Characterization of the cell kinetics and growth properties of cystic fibrosis diploid fibroblasts in vitro.

The population growth kinetics of human diploid skin fibroblasts derived from cystic fibrosis homozygotes and heterozygotes and from normal subjects were investigated. Our data suggest the following: 1. Population doubling times increase with time in culture, with no significant differences observed among the three genotypes tested, when data were compared at the same subculture times or phases of growth. 2. The fraction of dividing cells in a population decreased with the duration in which the cells were in culture. No significant differences were obtained for cells derived from the three genotypes. 3. Cell cycle times were very similar (18-20 hr) when comparing the normal and cystic fibrosis lines or when comparing cystic fibrosis lines in phases 1 and 2 of growth. 4. No significant variations in population doubling times or growth fractions could be attributed to age or sex of the biopsy donor. 5. Variability in growth fractions and doubling times was minimal through the eighth subculture period but was very great in older cultures (tenth subculture). 6. Changes in growth fractions and doubling times appear to be due to the possibility of "aging" of human diploid fibroblasts in culture rather than to the presence or absence of genes for cystic fibrosis. 7. It is strongly indicated that differences in cell kinetic parameters cannot be used as the basis for differentiation between cells derived from normal or cystic fibrosis genotypes.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

Differences in agglutinability of adult and fetal human fibroblasts using phytohemagglutinin

Whereas Concanavalin A (Con A) and Wheat Germ Agglutinin (WGA) detect differences in the agglutinability of transformed, established and secondary cultures, Phytohemagglutinin (PHA) detects differences between cultured adult and fetal human fibroblasts. Adult cells agglutinate with PHA to the same extent as transformed cells, whereas fetal cells show significant agglutination only after trypsinization. Differences in cell size, growth rate, surface architecture or binding of fluorescent PHA could not be demonstrated between adult and fetal cells. Although the basis for this apparent difference in agglutinability remains unknown, it is the first demonstration that fetal cells (even after prolonged in vitro culture) retain at least some surface properties not shared by adult or transformed cells.     

Differential effects of hydrocortisone on both growth and collagen metabolism of human fibroblasts from normal and keloid tissue

Cultured fibroblasts isolated from normal and keloid tissue do not differ in their growth characteristics or in the rate of collagen synthesis under routine culture conditions. The addition of hydrocortisone to the culture media results in significant differences in both growth and collagen synthesis between these cell types. Collagen syntehsis is inhibited 60% in normal cultures by hydrocortisone (0.5 μg/ml) and the population size at which density-dependent growth inhibition is achieved is increased. Keloid-derived fibroblasts grow to a lower maximum density in the presence of hydrocortisone, while their rate of collagen syntehsis is not significantly reduced. The rate of non-collagen protein synthesis is increased significantly by hydrocortisone in both cell types. Comparison of normal and keloid-derived cultures obtained from a single individual suggests that the keloid phenotype with respect to both growth and collagen synthesis is restricted to the fibroblasts isolated from the keloid nodule.     

Insulin receptors in normal and transformed fibroblasts: relationship to growth and transformation

The insulin receptors in normal and transformed lines of mouse Balb/3T3 fibroblasts have been studied. In the normal fibroblasts, the binding of insulin was low in growing cells and increased 2–9 fold in confluent stationary cells. Insulin binding was increased whether growth arrest was due to contact inhibition of growth or serum starvation. When serum-starved cells were stimulated to grow by the addition of fresh serum, insulin binding declined. In cells transformed by simian virus 40, Kirsten, Moloney, and Harvey sarcoma viruses, methylcholanthrene, X rays, or spontaneously, the binding was low, in the same range as growing normal cells. In simian virus 40-transformed cells, insulin binding increased 4 fold as the cells reached higher densities in culture. No relationship to changes in cell size was found. The differences in binding were due to changes in the concentration of the receptors, without changes in their affinity for the hormone.     

