Influence of fetal bovine serum and hormones on primary vs. long-term cultures of human breast cancers

Summary We investigated the influence of fetal bovine serum, complemented or otherwise with estradiol or insulin or both, on the proliferation of mammary cancer cells from long-term and primary cultures. The long-term culture corresponded to mouse MXT and MCF-7 cell lines whereas the primary culture corresponded to primitive breast cancers squashed onto histologic slides and maintained in cultures for between 12 and 48 h. Cell proliferation was evaluated by means of digital cell image analysis of Feulgen-stained nuclei. Our results show that the addition of estradiol and insulin slightly but nevertheless significantly increases the proportion of cells still living at Hour 48 of culture. Fetal bovine serum, necessary for the growth of MXT and MCF-7 mammary cells, was highly cytotoxic with respect to the primary cultures of the 20 breast cancers under study. We are now conducting new experiments using chemically defined media to study the influence of new antineoplastic compounds on primary cultures of breast cancers.     

The effects of maternal undernutrition on maternal and fetal serum insulin-like growth factors, thyroid hormones and cortisol in the guinea pig.

The insulin-like growth factors (IGF-I and -II) are potential mediators of the effects of maternal undernutrition on fetal growth and muscle development. The effects of a 40% reduction in maternal feed intake on serum levels of the IGFs, the thyroid hormones and cortisol, were investigated for the last two trimesters (day 25 to birth). This level of undernutrition is known to cause a 35% reduction in fetal and placental weights, and a 20-25% reduction in muscle fibre number. Maternal IGF-I level was greater than non-pregnant levels on day 25 gestation, in both control and restricted dams, and declined with gestational age. The increase in IGF-I level in the 40% restricted group was approximately two-thirds that of control animals. Fetal serum IGF-I was also reduced in undernourished fetuses throughout gestation. Maternal IGF-II did not change with gestational age and was unaffected by undernutrition. Fetal IGF-II reached a peak at day 55 of gestation, this peak was greatly diminished by maternal feed restriction. Both IGF-I and IGF-II tended to be related to fetal, placental and muscle weights at day 65 of gestation. Thyroid hormone concentration declined in maternal serum and increased in fetal serum with increasing gestational age. Levels were not significantly affected by undernutrition. Both triiodothyronine (T3) and thyroxine (T4) were correlated with IGF-I in maternal serum (P < 0.05), but not in fetal serum. Cortisol levels were elevated by undernutrition in both maternal and fetal serum, and increased with gestational age. Cortisol was inversely correlated with serum IGF-I in both maternal and fetal serum. Maternal serum IGF-I may mediate the effects of undernutrition on fetal growth by affecting the growth and establishment of the feto-placental unit in mid-gestation. Fetal IGF-I may mediate the effects on muscle growth, whereas IGF-II seems to be related to hepatic glycogen deposition. Cortisol may play a role via its effect on the IGFs, but the thyroid hormones are unlikely to be important until the late gestation/early postnatal period.     

Effects of steroid hormones in fetal bovine serum on plating ang cloning of human cells in vitro.

Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-beta-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 mug per ml of progesterone. The addition to non-supporative sera of 0.1 mug per ml of 17-beta-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro.     

Obstetric importance,diagnosis, and management of fetal tachycardias.

During 1980-7, 23 pregnancies of 22-38 weeks'' duration were investigated for fetal tachycardia. Twelve were cases of supraventricular tachycardia, eight of atrial flutter, and three cases in which the rhythm varied between supraventricular tachycardia and atrial flutter. In 11 cases the fetus had developed non-immune fetal hydrops before referral; 12 cases were non-hydropic at referral but one of this group of fetuses became hydropic during treatment. No relation was found between the rate or type of arrhythmia and the presence or absence of intrauterine heart failure. One non-hydropic infant was delivered electively prematurely. Maternal antiarrhythmic treatment was instituted in the remaining 22 cases. Conversion of the arrhythmia was achieved with digoxin alone in five cases and with a combination of digoxin and verapamil in nine. Control of the arrhythmia was achieved in seven of the 10 non-hydropic fetuses, and all were delivered at term with no deaths. Of the 12 hydropic fetuses, control was achieved in seven. Only three of the hydropic fetuses were delivered close to term. There were two deaths, both in the hydropic group. Of the whole group, five neonates suffered severe complications of prematurity. In this series the main benefit of treatment appeared to be in prolonging gestation of those hydropic fetuses in which conversion was achieved.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum.

Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58 x 10(-6) M and a Vmax of 0.53 nmol per hr per 50 microliter serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8 x 10(-8) M. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 microliter serum, and the KM for putrescine, 50.3 x 10(-6) M. The K1 of the ABS putrescine oxidase for aminoguanidine was 41 x 10(-6) M. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56 degrees C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed.     

Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro

Summary Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro. Supported in part by NIH-NCI-EC2074.     

Placental hormones,genomic imprinting,and maternal—fetal communication

Placental hormones are produced by one genetic individual (the fetus) to act on the receptors of another genetic individual (the mother). Mothers are probably able to extract some information from placental hormones, but this information may be limited to a crude measure of fetal vigor. Placental hormones are most easily interpreted as fetal attempts to manipulate maternal metabolism for fetal benefit. An evolutionary model is presented for a hypothetical hormone that increases the nutrient content of maternal blood. The model predicts that, at an evolutionary equilibrium, the hormone will be produced solely by the mother or solely by the placenta, but not by both. If the gene for the hormone is subject to genomic imprinting, the paternally-derived allele will be active and the maternally-derived allele will be silent. Hormone production benefits the members of the mother's current litter at some cost to future litters. Therefore, paternity changes between litters increase the level of hormone production. On the other hand, offspring that produce less of the hormone than litter-mates share the benefits but have lower costs. Therefore, multiple paternity within litters reduces the level of hormone production.     

Amniotic fluid and plasma glycine/valine ratios in substrate deprived growth retarded fetal rats.

Characteristic profiles of the free amino acid concentration in umbilical cord blood of growth retarded newborns have been observed. We hypothesized that the amniotic fluid of growth retarded fetal rats would show an increase in the ratio between glycine and valine which would parallel the pattern observed in the cord blood of growth retarded neonates, thus providing an index for the antepartum identification of the substrate deprived growth retarded fetus. Six test and 6 control dams were tested. Four fetuses per dam, matched for uterine location were examined. Test animals were fasted for 72 hours. Sampling was performed on day 21 under anaesthesia. Fetal size was significantly reduced (P < 0.0001) in the test group. [T = 2.68 gs. +/- 0.28 vs. C = 3.67 gs. +/- 0.25]. Fetal plasma concentrations of glycine showed an increase in test animals (P < 0.01) while valine showed a significant reduction (P < 0.0001). Glycine (pm/microliters) T = 308 +/- 64 vs. C = 269 +/- 47, valine (pm/microliters) T = 424 +/- 79 vs. C = 671 +/- 218]. Amniotic fluid concentrations for both glycine and valine were significantly decreased (P < 0.0001) in test animals. [Glycine (pm/microliters) T = 710 +/- 124 vs. C = 931 +/- 178; valine (pm/microliters) T = 845 +/- 169 vs. C = 1,339 +/- 234]. The glycine/valine ratio was significantly increased (P < 0.01) in both fetal plasma and amniotic fluid in test animals [Plasma T = 0.74 +/- 0.18 vs. C = 0.43 +/- 0.13. Amniotic fluid T = 0.85 +/- 0.08 vs. C = 0.69 +/- 0.09]. Consistent with our hypothesis, the amniotic fluid concentrations generally parallel the observations made in the plasma. This finding could enhance the antepartum identification of the substrate deprived growth retarded fetus.     

Amniotic fluid steroid levels and fetal adrenal weight in congenital adrenal hyperplasia

The concentration of 17-OH-progesterone was determined in second trimester amniotic fluid collected from 58 pregnancies at risk for fetal 21-hydroxylase deficiency. The prediction was incorrect in 1 male nonsalt-loser who had an increased plasma 17-OH-progesterone concentration at 3 months of age. All 11 infants predicted to be affected were salt-losers. The adrenals from 2 affected fetuses available for study were significantly enlarged in comparison with adrenal size in 84 normal fetuses from 15 to 26 weeks' gestation. Amniotic fluid steroid analysis reliably predicts the fetus with 21-hydroxylase deficiency most at risk in early infancy. There is no evidence from this study to indicate that ACTH is not the main trophic factor for fetal adrenal growth and steroidogenesis.     

Prenatal diagnosis and significance of fetal infections.

