Charcoal-treated Fetal Calf Serum Research Articles

A Novel Pathway to Enhance Adipocyte Differentiation of 3T3-L1 Cells by Up-regulation of Lipocalin-type Prostaglandin D Synthase Mediated by Liver X Receptor-activated Sterol Regulatory Element-binding Protein-1c

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is expressed in adipocytes and is proposed to be involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes, because L-PGDS gene knock-out mice show abnormalities in these functions. However, the role of L-PGDS and the regulation mechanism governing its gene expression in adipocytes remain unclear. Here, we applied small interference RNA of L-PGDS to mouse 3T3-L1 cells and found that it suppressed differentiation of these cells into adipocytes. Reporter analysis of the mouse L-PGDS promoter demonstrated that a responsive element for liver receptor homolog-1 (LRH-1) at -233 plays a critical role in preadipocytic 3T3-L1 cells. Moreover, we identified two sterol regulatory elements (SREs) at -194 to be cis-elements for activation of L-PGDS gene expression in adipocytic 3T3-L1 cells. L-PGDS mRNA was induced in response to synthetic liver X receptor agonist, T0901317, through activation of the expression of SRE-binding protein-1c (SREBP-1c) in the adipocytic 3T3-L1 cells. The results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay revealed that LRH-1 and SREBP-1c bound to their respective binding elements in the promoter of L-PGDS gene. Small interference RNA-mediated suppression of LRH-1 or SREBP-1c decreased L-PGDS gene expression in preadipocytic or adipocytic 3T3-L1 cells, respectively. These results indicate that L-PGDS gene expression is activated by LRH-1 in preadipocytes and by SREBP-1c in adipocytes. Liver X receptor-mediated up-regulation of L-PGDS through activation of SREBP-1c is a novel path-way to enhance adipocyte differentiation.

Squalene Synthase, a Determinant of Raft-associated Cholesterol and Modulator of Cancer Cell Proliferation

Several cues for cell proliferation, migration, and survival are transmitted through lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol. Cells obtain cholesterol from the circulation but can also synthesize cholesterol de novo through the mevalonate/isoprenoid pathway. This pathway, however, has several branches and also produces non-sterol isoprenoids. Squalene synthase (SQS) is the enzyme that determines the switch toward sterol biosynthesis. Here we demonstrate that in prostate cancer cells SQS expression is enhanced by androgens, channeling intermediates of the mevalonate/isoprenoid pathway toward cholesterol synthesis. Interestingly, the resulting increase in de novo synthesis of cholesterol mainly affects the cholesterol content of lipid rafts, while leaving non-raft cholesterol levels unaffected. Conversely, RNA interference-mediated SQS inhibition results in a decrease of raft-associated cholesterol. These data show that SQS activity and de novo cholesterol synthesis are determinants of membrane microdomain-associated cholesterol in cancer cells. Remarkably, SQS knock down also attenuates proliferation and induces death of prostate cancer cells. Similar effects are observed when cancer cells are treated with the chemical SQS inhibitor zaragozic acid A. Importantly, although the anti-tumor effect of statins has previously been attributed to inhibition of protein isoprenylation, the present study shows that specific inhibition of the cholesterol biosynthesis branch of the mevalonate/isoprenoid pathway also induces cancer cell death. These findings significantly underscore the importance of de novo cholesterol synthesis for cancer cell biology and suggest that SQS is a potential novel target for antineoplastic intervention.

Oestrogen and progesterone receptors in colorectal cancer and human colonic cancer cell lines.

