Differences in the incorporation of thymidine into DNA of normal and cystic fibrosis fibroblasts in vitro.

Although similar fractions of cells were in the S phase of the cell cycle, normal human skin fibroblasts were shown to incorporate more than twice the 3HTdR into their DNA in vitro than did cells obtained from individuals with cystic fibrosis (CF). Obligate heterozygotes incorporated an intermediate amount of the DNA precursor. Studied were initiated to determine the basis of the differential incorporation of 3HTdR among the genotypes. An analog of thymidine, BUdR, produced varied effects on the growth kinetics of the three genotypes. The growth of cells in BUdR resulted in a 50% increase in the population doubling times of all three genotypes, and caused the cell morphology to change from a spindle shape to one in which the cells became broadened and flat, with numerous cytoplasmic projections extending for distances of several cell diameters. The activities of thymidine kinase and the participation of the exogenous and de novo pathways in the synthesis of TMP were found to be approximately the same in all three genotypes. The data suggest that an alteration in the transport of thymidine into the cells may account for the differences in TdR incorporation into DNA, and this may be associated with other changes in cystic fibrosis that are apparently membrane associated.     

Surface enzymes in cultured fibroblasts from cystic fibrosis patients.

Membrane function was examined in cultured cells from cystic fibrosis patients by assaying several enzymes on intact skin fibroblasts attached to culture dishes. This technique required few cells and minimized disruption of cellular organization. Comparison of enzyme activities of intact and broken cells showed that 12% of total glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, was measurable using intact cells, while all adenosine monophosphatase was measurable using intact cells. Alkaline paranitrophenylphosphatase activity was divided between the cell surface and interior. Substrate competition experiments indicated that substrate specificities for adenosine monophosphatase and paranitrophenylphosphatase activities were different. Adenosine monophosphatase activities of 2 control and 2 cystic fibrosis strains fluctuated similarly during the cell culture cycle. The apparent Km values relative to adenosine monophosphate were similar in all strains. A chromatographic fraction of serum from a cystic fibrosis patient that was inhibitory to oyster ciliary activity had no effect on adenosine monophosphatase activity of normal fibroblasts. Furthermore, fractions of media from cystic fibrosis homozygote and heterozygote fibroblast cultures were not inhibitory to adenosine monophosphatase activities of intact normal fibroblasts or of part iculate fractions prepared from them. In light of previous studies that showed that factors from cystic fibrosis serum of culture medium disrupted specific membrane activities, it is proposed that the cystic fibrosis factor interacts with the plasma membrane, interfering most conspicuously with the protein functions that are sensitive to changes in their membrane environment.     

Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions

A new culture model was developed to study the role of proliferation and apoptosis in the etiology of keloids. Fibroblasts were isolated from the superficial, central, and basal regions of six different keloid lesions by using Dulbecco's Modified Eagle Medium containing 10% fetal calf serum as a culture medium. The growth behavior of each fibroblast fraction was examined in short-term and long-term cultures, and the percentage of apoptotic cells was assessed by in situ end labeling of fragmented DNA. The fibroblasts obtained from the superficial and basal regions of keloid tissue showed population doubling times and saturation densities that were similar to those of age-matched normal fibroblasts. In contrast, the fibroblasts from the center of the keloid lesions showed significantly reduced doubling times (25.9 +/- 6.3 hours versus 43.5 +/- 6.3 hours for normal fibroblasts) and reached higher cell densities. In long-term culture, central keloid fibroblasts formed a stratified three-dimensional structure, contracted the self-produced extracellular matrix, and gave rise to nodular cell aggregates, mimicking the formation of keloid tissue. Apoptotic cells were detected in both normal and keloid-derived fibroblasts, but their numbers were twofold higher in normal cells compared with all keloid fibroblasts. To examine whether apoptosis mediates the therapeutic effect of ionizing radiation on keloids, the cells were exposed to gamma rays at a dose of 8 Gy. Under these conditions, a twofold increase in the population of apoptotic cells was detected. These results indicate that the balance between proliferation and apoptosis is impaired in keloid fibroblasts, which could be responsible for the formation of keloid tumors. The results also suggest that keloids contain at least two different fibroblast fractions that vary in growth behavior and extracellular matrix metabolism.     

