Calcium,magnesium, and serum factors in multiplication of normal and transformed human lung fibroblasts

Summary Serum factors determine the extracellular requirement for both Ca²⁺ and Mg²⁺ for multiplication of normal human lung fibroblasts in vitro. Serum factors also affect the extracellular Ca²⁺ requirement for transformed fibroblasts but to a different extent than for normal cells. Transformed cells exhibit a reduced requirement for both Ca²⁺ and Mg²⁺ for multiplication. The apparent reduction in Ca²⁺ requirement of transformed cells is dependent on the level of serum factors in the medium. The reduced Mg²⁺ requirement for transformed cells is more striking than the loss of Ca²⁺ and independent of the level of serum factors in the medium. A sequential effector relationship among serum factors, Ca²⁺ and Mg²⁺, in a proliferative control system for normal cells is proposed. Alteration or bypass of an intracellular Mg²⁺-requiring process is proposed as a major lesion in the transformed cells. This alteration causes an observed loss of requirements for both Ca²⁺ and serum factors for the multiplication of transformed cells. This work was supported by Grant CA-15305 from the National Cancer Institute, Contract 223-74-1156 from the Bureau of Biologics, Food and Drug Administration, HEW Biomedical Research Support Grant S07RR05800, and the W. Alton Jones Foundation.     

Effect of retinoids on growth factor-induced anchorage independent growth of human fibroblasts

Summary We have studied the effects of all-trans retinol, all-trans retinoic acid, and anhydroretinol, a biologically inactive retinoid, on anchorage-independent growth of human fibroblasts induced by purified growth factors. The anchorage-independence assay was, conducted in medium supplemented with serum that had had its peptide growth factors inactivated by treatment with dithiothreitol and iodoacetamide. Physiologic concentrations of either all-trans retinol (0.5 μM) or all-trans retinoic acid (1.0 nM) but not anhydroretinol (0.5 μM) reduced the frequency of anchorage-independent growth of normal human fibroblasts induced by platelet-derived growth fator (PDGF). All-trans retinol was also tested for its effect on the frequency of anchorage-independent growth induced by basic fibroblast growth factor (bFGF) and was found to decrease this growth. All-trans retinol also reduced the frequency of anchorage-independent growth of the human fibrosarcoma-derived cell, line, HT1080, which grew in semisolid medium without added growth factors. Inasmuch as these retinoids reduced the frequency of anchorage-independent growth induced by either PDGF or bFGF and because PDGF and bFGF bind to independent cell membrane receptors and are known to stimulate different pathways leading to DNA synthesis, the data suggest that physiologically active retinoids, have an effect on a step that is common to both signal pathways. This research was supported in part by Department of Energy, Washington, DC, grant DE-F602-87ER-60524 and by DHHS grants CA21289 and, CA32490 from the National Cancer Institute, Bethesda, MD.     

Characterization of growth factors in human cartilage

Growth factor activity has been identified in the chondrocytes and extracellular matrix (ECM) fractions of human costal cartilage. There was about five times more growth factor activity in the ECM than was found to be associated with the chondrocytes. The growth factor activity in chondrocytes was found to be associated with chromatin. Both the chromatin-associated growth factor (CAGF) activity and extracellular matrix growth factor (EMGF) activity were characterized for molecular weight, charge, and the effect of reduction by sulfhydryl reducing reagents. Biorex cation exchange chromatography showed that both CAGF and EMGF were cationic. CAGF and EMGF have molecular weights between 15,000 and 18,000 as determined by size exclusion chromatography on HPLC TSK 3000 columns equilibrated with guanidine-HCl and dithiothreitol.     

Effect of placental factors on growth and function of the human fetal adrenal in vitro

Conditioned medium from human placental monolayer cultures (PM) had a marked stimulatory effect on proliferation (3H-thymidine uptake) of human fetal zone adrenal cells in primary monolayer culture, even in the absence of serum. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) also significantly stimulated fetal adrenal cell growth. However, the effects of PM differed from those of EGF and FGF in several respects: 1) maximal response to PM was 2-5 times greater; 2) mitogenic effects of EGF and FGF were suppressed by adrenocorticotropic hormone (ACTH), whereas that of 50% PM was not; 3) PM inhibited ACTH-stimulated steroidogenesis (dehydroepiandrosterone sulfate and cortisol), but EGF and FGF did not. Preliminary characterization studies have indicated that approximately half of the placental growth-promoting activity is heat resistant and sensitive to bacterial proteases, and that 50-60% of the activity is lost after dialysis with membranes having a molecular weight cutoff of 3500. These findings suggest a role for the placenta in the growth and differentiated function of the human fetal adrenal gland.     