Receptors for oestrogen (ER) and progesterone (PR) were assayed in tissue from 17 patients with colorectal cancer and five colonic cancer cell lines using enzyme immunoassays. ERs and PRs were detected in 15 and 17 cancers respectively, although the levels detected were low: median (range) ER 1.3 (0-11.3) and PR 3.9 (0.3-10.2) fmol per mg protein. These values were not significantly different from median (range) levels of ER (1.1 (0.6-3.0) fmol/mg) and PR (1.9 (0.5-3.2) fmol/mg) detected in normal mucosa. There were significant positive correlations between the levels of ER and PR for cancer tissue (tau = 0.56, P < 0.005; r(log transform) = 0.68, P < 0.003; n = 17) but not for mucosa, and between levels of ER in cancer tissue and mucosa (tau = 0.55, P < 0.05; r(log transform) = 0.70, P < 0.025; n = 10) but not between the corresponding PR values. In maintenance media, which contained phenol red and unstripped fetal calf serum, the median (range) concentration of ER was 1.9 (1.2-10.4) fmol/mg and that for PR 24.3 (9.1-63.2) fmol/mg in the five cell lines studied (HT-29, LS174T, SW620, LoVo, COLO 320DM). The addition of oestradiol (10 nmol/l) to phenol red-free medium containing 5 per cent dextran-coated charcoal-treated fetal calf serum had little effect on the concentration of ERs or PRs in SW620, LoVo and COLO 320DM cells after 7 days' culture. It is concluded that ERs and PRs are expressed in malignant and normal colonic mucosa. ERs appear to be a feature of the colonic mucosa rather than the malignant process, but in carcinoma may regulate synthesis of PRs, suggesting a degree of oestrogen responsiveness.

Effects of a novel co-culture system on development, metabolism and gene expression of bovine embryos produced in vitro

An in vitro model using co-culture of bovine in vitro-produced (IVP) embryos and bovine oviduct epithelial cells (bOECs) was established to study embryo-maternal interactions in the oviductal environment. In vitro conditions maintaining differentiated growth of oviductal cells were determined by evaluating several media supplemented with different sera at various concentrations. Morphological features were used as indicators of physiological growth, and it became obvious that synthetic oviduct fluid (SOF) supplemented with either oestrous cow serum (OCS) or dextran-coated charcoal-treated fetal calf serum (DCC-FCS) helped to prevent dedifferentiation of bOECs (Expt 1). RT-real-time-PCR analysis revealed an increased mRNA content of the oviduct-specific glycoprotein GP 85-97 when using lower serum concentrations (2 and 5% compared with 10%; Expt 2). In subsequent experiments in which cell-free cultured controls and co-cultured embryos were compared, co-cultured embryos showed an increased rate of cleavage (P < 0.05) after 3 days. Successive cell-free culture until day 8 resulted in a lower rate of blastocyst development (P < 0.05) and reduced ATP content (P < 0.05) of co-cultured versus control embryos (Expt 3). Long-term co-culture (8 days) in SOF with 5% OCS increased the expression of developmentally relevant genes (glucose transporter 1 (Glut-1) and heat shock protein (HSP 70)) in co-cultured versus control embryos (Expt 4). Higher embryonic Glut-1 mRNA expression after co-culture was obvious when using 10% DCC-FCS, but was not significant when culture medium was supplemented with 10% rather than 5% OCS (Expt 5). In conclusion, SOF with 5% OCS supports differentiated growth of bOECs. Co-culture under these conditions improves early cleavage rate, but not blastocyst development, and increases the expression of developmentally relevant genes influenced by type of serum and serum concentration.

Effects of a novel co-culture system on development, metabolism and gene expression of bovine embryos produced in vitro