Pygmy mouse fibroblasts in tissue culture: evaluation of multiplication stimulating activity (MSA) receptors and growth responses to MSA

The pygmy mouse has been proposed as a model for growth hormone resistance; it has normal serum somatomedin levels and does not respond to growth hormone treatment. In order to determine if the growth impairment is caused by a defect in somatomedin binding or in postreceptor action of somatomedin we compared fibroblasts derived from pygmy mice with those from normal appearing littermates. Using multiplication-stimulating activity (MSA) as a model somatomedin we found a normal Ka of binding to the cell surface MSA receptor but a significantly increased number of MSA receptors on the fibroblasts derived from pygmy mice. Studies of thymidine incorporation into DNA failed to demonstrate a difference between pygmy and normal fibroblasts in their responses to MSA alone, but there was a significantly greater thymidine incorporation into the DNA of normal fibroblasts when both competence factor (platelet-derived growth factor) and progression factors (somatomedins and growth hormone deficient platelet-poor plasma) were present in the test medium. On the other hand, cell proliferation studies did not demonstrate a consistent difference in the growth rate of normal versus pygmy fibroblasts. The data support the conclusion that the imparied growth of the pygmy mouse in vivo may be caused by factors which lie outside of the growth hormone-somatomedin pathway.     

Induction of proteins and mRNAs after uv irradiation of human epidermal keratinocytes

uv sensitivity of cultured human epidermal keratinocytes was analyzed at different growth conditions and compared with the sensitivity of dermal fibroblasts derived from the same skin specimen. No significant differences in survival curves were found between these two cell types, although keratinocytes grown under standard conditions were slightly more resistant to uv irradiation than fibroblasts. The extracellular concentration of calcium appeared to be critical not only in the regulation of keratinocyte proliferation and differentiation, but also in the uv sensitivity of these cells: keratinocytes grown under conditions which favor cell proliferation (low calcium concentration) are more resistant to uv irradiation than those grown under conditions favoring differentiation (high calcium concentration). Two-dimensional protein gel electrophoresis was used to detect a possible effect of uv irradiation on the accumulation of specific mRNAs in the cytoplasm and/or on the synthesis of specific proteins. Proteins were pulse labeled in vivo with [35S]methionine or synthesized in vitro in rabbit reticulocyte lysates on mRNA isolated from keratinocytes that were irradiated with different uv doses at different periods of time prior to isolation. Alterations in expression were demonstrated for several proteins in both in vivo and in vitro experiments.     

Effect of azelastie on PGE2 production in fibroblasts in normal skin. the possibility of inhibition of inducible cyclooxygenase by azelastine

The effects of azelastine on prostaglandin E2 (PGE2) production were investigated by using cultured normal dermal fibroblasts obtained from the same traumatic region of 3 patients and the CRL-1475 cell line, lnterleukin-1 β (IL-1) enhanced PGE2 production in cultured normal fibroblasts and CRL-1475 cells. 10⁻⁶ M azelastine inhibited PGE2 production in these IL-1-stimulated fibroblasts. However, the drug did not influence spontaneous PGE2 production in cultured CRL-1475 fibroblasts not stimulated with IL-1 but slightly it increased in cultured normal dermal fibroblasts under the same conditions. These results suggest that azelastine either regulates synthesis of an inducible cyclooxygenase protein or inhibits PGE2 production as an inducible cyclooxygenase inhibitor.     

Growth characteristics and protein content of tissue-cultured fibroblasts from cystic fibrosis patients.

Proliferation rates and cellular protein content have been measured in cultured fibroblasts derived from the skin of normal volunteers and cystic fibrosis patients. Three methods of measuring growth indicated that under our conditions, CF fibroblasts divide normally with a mean doubling time of 29 hr. During the logarithmic growth phase, however, lower cell protein/DNA ratios were observed consistently in CF cultures. This difference was not present in contact-inhibited, confluent fibroblasts. The finding of an apparent reduction in protein synthesis during rapid division, coupled with an observation by others that CF fibroblasts fail to normally induce collagen formation, suggests the possibility of a disturbance in the biochemical regulation of protein synthesis.     

Characterization of proteoglycans in Alzheimer's disease fibroblasts.

