Effects and interactions of epidermal growth factor, insulin, hydrocortisone, and estradiol on the cloning of human tumor cells

We investigated the effects and interactions of epidermal growth factor (EGF), insulin, hydrocortisone, and estradiol on the growth of 18 freshly obtained human tumors in our human tumor stem cell assay (HTSCA) cultured at a reduced serum concentration (8.5% ml). All possible combinations of these four supplement factors were added to the assay to determine the ability of each component to enhance colony formation. We found that hydrocortisone was the most effective single supplement in stimulating colony growth in the HTSCA. Supplementation with insulin, estradiol, or both had some growth-promoting effect but not as great as hydrocortisone. Moreover, the addition of insulin, estradiol, or both often demonstrated a negative interaction with hydrocortisone. EGF supplementation alone; in dual combination with insulin, estradiol, or hydrocortisone; or in combination with estradiol and insulin in the assay did not significantly increase colony formation. However, EGF added to the cultures containing hydrocortisone with insulin and/or estradiol significantly increased colony formation and reversed the negative effect of insulin and estradiol on hydrocortisone activity. Thus, under conditions of our assay, the most effective combination in promoting colony growth contained all four factors.     

Effect of Epidermal Growth Factor on Follicular Development and Steroidogenesis in Perfused Rat Ovary

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Role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies

The biological role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-alpha and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E2)-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control). E2-stimulated colony growth in 5 tumors (167% of control) and the antibody blocked E2-stimulation in 3 of the 5. Inhibition of E2-stimulated growth in 3 and basal growth in 4 other tumors by the EGF receptor antibody suggest that endogenously secreted TGF-alpha has a role as an autocrine/paracrine growth factor in constitutive and E2-stimulated tumor cell proliferation in a majority of human tumors. A polyclonal antibody against TGF-alpha was used to study the role of TGF-alpha in E2-, prolactin(Prl)- and progesterone(Prog)-stimulated proliferation of NMU(nitrosomethylurea)-induced rat mammary tumor cells under similar culture conditions. TGF-alpha, E2, Prl and Prog stimulated colony growth equally to 176, 187, 168 and 181% of control. The antibody produced significant and similar inhibition of TGF-alpha and E2-stimulated growth (95 and 83%). In contrast, inhibition of Prl- and Prog-stimulated growth by the antibody was only 24 and 37%. The TGF-alpha ligand antibody did not have an agonist or antagonist effect when added alone. Thus, TGF-alpha seems to be a major stimulatory growth factor mediating E2-induced tumor cell proliferation in rat mammary tumors. It is less important in Prl- and Prog-induced tumor growth and not essential for basal growth in these tumors. We conclude that TGF-alpha is a biologically important autocrine/paracrine growth factor in primary human breast cancer cell proliferation and in E2-induced rat mammary tumor growth.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Ovarian steroid synthesis in tissue culture after administration of hormones and epidermal growth factor

The features of steroidogenesis of immature mouse ovaries in culture under the influence of follicle-stimulating hormones (FSH), human chorionic gonadotropin (hCG), epidermal growth factor (EGF) and insulin have been investigated during the period of reinitiation of meiosis in the oocytes. Secretion of progesterone is stimulated after addition of FSH, hCG and of insulin and EGF combination to the medium. EGF increases FSH-stimulated progesterone secretion and inhibits estradiol secretion. The ratios progesterone/estradiol and testosterone/estradiol increase, when EGF is added to the culture medium. It is analogous to the action of hCG. It is suggested that EGF may be an intrafollicular EGF regulator of luteinizing hormone action on the sex and somatic cells of the mammalian ovaries.     

Three novel hormone-responsive cell lines derived from primary human breast carcinomas: functional characterization

Human breast cancer primary cultures are useful tools for the study of several aspects of cancer biology, including the effects of chemotherapy and acute gene expression in response to different hormonal/chemotherapy treatments. The present study reports the conditions for primary culture of breast cancer samples from untreated patients and the most effective collagenization method to dissociate human samples consisting in an overnight incubation with 1 mg/ml types II or IV collagenase and further incubation in DMEM:F12 (1:1) medium supplemented with glutamine, bovine insulin, penicillin-streptomycin, HEPES, estradiol, cortisol (F), tri-iodothyronine (T(3)), transferrine (TR), and 10% fetal calf serum (FCS). These conditions proved to be appropriate for both primary culture and the development of stable cell lines. Of the seven cell lines obtained, three fast growing and estrogen receptor (ER)+/progesterone receptor (PgR)+/EGF receptor (EGFR)+ have been characterized. The cells are able to grow both in soft agar and in nude mice, and express cytokeratins, all parameters characteristic of malignant epithelial cell lines. The cells also exhibit an increased proliferation rate in the presence of estradiol, progesterone, and EGF, suggesting the presence of the corresponding receptors. The mRNA expression of type alpha- and beta-ER as well as EGFR, was confirmed by RT-PCR. In conclusion, the novel cell lines described, arose from primary tumors and are sensitive to estradiol, progesterone, and EGF. This not only expands the repertoire of breast cancer cells available as potentially useful tools for examining most parameters in breast cancer "in vitro", but also provides unique new models to explore the complex regulation by steroids as well as growth factors in such cells.     