An in vitro model using co-culture of bovine in vitro-produced (IVP) embryos and bovine oviduct epithelial cells (bOECs) was established to study embryo-maternal interactions in the oviductal environment. In vitro conditions maintaining differentiated growth of oviductal cells were determined by evaluating several media supplemented with different sera at various concentrations. Morphological features were used as indicators of physiological growth, and it became obvious that synthetic oviduct fluid (SOF) supplemented with either oestrous cow serum (OCS) or dextran-coated charcoal-treated fetal calf serum (DCC-FCS) helped to prevent dedifferentiation of bOECs (Expt 1). RT-real-time-PCR analysis revealed an increased mRNA content of the oviduct-specific glycoprotein GP 85-97 when using lower serum concentrations (2 and 5% compared with 10%; Expt 2). In subsequent experiments in which cell-free cultured controls and co-cultured embryos were compared, co-cultured embryos showed an increased rate of cleavage (P < 0.05) after 3 days. Successive cell-free culture until day 8 resulted in a lower rate of blastocyst development (P < 0.05) and reduced ATP content (P < 0.05) of co-cultured versus control embryos (Expt 3). Long-term co-culture (8 days) in SOF with 5% OCS increased the expression of developmentally relevant genes (glucose transporter 1 (Glut-1) and heat shock protein (HSP 70)) in co-cultured versus control embryos (Expt 4). Higher embryonic Glut-1 mRNA expression after co-culture was obvious when using 10% DCC-FCS, but was not significant when culture medium was supplemented with 10% rather than 5% OCS (Expt 5). In conclusion, SOF with 5% OCS supports differentiated growth of bOECs. Co-culture under these conditions improves early cleavage rate, but not blastocyst development, and increases the expression of developmentally relevant genes influenced by type of serum and serum concentration.

ICI 182,780 Stimulates the Growth of a Uterine Cell Line Derived from p53-Deficient Mice

Abstract UE8 is a uterine epithelial cell line established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture and is strongly positive for cytokeratin, but negative for vimentin in immunocytochemistry. UE8 shows an active growth in a phenol red free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 supplemented with 3% heat-inactivated and dextran-coated charcoal-treated fetal calf serum. Both immunocytochemistry and immunoblot analyses confirmed that UE8 was negative for the estrogen receptor. Diethylstilbestrol added to the medium at concentrations between 10−6 and 10−8 M had no significant effect on the proliferative rate, and estradiol-17β at 10−6 M was slightly inhibitory. Unexpectedly, however, the“pure anti-estrogen”ICI 182,780 at 10−6 M significantly enhanced cell proliferation. This is the first observation that the“pure anti-estrogen”ICI 182,780 stimulates the growth of uterine epithelial cells without estrogen receptors.

Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer

Supplementation of synthetic oviduct fluid (SOF) medium plus amino acids and bovine serum albumin (BSA) with either fetal calf serum (FCS) or charcoal-treated FCS (CT-FCS) from Day 5 of development was investigated to determine if either in vitro or post-transfer development was altered. Development to the compact morula stage or beyond was similar for all 3 treatments. However, blastocyst development at Day 7 was accelerated when serum was added to the medium (21.6, 40.1 and 39.4% blastocysts from cleaved embryos for BSA, FCS and CT-FCS, respectively; P<0.01), but cell number of the resulting embryos was unaffected. Furthermore, addition of CT-FCS decreased the between replicate variation in embryo development and produced more Grade 1 and 2 quality embryos (25.8%) than BSA supplementation (18.1%; P<0.05). The transfer of Grade 1 and 2 embryos at Day 7 following culture resulted in similar pregnancy and embryo survival rates for the 3 treatments, with a tendency for lower embryo survival of embryos cultured in FCS (embryo survival at Day 50 = 37.7% vs 53.3% and 57.6% for FCS, BSA and CT-FCS, respectively; P ∼ 0.1). Significant fetal loss from Day 50 to term occurred within all 3 treatments. There were no birth weight differences for calves amongst the 3 culture treatments; however, one of the sires produced calves that were significantly heavier than expected, suggesting a possible sire-by-embryo interaction. These results demonstrate that addition of FCS may promote blastocyst development; however, there was also a tendency for lower embryo survival. Thus charcoal treatment of FCS is recommended, because it decreases variability in embryo development between runs and results in embryo survival rates to term similar to that of BSA-supplemented media.