Skin fibroblasts lines established from patients with Alzheimer's disease and old normal individuals were cultured with 35S-sodium sulfate and 3H-glucosamine. Proteoglycans were isolated and characterized. Sulfate incorporation into proteoglycans increased in Alzheimer's disease fibroblasts relative to normal controls. These increases changed the ratio of chondroitin sulfate to heparan sulfate proteoglycan from 1.4 to 1.7 (p = 0.0012) and decreased the ratio of cell to medium proteoglycans from 0.32 to 0.26 in normal and Alzheimer fibroblasts (p = 0.006), respectively. HPLC analysis of the disaccharides produced by chondroitinase ABC revealed no differences in composition between proteoglycans of Alzheimer and normal fibroblasts in either the cell or medium fraction. However, analysis of disaccharides produced by heparinase plus heparitinase showed differences in composition in the medium but not the cell fraction. delta UA-GlcNS was increased by 30% while delta UA-GlcNS-6S was reduced by 40% in Alzheimer's disease.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

Behaviour of Duchenne dystrophy fibroblasts in collagen gels

In order to clarify the possible involvement of the cell surface in the pathogenesis of Duchenne muscular dystrophy, we have examined the behaviour of fibroblasts cultured from Duchenne patients in hydrated collagen lattices. No differences could be found between Duchenne and normal skin fibroblasts, either after initial seeding or following prolonged culture within the collagen gel.     

Serial cultivation of epithelial cells from human and macaque salivary glands

Summary To study the regulation of human salivary-type gene expression we developed cell culture systems to support the growth and serial cultivation of salivary gland epithelial and fibroblastic cell types. We have established 22 independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 fibroblast feeder layer in a mixture of Ham’s F12 and Dulbecco’s modified Eagle’s media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal growth factor. Salivary gland epithelial cells cultured under these conditions continued to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in fibroblasts isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata. The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression.     

The pathophysiology of neurofibromatosis. I. Resistance in vitro to 3-nitrotyrosine as an expression of the mutation

The in vitro expression of the autosomal dominant mutation responsible for neurofibromatosis was probed using the amino acid analogue 3-nitrotyrosine as a cell culture selective agent. The presence of 3-nitrotyrosine in culture medium led to inhibition of growth and cell death among normal skin fibroblasts in log phase growth, whereas cell strains derived from six different patients' neurofibromas or skin cells, or both, exhibited a consistently enhanced ability to survive under the same conditions. At 0.8 mM 3-nitrotyrosine, four patient-derived skin fibroblast strains could be differentiated from five strains of control skin fibroblasts with a high level of confidence (P < 0.0000). In the same way four neurofibroma-derived fibroblast strains were differentiated from control skin fibroblasts (P < 0.0022). Neurofibroma-derived cells were not different from control cells when treated with 5-fluorotryptophan or p-fluorophenylalanine.     

Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon.

Periostin is a unique extracellular matrix protein, deposition of which is enhanced by mechanical stress and the tissue repair process. Its significance in normal and neoplastic colon has not been fully clarified yet. Using immunohistochemistry and immunoelectron microscopy with a highly specific monoclonal antibody, periostin deposition was observed in close proximity to pericryptal fibroblasts of colonic crypts. The pericryptal pattern of periostin deposition was decreased in adenoma and adenocarcinoma, preceding the decrease of the number of pericryptal fibroblasts. Periostin immunoreactivity appeared again at the invasive front of the carcinoma and increased along the appearance of cancer-associated fibroblasts. ISH showed periostin signals in cancer-associated fibroblasts but not in cancer cells. Ki-67-positive epithelial cells were significantly decreased in the colonic crypts of periostin-/- mice (approximately 0.6-fold) compared with periostin+/+ mice. In three-dimensional co-culture within type I collagen gel, both colony size and number of human colon cancer cell line HCT116 cells were significantly larger ( approximately 1.5-fold) when cultured with fibroblasts derived from periostin+/+ mice or periostin-transfected NIH3T3 cells than with those from periostin-/- mice or periostin-non-producing NIH3T3 cells, respectively. Periostin is secreted by pericryptal and cancer-associated fibroblasts in the colon, both of which support the growth of epithelial components.     

Effect of fibroblast donor cell age and cell cycle on development of bovine nuclear transfer embryos in vitro

The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6-15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2-3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.     