Sensitivity and specificity of the bioassay of estrogenicity in mammary gland and seminal vesicles of male mice

Young intact (18 days old) and adult castrated males of CBA and C3H/Di mice were used for measuring the estrogenicity on the basis of growth response of mammary epithelial structures and the weight of seminal vesicles. It was demonstrated that heavier young males had disproportionally heavier seminal vesicles (sex steroid-responsive organs) than small animals at day 33 of age (that is on the day when experimental animals were killed and organs dissected). However, the weight of the spleen (sex steroid-nonresponsive organ) was proportionally related to body weight. To minimize variability in hormone responsiveness, all animals were weighed at the age of 18 days and only males weighing 8+/-1 g were used for hormone treatment. The percentage area of mammary fat pad occupiedby mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.01 microg x d(-1). The maximum effective dose of estradiol was 0.1 microg x d(-1) and dose 10 microg x d(-1) of estradiol decreased mammary size to control level (inverted-U-shaped dose-response curve). Progesterone alone stimulated mammary growth only in high doses (500 microg x d(-1) and higher) in young intact males, but had no effect on mammary growth in adult castrated animals. In young intact males, estradiol alone, or progesterone alone decreased the weight of seminal vesicles. No such inhibitory effect of these hormones was noted in adult castrated males. Progesterone acted synergistically with estradiol to produce higher mammary growth compared to that in males treated with estradiol alone. In the presence of progesterone seminal vesicles weight was decreased by estradiol given in such low doses as 0.001 microg x d(-1) of estradiol, which is 10 times lower than that effective in animals treated with estradiol alone. On the other hand, in the adult castrated males a combination of estradiol plus progesterone stimulated seminal vesicles weight. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate (a synthetic steroid exhibiting progestantial and estrogenic activities) and inhibited by both testosterone and cortisol. Estradiol, progesterone, norethindrone acetate, or testosterone did not affect spleen weight and size of mammary lymph nodes.However, cortisol significantly decreased not only spleen weights but also size of mammary lymph nodes. These results showthat simultaneous evaluation of mammary gland growth, seminal vesicles, and the spleen weight in the same animal is suitable for bioassay of estrogenicity as well as for detection of androgenic and antiandrogenic activities.     

Sensitivity and specificity of bioassay of estrogenicity on mammary gland and uterus of female mice

Young intact (18 days of age) and adult ovariectomized (OV-X, ovariectomized between 21 to 24 days of age) C3H/Di mice were used to measure the estrogenicity on the basis of the growth response of mammary epithelial structures and weight of the uterus. The percentage area of the mammary fat pad occupied by mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.001 microg.d(-1). The maximum effective dose of estradiol was 0.01 microg.d(-1) and the dose 10 microg.d(-1) of estradiol decreased mammary size to control levels (inverted-U-shaped dose-response curve). Progesterone alone progressively stimulated mammary growth in young intact females from dose 125 microg.d(-1), in adult OV-X animals from dose 1000 microg.d(-1). Both in young intact and adult OV-X animals, uterine weight progressively increased during estradiol treatment. Progesterone alone had no effect on uterine weight in young intact animals; in adult OV-X animals, uterine weight was increased starting from dose 250 microg.d(-1). Progesterone acted synergistically with estradiol to produce higher mammary growth than that in females treated with estradiol alone. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate and inhibited by cortisol in both young intact and adult OV-X animals. Testosterone inhibited estradiol plus progesterone stimulated growth of mammary gland only in OV-X animals, but stimulated uterine weights in both young intact and adult OV-X animals. Spleen weight and size of mammary lymph nodes were not affected by estradiol, progesterone, norethindrone acetate or testosterone, but were decreased by cortisol. Cortisol also decreased the percent area of the mammary fat pad occupied by mammary epithelial structures, but had no effect on weight of the uterus. These results show that bioassay of estrogenicity in females is not specific. Mammary and uterine growth is stimulated not only by estrogens but also by progesterone and testosterone, respectively.     