Dexamethasone Inhibits the Growth of a Uterine Cell Line Derived from p53-Deficient Mice

UE8 is one of several uterine cell lines established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture; it is strongly positive for cytokeratin, but negative for vimentin. Immunoblot analysis confirmed that UE8 has glucocorticoid receptors. It grows actively in medium supplemented with 3% heat-inactivated, dextran-coated charcoal-treated fetal calf serum. Dexamethazone (DEX) inhibited its growth dose-dependently at concentrations between 10−6and 10−9 M. The inhibition by DEX was further examined in chemically defined medium (CDM): DMEM/F12 containing transferrin (10 μg/ml), selenium (10−8 M) and 0.1% bovine serum albumin. DEX at 10−6 M inhibited the growth of UE8 in CDM or in CDM supplemented with insulin-like growth factor-1 (IGF-1: 10 ng/ml), but there was no inhibition by DEX(10−6M) in the presence of epidermal growth factor (10 ng/ml). Thus, DEX inhibits the growth of a uterine epithelial cell line derived from p53-deficient mice in vitro, and also suppresses IGF-1-induced proliferation.

Hormonal Regulation of the p53 Tumor Suppressor Protein in T47D Human Breast Carcinoma Cell Line

Under normal culturing conditions, the T47D human breast cancer cell line expresses progesterone receptor constitutively and is responsive to estrogen. Because the tumor suppressor protein p53 plays a central role in determining genetic stability and cell proliferation, we have examined the effects of 17 beta-estradiol, the synthetic progestin R5020, and the antiprogestin RU486 on the levels of this protein in T47D cells. Western blot analysis of cellular extracts, performed with a monoclonal antibody capable of quantitatively supershifting a specific p53-p53 response element complex in a gel mobility shift assay, detected a single immunoreactive band representing p53. When cells were grown for 4-5 days in culture medium containing charcoal-treated fetal calf serum, p53 levels declined to 10% of the level seen in the control (no charcoal treatment) group. Supplementation of culture medium containing charcoal-treated calf serum with 0.1-1 nM 17 beta-estradiol restored p53 to its normal levels. A 4-day treatment of cells with R5020 or RU486 lowered the p53 levels in cells grown in normal culturing conditions to 15 and 30% of control levels, respectively. R5020 and RU486 treatments also caused down-regulation and/or hyperphosphorylation of the progesterone receptor, which correlated with the down-regulation of p53. These observations by estradiol while R5020 down-regulates this protein. Since estradiol is known to promote cell proliferation, the induction of p53 observed in this study leads us to propose that estradiol stimulates p53 to regulate proliferation of T47D cells in culture.

Glucocorticoids enhance the potency of Schwann cell mitogens

Previous studies have documented that cultured Schwann cells require serum-containing medium to respond maximally to mitogens. We now report that Schwann cells are able to proliferate to a mitogenic response in a serum-free defined medium termed oligodendrocyte defined media (ODM). Glucocorticoids are the essential component of ODM which allow Schwann cell proliferation in the serum-free medium. Charcoal treatment of the fetal calf serum decreases the mitogenic potency of the axolemma-enriched fraction (AEF) by 50%. The addition of 2 microM hydrocortisone to charcoal-treated fetal calf serum restores 75% of the lost mitogenicity. These observations are consistent with the view that glucocorticoids present in fetal calf serum are potent co-mitogens essential for AEF-induced Schwann cell proliferation. The synthetic glucocorticoid, dexamethasone, is a more potent co-mitogen than hydrocortisone, with a maximal effect at concentrations less than 10 nM. In contrast, other steroids including aldosterone, progesterone, testosterone, and 17 beta-estradiol have no effect on enhancing the mitogenic response of Schwann cells to the AEF. The glucocorticoid antagonists RU 486 and dehydroepiandrosterone (DHEA), but not the antiestrogenic compound tamoxifen, block AEF-induced Schwann cell proliferation. These results suggest that glucocorticoid-induced Schwann cell proliferation is mediated through a glucocorticoid receptor (GR) mechanism. We detected immunoreactivity to the GR in the cytoplasm, but not in the nuclei of Schwann cells grown in ODM lacking dexamethasone. The addition of 100 nM dexamethasone to these cultures resulted in immunoreactivity in the nucleus. This data suggests that glucocorticoids working through the GR are potent co-mitogens for Schwann cell proliferation.

Thyrotropin-releasing hormone gene expression in the anterior pituitary. II. Stimulation by glucocorticoids.