Atmospheric air vs. normal middle ear gas: Effects on in vitro growth and collagen synthesis in normal middle ear fibroblasts

Summary The present study was undertaken to quantitate the effects of atmospheric air and normal middle ear gas on cultured fibroblasts obtained from normal rabbit middle ear mucosa. The cells were exposed to three different gas compositions: 7% O₂:5% CO₂:88% N₂, 21% O₂:5% CO₂:74% N₂, and 75% O₂:5% CO₂:20% N₂. The growth was monitored by measuring the total content of cell protein, the amount of DNA, and the cell division activity. The activity of the synthetic apparatus was determined by the collagen synthesis. For comparison, rabbit skin fibroblasts were grown under identical conditions. The results demonstrated significantly higher replication rate of middle ear fibroblasts at 7% oxygen than at atmospheric air whereas the collagen synthesis was significantly lower at 7%. Furthermore, the responses varied significantly between rabbit middle ear and rabbit skin fibroblasts. Thus the present study substantiates the hypothesis of an influence of atmospheric air on the middle ear mucosa which might be of importance, e.g., in relation to insertion of ventilation tubes or longstanding perforations of the tympanic membrane in otitis media.     

Effect of growth arrest on the doubling potential of human fibroblasts in vitro: A possible influence of the donor

Summary Population doublings versus time in culture were compared in human postnatal skin fibroblasts from normal donors, a cancer patient, and from donors suffering from Cockayne syndrome, Ataxia telangiectasia, and Fanconi’s anemia (FA). Confluent cultures were maintained in a nonproliferating state for 14 to 27 d in 0.5% serum medium. The results show that the ability of cells to resume division after a resting stage can be influenced by pathologic conditions. In arrested FA cell populations an increase of the population doublings and of the calendar time were observed. It is possible that in some cell populations the resting stage favors the expression of growth potentialities related to an instability of the cells. This work was supported by contract CRL 802030 from the I.N.S.E.R.M. and by Euratom.     

Survival of normal colonic epithelial cells from both rats and humans is prolonged by coculture with rat embryo colonic fibroblasts

Primary cultures of normal colonic epithelial cells from both humans (HCEC) and rats (RCEC) have been established using coculture with colon fibroblasts isolated from rat term embryos. While no other factors we have analyzed had any effect on the survival of epithelial cells, which is normally 3–4 days, coculture with viable fibroblasts extended this period to at least 2 weeks. The effects depended on early passages and low seeding densities of the fibroblasts and on direct cell–cell contact. We have obtained cultures of epithelial cells expressing keratin, laminin, and uvomorulin, displaying a polygonal, epithelial morphology and forming microvilli. DNA synthesis as measured by BrdU uptake into DNA varied widely between colonies of the same culture depending on cell morphology: flat colonies of RCECs contained 5.7%±0.56% BrdU-positive cells, while the proportion in dense three-dimensional colonies reached 50.3%±2.6%. In HCECs the growth fraction was lower, but showed the same distribution between classes of colonies. In the presence of rat embryonic colon fibroblasts, growth factors exerted survival activity on colonic epithelial cells. Consecutive addition of insulin and epidermal growth factor/fibroblast growth factor (EGF/FGF) increased colony number (15.0±1.0 and 23.0±2.0 colonies/well respectively; p0.05 increased above control) and size (1022±155 and 1207±158 cells/colony respectively; p0.05 increased above control) compared to serum-free control medium and basic MEM without growth factors. BrdU labeling index was not increased, however: EGF/FGF actually decreased BrdU labeling from 33.2%±3.9% in controls to 21.3%±3.8% in the EGF/FGF group (p0.05) owing to the high proportion of flat colonies consisting of resting cells.The newly established culture model can now be used to investigate growth control mechanisms in colonic mucosa and the effects of toxic and/or tumor-promoting substances on these mechanisms.     