Effect of growth factors, EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes

The objective of these experiments was to determine the effect of exogenous addition of insulin-like growth factor-I (IGF-I, 100 ng/mL), epidermal growth factor (EGF, 10 ng/mL) and estradiol (E2, 100 ng/mL) to the maturation medium of sheep oocytes on their subsequent development in vitro. Addition of IGF-I to the maturation medium did not improve nuclear or cytoplasmic maturation of sheep oocytes at the concentration tested. However, EGF improved significantly the resumption of meiosis (84% oocytes in metaphase II stage after IVM vs. 59% in medium alone). Cleavage rate and blastocyst development rates were improved (P<0.01) after addition of EGF (60% and 29%, respectively), as compared with maturation in TCM 199 alone (39% and 19%, respectively), but remained lower than rates observed after maturation in complete medium containing follicular fluid (FF, 10%) and FSH (81% and 35%, respectively). No additive effect of EGF over FSH was observed during these experiments. Addition of FF to FSH containing maturation medium improved significantly both cleavage (P<0.001) and blastocyst rates (P<0.05). Addition of E2 to the IVM medium is not required when medium already contains FF. However, in defined conditions supplementation of maturation medium with E2 had a positive effect. These results suggest that EGF, FSH and E2 may play an important role in the nuclear and cytoplasmic maturation of sheep oocytes in vitro.     

The interaction of testosterone and gonadotropins in stimulating estradiol and progesterone secretion by cultures of corpus luteum cells isolated from pigs in early and midluteal phase.

The first objective of this research was to define the capacity of corpora lutea of pig to secrete estradiol in the presence of an androgen substrate which was testosterone. The second objective was to define the synergism between gonadotropic hormones such as LH, FSH, and PRL and testosterone as measured by estradiol and progesterone secretion by two types of porcine luteal cells. Luteal cells were collected from newly forming corpora lutea (0-3 days after ovulation) and from mature corpora lutea (8-10 days after ovulation). After dispersion, luteal cells were suspended in medium M199 supplemented with 10% of calf serum and grown as monolayers at 37 degrees C. Control cultures were grown in medium alone while other cultures were supplemented with either testosterone alone at a concentration of 1 x 10(-7) M or with 10, 100, 500 ng LH plus testosterone, 10, 100, 500 ng FSH plus testosterone or 10, 100, 500 ng PRL plus testosterone. After 2 days of cultivation all cultures were terminated and media were frozen at 20 degrees C for further steroid analysis. Testosterone added to the culture medium in the absence of gonadotropins was without effect on estradiol and progesterone secretion by luteal cells collected in the corpora lutea of the early luteal phase. On the other hand testosterone added to the medium significantly increased progesterone and estradiol secretion by cultured luteal cells collected in the midluteal phase of the cycle. No additive stimulatory action of gonadotropins and testosterone on progesterone secretion was observed in cultures of luteal cells from the early luteal phase but this was not the case in cultures of luteal cells from the midluteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of progesterone and estrogen in development of uterine tone in mares

In two experiments (30 mares/experiment), the uterus was recorded as having flaccid tone characteristic of estrus or seasonal anestrus (tone score 1), intermediate tone characteristic of diestrus (tone score 2), or increased or maximal tone characteristic of early pregnancy (tone score 3 or 4). In Experiment I (five mares/group), uterine tone in seasonally anovulatory mares was not altered significantly from the flaccid state by daily administration of 100 mg progesterone plus 1 mg estradiol 17beta or 1 mg estradiol 17beta alone. Uterine tone in seasonally anovulatory mares receiving 100 mg progesterone alone increased to intermediate level (score 2; P<0.05) and remained there throughout the treatment period. Tone scores in the group receiving a 14-d progesterone priming period followed by progesterone plus estradiol were higher (P<0.02) on Days 16 to 28 than scores in the group receiving progesterone alone throughout the treatment period. In Experiment II, (five mares/group), steroid treatments were begun on Day 10 postovulation. The combination of 1 mg exogenous estradiol plus progesterone produced greater uterine tone than exogenous progesterone alone. There were no significant differences between the pregnant control group and the group receiving progesterone plus 1 mg estradiol. There were no significant differences between the group receiving progesterone alone and the group receiving progesterone plus 5 mg estradiol. Results supported the hypothesis that the maximum uterine tone of early pregnancy is caused by progesterone priming followed by exposure to low levels of estradiol plus continued exposure to progesterone.     