The present studies were undertaken to determine whether glucocorticoids (GC) regulate TRH gene expression in cultured anterior pituitary (AP) cells. AP cells derived from 15-day-old male rats were cultured for up to 18 days in Dulbecco's Modified Eagle's Medium-L-15 medium supplemented with 1) fetal calf serum (FCS), 2) charcoal-treated FCS, 3) normal rat serum, or 4) serum from rats that were adrenalectomized, rendered hypothyroid, and gonadectomized (ATG rat serum). Dexamethasone (Dex) or corticosterone (Cort) was added to the culture medium at various concentrations with exposure times ranging from 4-18 days. TRH and prepro-TRH-(25-50) in cellular extracts and release media were measured by RIA, and pro-TRH mRNA was determined by Northern blot analysis and in situ hybridization. Dex substantially stimulated cellular TRH and prepro-TRH-(25-50) accumulation under all culture conditions investigated, i.e. in medium supplemented with any of the four sera. TRH gene expression did not occur in medium supplemented with charcoal-treated FCS or ATG rat serum. Pretreatment with 10(-8) M Dex caused a significant increase in basal as well as cAMP- or phorbol ester-stimulated release of the peptide. Steady state pro-TRH mRNA levels rose 6.8- and 4.2-fold (both P < 0.01) after treatment with 10(-8) M Dex for 4 and 12 days, respectively. In situ hybridization experiments revealed that this rise in pro-TRH mRNA levels was probably the result of an increase in the number of AP cells expressing pro-TRH. Both Dex and Cort caused a dose-dependent increase in TRH accumulation, but Cort was approximately 40 times less potent than Dex. These results indicate that GC stimulate TRH gene expression in cultured AP cells. The presence of GC in culture medium is a prerequisite for the occurrence of TRH gene expression in the AP. As GC have been reported to reduce pro-TRH mRNA levels in the hypothalamus in vivo, our results may provide an example of the tissue-specific effects of GC on TRH gene expression.

Interference between estradiol and L-triiodothyronine in the control of the proliferation of a pituitary tumor cell line

We showed that the proliferation of F 4Z 2 cells, established from a rat pituitary tumor, was stimulated by L-triiodothyronine (LT 3) in the presence of 3 or 0.1% charcoal treated-fetal calf serum (CT-FCS), and by estradiol (E 2) in the presence of 3% CT-FCS only ( Cancer Res., 50, 1990, 3786). Here we report on the consequences of the simultaneous addition of these hormones. In the presence of 0.1% CT-FCS, E 2, and with a lower potency, estrone and estriol inhibited dose-dependently stimulation by LT 3. The results were more complex with 3% CT-FCS: the LT 3 effect was blunted by E 2 > 0.1 nM (and vice versa) and the hormone effects were additive at lower concentrations. E 2, at concentrations antagonistic to the effects of LT 3, decreased LT 3 binding and LT 3 receptor mRNA without modifying the effect of LT 3 on these mRNAs. In addition, E 2 blocked both the LT 3-induced increases of insulin-like growth factor-I (IGF-I) secretion and IGF-I mRNA concentration, and the LT 3-induced decrease of IGF-binding proteins in conditioned culture medium. We propose that E 2 prevents LT 3 stimulation of F 4Z 2 cell proliferation by blunting the LT 3-induced accumulation of unbound IGF-I in the culture medium.

Sex steroids induced up-regulation of 1,25-(OH) 2 vitamin D 3 receptors in T 47D breast cancer cells

There is evidence indicating that 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] through binding to its specific receptor (VDR) exerts an antiproliferative effect on breast cancer cells. Considering the importance of receptor regulation in modulating the target cell responsiveness to hormones, the effect of dihydrotestosterone (DHT) and estradiol-17β(E 2) on the regulation of VDR number was investigated in T 47D human breast cancer cells that also express androgen and estrogen (ER) receptors. T 47D cells were grown in RPMI medium containing 10% charcoal-treated fetal calf serum and the receptor content was determined in cells at confluence. Whole cell binding studies confirmed the presence of highly specific, saturable (4.01 ± 1.82 fmol/10 6 cells), high affinity ( K d = 0.079 ± 0.058 × 10 −9 M) 1,25(OH) 2D 3 receptors in control cells. Exposure to 10 −7 M DHT for 72 h resulted in a significant increase in VDR levels. Similar results were obtained with 10 −7M E 2. DHT- and E 2-induced up-regulation was completely suppressed by 10 −6 M tamoxifen (TAM) addition but unaffected by 10 −6 M flutamide. TAM treatment alone produced a significant dose-dependent increase in VDR content, that was maximal at 10 −6 M. Our data strongly suggest, for the first time, an up-regulation of VDR by DHT and E 2 via an ER-mediated mechanism.