Effect of hormones and growth factors on in vitro development of sheep preantral follicles

The present investigation attempts to improve the frequency of in vitro maturation of oocytes by culturing small (150–250 μm) and large (>250–400 μm) preantral follicles (PFs) of sheep for 6 days in various combinations/sequences of thyroxin (T₄), FSH, LH, transforming growth factor alpha (TGF-), epidermal growth factor (EGF) and heat-treated foetal calf serum (FCS). Bicarbonate-buffered tissue culture medium 199, supplemented with 50 μg ml⁻¹ gentamicin sulphate, served as the control medium. In vitro development was initially assessed by the proportion of PFs exhibiting an increase in size, mean increase in diameter and antrum formation. Nuclear maturation to the metaphase II stage of the oocytes isolated from cultured PFs, after an additional 24-h in vitro maturation, indicated success. A total of 15% of oocytes from small PFs and 55% from large PFs, cultured in T₄ + FSH, matured to metaphase II. Culture of PFs in other combinations/sequences of hormones and growth factors, including the control medium, supported a significantly lower proportion of oocytes maturing to metaphase II stage. It is concluded that 6-day in vitro culture of sheep PFs in thyroxin and FSH greatly improves the frequency of oocyte maturation to metaphase II stage.     

Insulin-like growth factors and binding proteins in the fetal rat: Alterations during maternal starvation and effects in fetal brain cell culture

Maternal malnutrition adversely affects fetal body and brain growth during late gestation. We utilized a fetal brain cell culture model to examine whether alternations in circulating factors may contribute to reduce brain growth during maternal starvation; we then used specific immunoassay and western blotting techniques, and purified peptides to investigate the potential role that altered levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) may play in impaired growth during maternal nutritional restriction.Fetal, body, liver, and brain weight were reduced after 72 hr maternal starvation, and plasma from starved fetuses were less potent than fed fetal plasma in stimulating brain cell growth. Circulating levels of IGF-I were reduced in starved compared to fed fetuses, while levels of IGF-II were similar in both groups. In contrast, [¹²⁵I]-IGF-I binding assay demonstrated an increase in the availability of plasma IGFBPs following starvation. Western ligand blotting and densitometry indicated that levels of 32 Kd IGFBPs were 2-fold higher in starved compared to fed fetal plasma. Immunoblotting and immunoprecipitation with antiserum against rat IGFBP-1 confirmed that heightened levels of immunoreactive IGFBP-1 accounted for the increase in 32 Kd IGFBPs in starved plasma. Levels of 34 Kd BPs, representing IGFBP-2, were unaffected by starvation. Reconstitution experiments in cell culture showed that IGF-I promoted fetal brain cell growth, and that when they were supplemented with IGF-I, the growth promoting activity of starved fetal plasma was restored to fed levels. These changes were measured using MTT to assess mitochondrial reductase activity. Conversely, addition of physiological amounts of rat IGFBP-1 inhibited the effects of fed fetal plasma on brain cell growth, and bioactivity was reduced even further with higher concentrations of IGFBP-1. Based on these results, we conclude that reciprocal changes in circulating levels of IGFBP-1 (increased) and IGF-I (decreased) may combine to reduce the availability of IGF-I to this tissue and limit fetal brain cell growth when maternal nutrition is impaired.     

In vitro properties of various growth factors and in vivo effects

This article summarizes some of the data that have been accumulated on several growth factors. Biochemical and biological properties of the Epidermal, Fibroblast, Astrocytes and Tumor growth factors (EGF, FGF, AGF, TGF) and those of growth factors derived from Platelets (PDGF), Brain (BDGF, ECGF), Eye (EDGF) and Cartilage (CDGF) are reviewed, as well as the in vitro mechanism of action of EGF and PDGF. The in vivo effects of these growth factors, particularly the experiments achieved to understand the physiological or physiopathological significance are described. The potential interest of these molecules in pharmacology and their use as wound healing agents is discussed.     

Evaluation of growth hormone production from GH1 cells in vitro: Effect of culture media and time in culture

Summary Growth hormone production by a rat pituitary tumor cell line (GH1) was measured during lag, exponential, and plateau phases of growth in different culture media. Growth hormone secretion was low during lag and early exponential phase; it increased late in the exponential phase and continued to increase during the plateau phase. This biphasic pattern of growth hormone production was observed in all media and sera utilized. Both the doubling time and growth hormone production were influenced by the choice of media and sera. In addition, the length of time in culture affected the growth fraction with passage level 40 GH1 cells having a 79% growth fraction, whereas the growth fraction of passage level 100 cells was 95%. Using the population doubling time as a criterion for a choice of medium, F-10 medium supplemented with 20% fetal bovine serum consistently yielded the most rapid doubling time (32 hr), whereas Dulbecco's MEM supplemented with 15% horse serum and 2.5% fetal bovine serum yielded the greatest plateau cell density. Growth hormone secretion and the population doubling times were directly related to culture conditions including length of time in culture, choice of tissue culture media, choice of sera, and the phase of cell growth (lag, exponential or plateau).     