The influence of progressive growth on the specific catalase activity of human diploid cell strains. I. Effect of cellular genotype: Homozygous strains

The specific catalase activity of human diploid cell strains increases with progressive growth of the culture, and falls again following subculture. Although the increase is small, it is readily demonstrable, and is exponential with time. The response of catalase activity to proggressive growth of the culture was studied in three abnormal human cell lines. A diploid cell strain, developed from a patient homozygous for the gene causing acatalasia I, had no detectable catalase activity throughout the life cycle of the culture. Another diploid cell strain, developed from a patient homozygous for the gene causing acatalasia II, had about 5% normal catalase activity, but the proportionate increase in specific activity as the culture grew was the same as for normal cells. Thus the mutation causing acatalasia II does not change the responsiveness of the cell in terms of catalase activity to progressive growth of the culture. The behavior of a heteroploid line was similar to that of the normal diploid strains, but when the growth of the heteroploid cultures reached a plateau, their population densities were four times higher than those of the diploid strains and they had about twice the specific catalase activity.     

Kinetics of chick embryo cell types in culture

The growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 +/- 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 +/- 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium-labeled nuclei decreases, and differs significantly as doubling level increases. This age-associated decline is due to an extension in the population doubling time. Direct cell-cycle analysis shows this increase to occur in the G1 phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density. When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 +/- 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 +/- 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 +/- 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells in culture.     

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.     

Analysis of population cytokinetics of chick myocardial cells in tissue culture

The growth of embryonic chick cardiac myocytes and fibroblasts in tissue culture was evaluated by the kinetics of nuclear labeling during continuous exposure to [3H]thymidine. The fraction of mitotically active cells, the mean intermitotic period and the population doubling times were determined in each cell type during 3 weeks in culture. After 24 hr in culture, 90% of the muscle cells were mitotically active with minimal population doubling times of 65 hr. By 17 days in culture only 5% of the myocytes continued to divide with population doubling times greater than 3000 hr. Primarily, the lengthening of doubling times was due to a withdrawal of cells from the mitotic cycle and much less to a lengthening of the intermitotic period. Growth of cardiac muscle cells from embryonic hearts from 4 to 10 days of development was also compared. Muscle cells from younger hearts displayed greater mitotic activity than those from older hearts at equivalent times in culture.     

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.     

Membrane fluidity in normal and cystic fibrosis fibroblasts

We have observed distinct differences in the polarization of fluorescence and temperature dependent emission intensity of the highly fluorescent phospholipid derivative (1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)--aminocaproyl phosphatidylcholine (NBD-PC), when incorporated in the plasma membranes of normal and cystic fibrosis fibroblasts. Fluorescence polarization measurements indicate that the fluorochrome has a much higher degree of rotational mobility in cystic fibrosis fibroblasts as compared with normal cells. Temperature dependent transitions in the emission intensity of NBD-PC incorporated in normal fibroblasts are indicated at 17.7 and 21.2° C while the abnormal cell membranes apparently undergo transitions at 8.7 and 13.5° C. These differences might be due to changes in plasma membrane composition and/or organization, in the case of the cystic fibrosis cells.     

Repair of X-ray-induced DNA damage in aging human diploid cells

Human diploid cells cultured in vitro provide an excellent model system for the study of aging. In this study, we examined the formation and rejoining of DNA single-strand breaks (SSBs) induced by X-rays in human lung diploid fibroblasts during senescence, by using a modified alkaline elution method. For detecting the formation and rejoining of DNA SSBs, conventional [¹⁴C]thymidine (TdR)-labeling and fluorometric methods were applied to dividing cells and to the whole cell population including non-dividing and slowly-dividing cells, respectively. We did not find any significant differences in the rejoining ability of X-ray-induced SSBs in human diploid cells at almost all population doubling levels, although only in terminally senescent cells the rejoining of SSBs seems to proceed more slowly. However, it was observed that the alkaline elution of DNA from unirradiated and X-irradiated cells seems to become faster with increasing in population doubling number, although there were no remarkable differences in the elution rates of DNA as measured by the [¹⁴C]TdR-labeling method and those measured by the fluorometric method. These results seem to suggest that the molecular size of DNA in human diploid cells in culture decreases with aging.     