Phorbol esters do not influence the maturation and the beta-adrenergic responsiveness of differentiated adipose precursor cells, cultured in a charcoal-treated serum medium.

Phorbol esters are a well defined group of tumor-promoting substances, which influence the proliferation, the differentiation and the hormonal responsiveness of a wide variety of cell types through the activation of protein-kinase C. Opposite effects have been described on the beta-adrenoceptors and the adenylate cyclase activity, depending on the cell type. Since there is no information about the influence of these substances on lipolysis in adipose precursor cells, the aim of the present study was to examine the effect of 7 days exposure to phorbol esters on the differentiation and the lipolytic responsiveness of rat preadipocytes, cultured in 10% charcoal-treated fetal calf serum. Our results demonstrate that prolonged exposure to these tumor promoters does not influence the differentiation, the beta-adrenoceptors number and the lipolytic response to adenylate cyclase or beta-adrenergic agonists in differentiated cells.

Multihormonal Control of Cell Proliferation: Opposite Effects of Two Stimulators (17β-Estradiol and L-Triiodothyronine) and One Inhibitor (Dexamethasone) on F4Z2Pituitary Tumor Cells*

From the MtTF4 tumor of rat pituitary origin we established the F4Z2 cell line whose growth is stimulated by 17 beta-estradiol (E2). Taking E2 actions as references we investigated actions of other effectors on the proliferation, protein, and insulin growth factor-I (IGF-I) secretions of F4F2 cells. Dexamethasone (Dex) and L-T3 were chosen because they have also intracellular receptors and they act in pituitary cells. Cells were cultured in 96-well plates in RPMI 1640 medium supplemented either with charcoal-treated fetal calf serum (CT-FCS) or with BSA and transferrin. Hormones were added at the time of seeding and cells were counted 2-10 days later without renewing the culture medium. The accumulation of immunoreactive IGF-I in conditioned medium was used as an index of IGF-I secretion. For studies on protein secretion, cells were incubated for 24 h with [35S]methionine and labeled proteins were separated by polyacrylamide gel electrophoresis. We found that: 1) L-T3, like E2, stimulated in a dose-dependent and specific manner the proliferation of F4Z2 cells cultured in the presence of 5% CT-FCS; EC50 was: 1 X 10(-11) M and 0.2 X 10(-11) M for L-T3 and E2, respectively. In contrast, L-T3 but not E2 remained active in serum-free medium; 2) Dex was a strong inhibitor of cell proliferation in serum-free medium and in medium supplemented with 5% CT-FCS (EC50: 5 X 10(-9) M). The antiglucocorticoid RU 38 486 prevented this inhibitory effect; 3) when a stimulator (E2 or L-T3) was simultaneously incubated with the inhibitor (Dex) the number of cells depended on the ratio of hormone concentrations. When there was no large excess of one effector this number was intermediary between those counted in the presence of each hormone separately and L-T3 was more potent than E2 in preventing Dex inhibition; 4) Dex, E2, and L-T3 modified the electrophoretic patterns of secreted proteins but there was no evidence for a correlation between these modifications and the inhibition or the stimulation of cell proliferation, and 5) the accumulation of immunoreactive IGF-I was insensitive to E2, increased by L-T3, and markedly decreased by Dex. L-T3 but not E2 prevented the effect of Dex.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of culture conditions on the estrogenic cell growth stimulation of human breast cancer cells

