pinocytosis rate. When confluent quiescent cultures in 2.5% serum were exposed to 10% serum, the resulting round of DNA synthesis was accompanied by enhanced pinocytosis per cell, even though the cells were incontact with one another. Furthermore, in a SV40-viral transformed 3T3 cell line, both the growth fraction and the pinocytosis rate per cell remained unchanged over a wide range of culture densities. These studies indicate that density-dependent inhibition of pinocytosis in 3T3 cells appears to be secondary to growth-inhibition rather than to any direct physical effects of cell–cell contact.