Viruses like rubella, cytomegalovirus, varicella-zoster virus and parasites like Toxoplasma gondii can be transmitted from a pregnant woman to her fetus and can affect fetal development. Several factors determine the likelihood of fetal infection and the risk of consequences for the fetus, such as the timing of transmission during gestation or the immunologic status of the mother. No single diagnostic modality can be applied to all infections. Knowledge of the diagnostic methods available is essential for accurate counseling and treatment of affected pregnant women.     

Relationship between adenohypophyseal and steroid hormones and variations in serum and urinary melatonin levels during the ovarian cycle, perimenopause and menopause in healthy women

Morning levels of serum melatonin, FSH, LH, prolactin (PRL), progesterone and estradiol were studied by RIA during the ovarian cycle, perimenopause and menopause in 79 healthy women. FSH and LH levels showed a slight nonsignificant increase from the fertile period to perimenopause, exhibiting a significantly greater increase during menopause. PRL, progesterone and estradiol showed parallel changes, reaching lower levels during menopause. Serum melatonin levels decreased with age, attaining minimum levels in menopause. FSH and estradiol were significantly correlated with melatonin in the follicular phase, while in the luteal phase a negative correlation was found between melatonin, progesterone and estradiol. No significant correlations were noted between serum hormone levels during the perimenopausal period. In menopause, as during the follicular phase, melatonin and FSH were negatively correlated. As expected, a significant positive correlation was found between morning serum levels of melatonin and nocturnal urinary excretion of this indoleamine in all groups studied.     

Amniotic fluid induces rapid epithelialization in the experimentally ruptured fetal mouse palate--implications for fetal wound healing

Cleft of the secondary palate is one of the most common congenital birth defects in humans. The primary cause of cleft palate formation is a failure of fusion of bilateral palatal shelves, but rupture of the once fused palate has also been suggested to take place in utero. The possibility of post-fusion rupture of the palate in humans has hardly been accepted, mainly because in all the cleft palate cases, the cleft palatal edge is always covered with intact epithelium. To verify whether the intrauterine environment of the fetus plays roles in wound healing when the once fused palate is torn apart, we artificially tore apart fetal mouse palates after fusion and cultivated them in culture medium with or without mouse or human amniotic fluid. We thereby found that the wounded palatal edge became completely covered with flattened epithelium after 36 hours in culture with amniotic fluid, but not in culture without amniotic fluid. Using histological and scanning electron microscopic analyses of the healing process, it was revealed that the epithelium covering the wound was almost exclusively derived from the adjacent nasal epithelium, but not from the oral epithelium. Such actions of amniotic fluid on the fetal wound were never simulated by exogenous epidermal growth factor (EGF), albumin, or both. In addition, the rapid epithelialization induced by amniotic fluid was not prevented by either PD168393 (an inhibitor of the EGF receptor-specific tyrosine kinase) or SB431542 (a specific inhibitor of TGFbeta receptor type I/ALK5). The present study provides new insights into the unique biological actions of amniotic fluid in the repair of injured fetal palate.     

Gender and ethnic differences in urinary stress hormones: the population-based Chicago Health, Aging, and Social Relations Study.

Gender and ethnic disparities in cardiovascular disease and mortality have spurred interest in the epidemiology of stress hormone production. Greater disease burden among men and blacks raises the possibility of gender and ethnic differences in stress hormone production. The purpose of this study was to determine whether urinary stress hormones were higher among men and blacks in a population-based sample. Urinary hormone analysis permits a time-integrated assessment of the stress response system. However, differences in collection and standardization strategies have led to inconsistent findings. Subjects were an ethnically diverse population-based sample of 229 men and women aged 50-67 yr who provided an overnight urine specimen. Urine concentration was standardized using a traditional creatinine-based approach as well as a new method that accounts for muscle mass. With the use of creatinine standardization, no gender or ethnic differences were noted in epinephrine or cortisol production. Norepinephrine levels were higher among women compared with men (P = 0.001), however. After accounting for muscle mass, we found that both epinephrine (P = 0.018) and norepinephrine (P = 0.033) levels were higher among men compared with women. No significant differences in cortisol production were found by gender or ethnicity. The consistency of these results with previous studies of 24-h urine samples suggests muscle mass should be accounted for when comparing overnight urinary hormone values across gender and ethnicity.     

Prenatal fetal karyotyping and maternal serum alpha-fetoprotein screening.