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

Human endometrial carcinoma cells release factors which inhibit the growth of normal epithelial cells in culture

Autocrine and paracrine interactions between cells are important homeostatic mediators in normal tissues. Alterations to growth factor signalling pathways are likely to play a role in multistep carcinogenesis. In this study normal human endometrial epithelial cells (NHEC) after 3 days in culture were treated with serum-free medium conditioned for 24 h by log phase or confluent cultures of established RL95-2, HEC1A, or AN3CA endometrial carcinoma (EC) cell lines. By day 4, NHEC treated with either log phase or confluent conditioned medium (CM) showed a significant decrease (50–90% of control) in [³H]thymidine ([³H]TdR) incorporation. DNA synthesis was inhibited more by confluent than by log phase CM. By day 7, NHEC treated with CM exhibited fewer colonies per culture, fewer cells per colony, and an increased percentage of single cells. Several growth-regulatory gene products found in the nucleus or at the cell membrane have been shown to be expressed differently in normal and transformed cells. We selected the p53 and c-Ha-ras p21 proteins to further investigate the mechanism of alteration of proliferation in cells treated with carcinoma CM. Thus, by day 7, the percentage of NHEC with nuclear localization of wild type p53 (wt p53) was elevated by treatment with CM. In contrast, CM-treated EC cells continued to proliferate, and showed a decrease in the percentage of cells expressing nuclear wt p53 and an increase in the cytoplasmic expression of c-Ha-ras p21. Our studies show that EC cell lines release factors which inhibit the proliferation of NHEC, thus favoring the proliferation of EC cells.Abbreviations CM conditioned medium - EC endometrial adenocarcinoma - NHEC normal human endometrial epithelial cells     

Effects of some antibiotics on the growth of human diploid skin fibroblasts in cell culture

Summary During serial subcultures 50 μg per ml gentamicin and penicillin (100 U per ml)-streptomycin (100 μg per ml) depressed cell growth significantly 2 weeks after the addition of the antibiotics; gentamicin, but not penicillin-streptomycin, stimulated cell growth before it became inhibitory. Removal of the antibiotics resulted in the cell yield returning to normal. The results show that these antibiotics can be harmful to cells even at concentrations thought to be safe.     

Estrogen mitogenic action. II. Negative regulation of the steroid hormone-responsive growth of cell lines derived from human and rodent target tissue tumors and conceptual implications

Summary In an accompanying report (Moreno-Cuevas, J. E.; Sirbasku, D. A., In Vitro Cell. Dev. Biol.; 2000), we demonstrated 80-fold estrogen mitogenic effects with MTW9/PL2 rat mammary tumor cells in cultures supplemented with charcoaldextran-treated serum. All sera tested contained an estrogen reversible inhibitor(s). The purpose of this report is to extend those observations to additional sex steroid-responsive human and rodent cell lines. Every line tested showed a biphasic response to hormone-depleted serum. Concentrations of ≤10% (v/v) promoted substantive growth. At higher concentrations, serum was progressively inhibitory. With estrogen receptor-positive (ER⁺) human breast cancer cells, rat pituitary tumor cells, and Syrian hamster kidney tumor cells, 50% (v/v) serum caused significant inhibition, which was reversed by very low physiologic concentrations of estrogens. This same pattern was observed with the steroid hormone-responsive LNCaP human prostatic carcinoma cells. Because steroid hormone mitogenic effects are now easily demonstrable using our new methods, the identification of positive results has nullified our original endocrine estromedin hypothesis. We also evaluated autocrine/paracrine growth factor models of estrogen-responsive growth. We asked if insulin-like growth factors I and II, insulin, transforming growth factor alpha, or epidermal growth factor substituted for the positive effects of estrogens. Growth factors did not reverse the serum-caused inhibition. We asked also if transforming growth factor beta (TGFβ) substituted for the serum-borne inhibitor. TGFβ did not substitute. Altogether, our results are most consistent with the concept of a unique serum-borne inhibitor as has been proposed in the estrocolyone model. However, the aspect of the estrocolyone model related to steroid hormone mechanism of action requires more evaluation. The effects of sex steroids at picomolar concentrations may reflect mediation via inhibitor “activated” intracellular signaling pathways.     