17β-Estradiol is a potent mitogen for hormone-dependent cell lines (MCF-7, T47D and ZR 75.1). However, the degree of hormone sensitivity is very much influenced by culture conditions. In order to understand which factors modulate estrogenic effects on cell growth, four different culture conditions were used: (a) medium with dextran-coated charcoal-treated fetal calf serum (DCC-FCS); (b) medium with dextran-coated charcoal-treated growth factor-inactivated serum (DCC-FCSd); (c) serum-free medium, after a 24-h incubation with serum to allow cell attachment; and (d) serum-free medium on collagen IV-treated plates. In all cell lines the highest cell growth stimulation was achieved when estradiol was added in the presence of 5% DCC-FCS, whereas reducing or removing serum from the culture medium resulted in a decrease in cell proliferation stimulation. We postulate that serum contains some still unknown components able to modulate the degres of estrogenic action in endocrine-dependent breast cancer cell lines.

Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures

Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady- state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units- erythroid (BFU-E) colonies. There was a high correlation of gamma- globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma- globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma- globin expression in adult erythroid progenitor cell cultures.

Fetal Calf Serum Contains Activities That Induce Fetal Hemoglobin in Adult Erythroid Cell Cultures

Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady-state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units-erythroid (BFU-E) colonies. There was a high correlation of gamma-globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma-globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma-globin expression in adult erythroid progenitor cell cultures.

Effects of 1,25‐dihydroxyvitamin D3 on cell‐cycle kinetics of T 47D human breast cancer cells

The replication of several human and animal cancer cell lines is regulated in vitro and in vivo by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonally active form of vitamin D3. We have examined the effects of concentrations of 1,25-(OH)2D3, which inhibit cellular replication, on the cell-cycle kinetics of a 1,25-(OH)2D3-responsive human breast cancer cell line, T 47D. After 6 or 7 days of treatment, a time period representing approximately five cell population doublings of control cultures, concentrations of 1,25-(OH)2D3 in the range 10(-9) M to 10(-6) M caused a time- and concentration-dependent decrease in cell numbers. Treatment of cells growing in charcoal-treated fetal calf serum with 10(-8) M 1,25-(OH)2D3 for 6 days reduced cell numbers to 49% +/- 9% (n = 9) of control, and this was associated with a marked increase in the proportion of cells in the G2 + M phase of the cell cycle from 9.7% +/- 0.5% (n = 11) to 19.6% +/- 2.3% (n = 9), significant by paired analysis (P less than 0.002). At higher concentrations of 1,25-(OH)2D3 (10(-7)-10(-6) M), there was a concentration-dependent decline in S phase and increases in both G0/G1 and G2 + M phase cells. Detailed analysis of the temporal changes in cell-cycle phase distribution following treatment with 2.5 X 10(-8) and 10(-7) M 1,25-(OH)2D3 showed an initial accumulation of cells in G0/G1 and depletion of S phase cells during the first 24 hr of treatment. This decline in S phase cells was not accompanied by a decline in % G2 + M indicating a transition delay in G2 or mitosis. At the lower dose these changes returned to control values at 48 hr and at later times were associated with a slight but consistent decline in G0/G1 phase and an increase in G2 + M. In contrast cells treated with 10(-7) M 1,25-(OH)2D3 had significantly elevated % G0/G1 cells at days 2 and 3, consistent with a transition delay through G1 phase. This was confirmed in stathmokinetic experiments which demonstrated an approximate sevenfold decrease in the rate of exit of cells from G0/G1 following 4 days of exposure to 10(-7) M 1,25-(OH)2D3. This accumulation of cells in G0/G1 was accompanied by a fall in % S phase cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Mitogenic effect of estradiol on MCF-7 human breast cancer cells can be modulated by serum.

The MCF-7 human breast cancer cell line responds to estradiol stimulation in vitro by increased proliferation only if prolonged subcultures in dextran-coated charcoal-treated fetal calf serum have been made previously. This growth stimulation is not obtained when cells are grown in medium containing 5% untreated fetal calf serum. We describe here the culture conditions under which we obtain a reproducible estradiol effect on cell growth.