Prenatal karyotyping was undertaken in 569 consecutive amniotic fluid samples where the indication for amniocentesis was two sequential raised maternal serum alpha-fetoprotein concentrations. In 475 successful cultures five chromosome abnormalities were found--four constitutional anomalies (47,XXY; 47,XYY; an inherited inv(8) (p23q11); and a de-novo translocation t(6;7) (p11;p22) and a culture-derived anomaly (trisomy 2) found in amniotic fluid cells but not in the fetus aborted because it had spina bifida. Of the pregnancies complicated by constitutional abnormalities, only the pregnancy in which the de-novo translocation was detected was terminated. No chromosome abnormalities were detected in the 17 pregnancies which miscarried after amniocentesis. These results provide little justification for including fetal karyotyping as an essential part of maternal serum alpha-fetoprotein screening programmes.     

Selective muscle hypertrophy, changes in EMG and force, and serum hormones during strength training in older women.

Effects of strength training (ST) for 21 wk were examined in 10 older women (64 +/- 3 yr). Electromyogram, maximal isometric force, one-repetition maximum strength, and rate of force development of the leg extensors, muscle cross-sectional area (CSA) of the quadriceps femoris (QF) and of vastus lateralis (VL), medialis (VM), intermedius (VI) and rectus femoris (RF) throughout the lengths of 3/12--12/15 (Lf) of the femur, muscle fiber proportion and areas of types I, IIa, and IIb of the VL were evaluated. Serum hormone concentrations of testosterone, growth hormone (GH), cortisol, and IGF-I were analyzed for the resting, preexercise, and postexercise conditions. After the 21-wk ST, maximal force increased by 37% (P < 0.001) and 1-RM by 29% (P < 0.001), accompanied by an increase (P < 0.01) in rate of force development. The integrated electromyograms of the vastus muscles increased (P < 0.05). The CSA of the total QF increased (P < 0.05) throughout the length of the femur by 5--9%. The increases were significant (P < 0.05) at 7/15--12/15 Lf for VL and at 3/15--8/15 Lf for VM, at 5/15--9/15 for VI and at 9/15 (P < 0.05) for RF. The fiber areas of type I (P < 0.05), IIa (P < 0.001), and IIb (P < 0.001) increased by 22--36%. No changes occurred during ST in serum basal concentrations of the hormones examined, but the level of testosterone correlated with the changes in the CSA of the QF (r = 0.64, P < 0.05). An acute increase of GH (P < 0.05), remaining elevated up to 30 min (P < 0.05) postloading, was observed only at posttraining. Both neural adaptations and the capacity of skeletal muscle to undergo training-induced hypertrophy even in older women explain the strength gains. The increases in the CSA of the QF occurred throughout its length but differed selectively between the individual muscles. The serum concentrations of hormones remained unaltered, but a low level of testosterone may be a limiting factor in training-induced muscle hypertrophy. The magnitude and time duration of the acute GH response may be important physiological indicators of anabolic adaptations during strength training even in older women.     

The midgestational human fetal pancreas contains cells coexpressing islet hormones.

In the fetal development of the mouse pancreas, endocrine cells have been found that express more than one hormone simultaneously. Our objective was to evaluate the existence of such cells in the human fetal pancreas. We found cells coexpressing two of the major pancreatic hormones (insulin, glucagon, and somatostatin) in sections of eight midgestational (12-18 weeks) pancreata and in 0-7% of cells in single-cell suspensions from midgestational pancreata. By electron microscopy, using granule morphology and immunoelectron microscopic techniques, we could confirm these findings and even detect cells containing three hormones. Morphologically different granules contained different immunoreactivities, suggesting parallel regulation of hormone production and packaging. In six newborn pancreata (born after 22-40 weeks of gestation), we could not find any multiple-hormone-containing cells. Subsequently, we evaluated whether multiple-hormone-containing cells proliferate by using pancreatic fragments and single-cell preparations at the light and electron microscopic level (six pancreata). No endocrine hormone-containing cells incorporated bromodeoxyuridine during a 1-hr culture period, indicating that these cells have lost the ability to proliferate under the conditions chosen. We conclude that, as in mice, the human fetal pancreas of 12-18 weeks of gestation contains endocrine cells that express multiple hormones simultaneously. These (multiple) hormone-containing cells do not seem to proliferate under basal conditions.