Growth of fetal rat gastro-intestinal epithelial cells is region-specifically controlled by growth factors and substrata in primary culture

The mammalian gastro-intestinal tract can be divided into three parts: esophagus and forestomach, glandular stomach, and intestine. We have previously reported primary culture systems for duodenal and glandular stomach epithelial cells in which the cells express tissue-specific marker proteins. However, the effects of growth factors and substrata on cell growth have not been fully investigated. In this study a primary culture system was established for forestomach epithelial cells and the mechanism by which the growth of gastro-intestinal epithelial cells is controlled in primary culture was examined. Forestomach, glandular stomach and duodenal epithelial cells proliferated rapidly in culture, increasing their numbers about 30-, 20-and 10-fold, respectively, in the first 5 days. Scanning electron microscopy showed that these three types of epithelial cells exhibited region-specific morphologies in culture. Results on the effects of growth factors and substrata on the proliferation of the epithelial cells revealed that the culture conditions required to induce maximal epithelial growth differed. Forestomach and glandular stomach epithelial cells required similar combinations of growth factors to proliferate, and these were quite different from those required for duodenal epithelial cells. Glandular stomach and duodenal epithelial cells could proliferate in a serum-free condition while forestomach epithelial cells could not. Thus, glandular stomach epithelial cells exhibited intermediate characteristics between forestomach and duodenal epithelial cells regarding their growth factor requirement. Glandular stomach and duodenal epithelial cells could not proliferate on plastic without collagen substrata while forestomach epithelial cells could. Duodenal epithelial cells proliferated faster on collagen gels than on collagen films, and forestomach epithelial cells faster on collagen films than on collagen gels. Glandular stomach epithelial cells proliferated similarly on both substrata. Thus again, glandular stomach epithelial cells exhibited intermediate characteristics between forestomach and duodenal epithelial cells regarding their substratum dependency. We conclude that the growth of gastro-intestinal epithelial cells is affected by both growth factors and substrata, and that glandular stomach epithelial cells exhibit intermediate characteristics between forestomach and duodenal epithelial cells in responding to these factors. These results suggest that a head-to-tail gradient exists in the gastro-intestinal tract which controls the epithelial response to growth factors and substrata.     

Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells

Summary We have investigated the response of several epithelial and fibroblastic cells to a mitogenic extract of bovine milk. Cation exchange chromatography was used to produce a mitogen-rich fraction from an industrial whey source that, although comprising only 0.5% of total whey protein, contained the bulk of the growth factor activity. This fraction was a source of potent growth promoting activity for all mesodermal-derived cells tested, including human skin and embryonic lung fibroblasts, Balb/c 3T3 fibroblasts, and rat L6 myoblasts. Maximal growth of all these cell types exceeded that observed in 10% fetal bovine serum. Feline kidney and baby hamster fibroblasts and Chinese hamster ovary cells were less responsive, achieving a maximal growth response of 50–75% that observed in 10% fetal bovine serum. Maximal growth achieved in whey-extract-supplemented cultures of Balb/c 3T3 and human skin fibroblasts, and L6 myoblast cultures exceeded that seen in response to recombinant acidic or basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, or epidermal growth factor. Importantly, addition of low concentrations of fetal bovine serum to the whey-derived mitogenic fraction produced an additive response. However, concentrated milk-derived factors were found to be inhibitory to the growth of all epithelial lines tested, including rat intestinal epithelial cells, canine kidney epithelial cells, and mink lung cells. It is concluded that industrial whey extracted in this form constitutes an important source of potent growth-promoting agents for the supplementation of mesodermal-derived cell cultures.     

Effects of fibroblast growth factor 2 and insulin-like growth factor II on the development of parthenogenetic mouse embryos in vitro

Most parthenogenetic embryos (PEs) in mammals die shortly after implantation, and this failure to develop is associated with genomic imprinting. We have examined the influence of human recombinant basic fibroblast growth factor 2 (FGF-2) and human recombinant insulin-like growth factor II (ICF-II) on the development of (CBA x C57BL/6)F1 parthenogenetic mouse embryos. Embryos were treated in vitro at the morula stage with different doses of FGF-2 and, after their development to blastocysts, transferred to pseudopregnant recipients. The optimal doses of FGF-2 did not affect the number of forming and implanting blastocysts, but increased, from 20 to 42%, the number of embryos developing to somite stages. PEs (18-21 somites) treated with an optimal dose of FGF-2 were explanted for further development in culture by treatment with the second growth factor, IGF-II. Eighty-three percent of those embryos cultured with IGF-II (2.5 microg/ml) developed to 35 or more somites, as compared with 36% of embryos cultured without any growth factors (P < 0.01). Also, a significantly higher proportion of PEs developed to 40-50 somites in this case. These results show that the in vitro treatment of PEs with FGF-2 at the morula stage increases the number of somite embryos, and the second treatment of somite PEs with IGF-II in culture medium prolongs their development significantly.