The effect of histamine on the growth of cultured fibroblasts isolated from normal and keloid tissue

Cultured fibroblasts derived from human keloid tissue are presented as a possible model system for studying the genetic regulation of cell growth. Histamine is shown to have a marked effect on the growth of cultured fibroblasts. A small increase in growth rate is seen during the log phase of the culture cycle and a 50% increase in cell number is observed during the plateau phase. Differences in the extent of growth stimulation are observed between strains isolated from different individuals. While most strains showed approximately 50% stimulation, a few were not stimulated and some strains gave a 100% or greater increase in cell number due to histamine. This phenotypic difference in extent of growth stimulation in response to histamine cannot be attributed to the gene or genes for keloid formation. However, elevated levels of histamine in vivo may be a contributing factor to the abnormal cell growth observed in this disorder. The extent of growth stimulation due to histamine decreases with repeated subculturing.     

Comparison of infarct-derived and control ovine cardiac myofibroblasts in culture: response to cytokines and natriuretic peptide receptor expression profiles

This study investigated whether gene expression profiles of myofibroblasts derived from infarcted myocardium differ from normal cardiac fibroblasts. We compared the expression of cytoskeletal proteins in cultured ovine cardiac fibroblasts derived from infarcted (ID) and noninfarcted ovine myocardium (NID) and the levels of expression of the natriuretic peptide receptors (NPR)-A and NPR-B in response to treatment with transforming growth factor (TGF)-beta1 and/or platelet-derived growth factor (PDGF). Transformation of cultured cardiac fibroblasts to myofibroblasts, as indicated by alpha-smooth muscle actin and vimentin expression, was independent of the presence of TGF-beta1, PDGF, or cell origin. ID fibroblasts had higher basal levels than NID fibroblasts of NPR-A (ID: 58.0 +/- 32.2 arbitrary density units, NID: undetectable), NPR-B (ID: 780 +/- 155, NID: 330 +/- 38 arbitrary density units) and collagen I (ID: 17.2 +/- 0.5, NID: 10.5 +/- 1.7 pg mRNA/mug total RNA, P < 0.05) but lower levels of alpha-SMa expression (ID: 50.2 +/- 7.9, NID: 76.9 +/- 3.2 fluorescence units, P < 0.05). NPR-A mRNA in ID fibroblasts showed a rapid fourfold increase in response to TGF-beta1 and/or PDGF at 4 and 2 h, respectively, followed by a profound decline; in NID cells, NPR-A mRNA was undetectable. In ID fibroblasts, cytokines reduced NPR-B mRNA below control levels; in NID fibroblasts, TGF-beta1 and PDGF elicited prompt increments in expression: a fourfold increase with TGF-beta1 at 8 h and a twofold increase with PDGF at 4 h (P < 0.05). In summary, transformation of cardiac fibroblasts to myofibroblasts in culture is independent of cytokine treatment. Moreover, whether the cultured cardiac fibroblasts are from infarct tissue is a major determinant of NPR expression levels and cytokine responses, even after four to five passages.     

Induction of alkaline phosphatase in cultured human fibroblasts. Comparison of normal cells and those from patients with cystic fibrosis.

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP. Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

Observatons on the growth and metabolic functions of cultured cells derived from human adipose tissue.

The growth and metabolic activity of cultured cells derived from human adipose tissue (CAT cells) were studied and compared to cultured skin fibroblasts. The morphological appearance of the CAT cells was distinctly different from that of fibroblasts. The growth rate of CAT cells as measured by 3H-thymidine incorporation was much slower than the fibroblast growth rate. Cultured CAT cells synthesized significantly 14C-glucose, while fibroblast cultures had a higher metabolic rate as measured by CO2 production. Insulin stimulated 3H-thymidine incorporation in both CAT and fibroblast cultures. The CAT cells did not show a consistent insulin response of lipid or CO2 production, but this may be a reflection of donor age or nutritional status. Even though the CAT cell may be a type of stromal cell peculiar to adipose tissue rather than a preadipocyte or adipocyte, it may prove useful in studies of human obesity.