Action of estradiol-17 beta on the expression of c-Fos gene in endometrial cells in cultured

The c-fos expression was investigated in primary culture of guinea-pig endometrial cells. Cells were made quiescent by serum depletion. Stimulation of these cells by estradiol (E2, 10(-8)M) alone or in combination with epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) failed to induce c-fos gene. The c-fos expression was early and transiently increased by fetal calf serum (15%) or estradiol plus EGF plus insulin. Protein synthesis inhibitors (cycloheximide or anisomycin) in association with E2 induced a superinduction of c-fos gene. In the same conditions puromycin had no effect. It appears that E2 acts in a multiple step process including an initial c-fos gene derepression by either EGF plus insulin or some protein synthesis inhibitors.     

卵泡刺激素和表皮生长因子对小鼠精原细胞增殖的影响

利用生殖细胞-体细胞体外无血清共培养模型研究了卵泡刺激素(FSH)和表皮生长因子(EGF)对小鼠A型精原细胞增殖的影响。精原细胞在ITS培养液(添加胰岛素、转铁蛋白和亚硒酸钠的DMEM)中培养24h后进行c-kit免疫细胞化学鉴定和EGF及其受体(EGFR)免疫细胞化学检测,72h后测定其形成集落数的情况。结果表明:ITS培养液能维持生殖细胞的活性,增殖细胞核抗原(PCNA)的表达增高。A型精原细胞呈c-kit阳性,EGF和EGFR主要表达于精原细胞。单独的FSH(1～100ng/ml)或EGF(1～10ng/ml)显著促进精原细胞集落数的增加。此外,EGF(0.1ng/ml)联合FSH(10ng/ml)具有加性效应,但更高剂量的EGF(1～10ng/ml)则降低了FSH的刺激作用。结果说明FSH可联合适量的EGF促进精原细胞的增殖。     

The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells

Summary The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5×10³/cm² in the presence of 2×10⁴/cm² lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10⁻¹⁰ M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.     

Modulation of 17 alpha-hydroxylase/C17,20-lyase activity in porcine theca cells

The major source of ovarian androgen is the theca cells. Androgens are produced by the conversion of progestins by the 17 alpha-hydroxylase/C17,20 lyase enzymatic system (lyase). The 3 beta-hydroxysteroid dehydrogenase and aromatase enzymes in the theca cells are modulated by gonadotropins as well as by steroids produced locally. Therefore, the combined effects of hCG plus progesterone, estradiol, or dihydrotestosterone (DHT) on microsomal lyase activity in theca cells from large and medium-sized follicles were determined. Theca cells (3 x 10(6) cells/6 ml/well) were cultured in Medium 199 (M199) containing only insulin (10 micrograms/ml) and transferrin (5 micrograms/ml). At 24 h, theca cells were treated with M199, hCG (15 ng/ml), progesterone, estradiol, or DHT (100 ng/ml) or a combination of hCG + one of the three steroids. Media were removed at various times of culture (27-72 h) and levels of androgen determined by RIA. Microsomes were incubated with 1 microCi [3H]progesterone +0.5 mM NADPH and radioactive conversion products were measured after purification by thin layer chromatography. Administration of progesterone, estradiol, or DHT alone had little effect on lyase activity in theca cells from medium-sized follicles whereas the addition of hCG alone significantly increased lyase activity in these cells. However, concomitant addition of any steroid with hCG inhibited the increase in lyase activity after the addition of hCG alone. Theca cells from large porcine follicles had a higher basal level of lyase activity compared to theca cells from the smaller follicles. Lyase activity in theca cells from large follicles was enhanced by progesterone; estradiol was inhibitory. DHT initially stimulated lyase activity in theca cells from large follicles, but was inhibitory later in culture. In contrast to its marked effect on theca cells from medium follicles, hCG had only a small effect on lyase activity in theca cells from large follicles. Thus, thecal lyase activity increased as the follicle matured, providing more androgen substrate for the production of estrogen. Lyase activity in theca cells of medium follicles appears to be regulated predominantly by gonadotropin from the pituitary while intraovarian regulation of lyase activity by steroids may be more important in larger follicles.     

Physiologic role of relaxin on mammary gland growth in rats

The role of relaxin in stimulating growth of the mammary gland was assessed in ovariectomized and intact male rats for a period of 20 days. In addition to relaxin alone, the ovarian mammogenic hormones estradiol and progesterone were used in combination with relaxin and with each other to evaluate responses of mammae. Indices for mammary growth included wet weight, dry fat-free tissue, DNA, RNA, total protein, and collagen. Quantitative estimates of DNA and collagen represented the best indicators of parenchymal and stromal growth, respectively. Because changes in body weights were significantly different among hormonally administered groups, these were included as well. In Ovariectomized young rats, relaxin alone and in combination with estradiol and progesterone increased all indices significantly (P less than 0.01). The collagenous portion of total protein was high for the group receiving relaxin alone (62%) compared with the control group (46%). Relaxin administered along with estradiol and progesterone increased collagen accumulation to 73%, compared with 54% in the estradiol + progesterone group. Relaxin did not significantly increase growth indices when administered to male rats at 10 and 20 micrograms/day, while 30 micrograms stimulated a significant increase in total protein (P less than 0.05), suggesting that 30 micrograms of relaxin/day may be considered the basal concentration needed to induce a physiologic response in males. Relaxin induced a growth effect on mammae by synergizing with progesterone and estradiol in order to stimulate parenchymal proliferation, as noted by a DNA increase, and to increase stromal distensibility of the mammary pad by invoking accumulation of collagen and total protein in substituting for mammary adipose tissue.     

Growth-promoting effects of progesterone in a human endometrial cancer cell line (Ishikawa-Var I).

Significant growth responses to progesterone of human endometrial adenocarcinoma cells (Ishikawa-Var I) were observed under in vitro culture conditions. Progesterone affected both the rate of exponential proliferation and cell population densities after the exponential phase. In the presence of the hormone, the doubling time of exponentially proliferating cells was reduced from 44 to 35.6 h and cell densities were increased by as much as 2-3 times over those of controls during approx. 2 weeks in culture. The effects of progesterone on cell population growth were dose dependent. Estradiol (10(-8) M) and testosterone (10(-6) M) did not affect cell densities and the effects of dexamethasone (10(-6) M) were small. In contrast, both progesterone and estradiol stimulated colony formation under anchorage-independent conditions in soft agar. These results suggest the possibility that growth of sensitive cell clones in endometrial tumors could be enhanced in some patients during adjuvant progestin therapy.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Distinctive effects of hydrocortisone on the modulation of EGF binding and cell growth in hela cells grown in defined medium

Hydrocortisone modulates the binding capacity of HeLa cells for ¹²⁵I-labeled epidermal growth factor (EGF). A twofold increase in ¹²⁵I-labeled EGF binding is observed within 24 hours after the addition of pharmacological concentration of hydrocortisone (5 × 10^?8?1 × 10^?6 M). This enhancement of binding is reversible, and occurs when the cells are cultured in either serum-supplemented or completely defined, serum-free, hormone-supplemented medium. Scatchard analysis of the binding data indicates that the number of ¹²⁵I-EGF binding sites is increased, and that no appreciable change in the affinity of the EGF receptor for labeled EGF occurs. In the serum-free condition hydrocortisone stimulates the growth of HeLa cells, but we have observed no connection between this growth stimulation and the enhancement of EGF binding. The growth response to hydrocortisone is independent of EGF, and the concentration dependency of the growth response to EGF is unaltered by the addition of hydrocortisone to the medium. Hydrocortisone elicits the growth response at a concentration as low as 5 × 10^?9 M, while a concentration higher than 5 × 10^?8 M is required to affect the binding capacity for ¹²⁵I-EGF. These effects are specific for glucocorticoid steroids. Similar concentrations of progesterone, testosterone, or estradiol produce no measurable response. Although the elevation of EGF receptor levels in the serum-supplemented medium is similar to that observed in the serum-free cultures, hydrocortisone is growth-inhibitory under these conditions. This growth inhibition occurs at pharmacological concentrations of hydrocortisone with a concentration dependency that is similar to that of the EGF receptor modulation.     

Epidermal growth factor effect on serum-free growth of primary and metastatic human tumors

The effect of epidermal growth factor (EGF) on the in vitro growth of human malignant tumors was compared in serum-supplemented (n = 54) and serum-free (N = 41) media at clonal density to determine the true EGF dependency of tumors. In the complete absence of serum at a 1,000 cells/cm2 seeding inoculation (approximately 100-200 adherent cells), EGF increased growth by greater than 50% in 27 of 41 specimens (66%), and growth increased by 100% or more in 18 of these EGF-sensitive tumors. In 12 serum-free cultures (29%), in vitro growth failed to occur without EGF. With 10% serum supplementation and a lower cell density (250 cells/cm2), EGF increased growth by greater than 50% in 34 of 54 specimens (63%), of which 25 had more than a 100% increase. The maximum growth induced by EGF in serum was usually seen in those tumors already capable of moderate in vitro growth. No difference in response to EGF was detected between specimens from primary tumors (n = 24) and those from metastases (n = 30). Under the stringent culture conditions of complete absence of serum and with tumors seeded at a low cell number, EGF stimulated most primary or metastatic human tumors to establish and sustain short-term in vitro growth successfully.