Fructose-induced enhanced mitogenicity of diploid human cells: Possible relationship with cell differentiation

Summary Fructose strongly stimulates the growth of normal diploid human skin fibroblasts (SFs) and induces marked changes in their morphology and lipid accumulation. This mitogenic effect occurs despite very low fructose consumption and depends on the presence of glutamine. The cell kinetics of cultured fructose-fed human skin fibroblasts were different from those fed on glucose: in the presence of fructose a high proliferative index persisted at Day 14 of culture and the duration of the total cell cycle and of the G1+1/2 M and S phases was slightly shorter. The mitogenic effect of fructose on SF was largest in the presence of human serum: it was small or undetectable when fibroblasts were cultured in media supplemented with dialyzed human serum, fetal bovine serum, or serum substitutes. This suggests that serum growth factor(s) mediate the mitogenic effect of fructose. Only normal diploid human cells seem to be sensitive to this mitogenic effect of fructose: the long-term growth of normal human liver cells on fructose was slightly better or similar to that on glucose. In contrast, fructose could only support limited growth of hamster fibroblastic Nil cells and of a transformed human fibroblastic line, which grew better with glucose.     

Dexamethasone-resistant cystic fibrosis fibroblasts show cross-resistance to sex steroids

Diploid skin fibroblasts derived from individuals with the autosomal recessive disease, cystic fibrosis (CF), were shown previously to be significantly more resistant to the cytotoxicity of dexamethasone, a glucocorticoid hormone, than were normal human fibroblasts. Here cystic fibrosis fibroblasts are also shown to be more resistant than normal human fibroblasts to the cytotoxic effects of the sex hormones, 17 beta-estradiol, dihydrotestosterone and progesterone. Since cells are believed to contain different receptors for each of the steroid hormones, it is not probable than the resistance of CF cells to these hormones results from a receptor deficiency. This was shown by the fact that CF cells were found to exhibit the same receptor activity as normal cells for 3-H-dexamethasone. Furthermore, neither normal human nor CF fibroblasts could be demonstrated to contain detectable receptor activity for 3H-17 beta-estradiol. In addition, the studies of fibroblast killing by hormones led to the further interesting observation that normal human diploid fibroblasts, regardless of the sex of the tissue donor, are sensitive to killing by each of the sex hormones. These findings suggest that the cytotoxic effects of the steroid hormones may be observed independently of the specific hormone receptors. The studies reported here thus suggest that the resistance of CF cells to the different steroid hormones is probably the result of a defect in a pathway in cellular steroid hormone metabolism other than that involving receptors.     

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.     

Modification of MCDB 110 medium to support prolonged growth and consistent high cloning efficiency of diploid human fibroblasts

In preparation for studies on the growth factor requirements of normal and transformed human fibroblasts, we have developed a serum-free medium that supports vigorous long-term serial subculture of diploid human fibroblasts and allows them to form large-sized colonies with high efficiency (40 to 60%) when plated at cloning density (2 to 5 cells/cm2). This medium, which is a modification of Ham's MCDB 110 base medium with its serum replacement supplements, is relatively easy to prepare and the cost of the serum replacements is approximately the same as that of fetal bovine serum supplied at 10%. The ingredients of "Supplement B" of MCDB 110 medium were added in an ethanol solution, rather than in the form of liposomes, and were combined with bovine serum albumin (0.5%), a lipid carrier. Gelatin and fetuin were included as attachment factors instead of polylysine. Bioassays indicated that none of the ingredients in the medium were contaminated with either epidermal growth factor or platelet-derived growth factor. In this modified serum-free medium, which we have designated McM+SR1, diploid human fibroblasts grew for 21 days at the same rate as in the base medium, McM, supplemented with 10% FBS (i.e., 21 population doublings). During the next 20 days, they underwent 15 population doublings which was 75% of the rate of cells growing in the medium containing serum.     

Immunochemical studies of the plasma and cultured fibroblast media fractions containing the cystic fibrosis ciliary inhibitor.

The cystic fibrosis ciliary inhibitor (CFCI) has been fractionated from plasma of cystic fibrosis (CF) homozygotes and from the media of cultured fibroblasts derived from CF homozygotes. Plasma and fibroblast media from normal controls have been fractionated in an identical manner. Fractions from plasma and fibroblast culture media that demonstrate ciliary inhibitory activity contain several proteins in a molecular weight range of approximately 5,000-11,000. These proteins have been partially characterized by immunochemical analysis with antisera to 33 human serum proteins. Immunological determinants of albumin, C3 (but not C3a), C4, C5, alpha1-lipoprotein, beta-lipoprotein, beta2-microglobulin and immunoglobulin light chains have been detected by hemagglutination in fractions of CF plasma that inhibited ciliary activity and in analogous fractions from normal sera. None of the proteins were detected in media of cultured fibroblasts from either genotype. Since the same proteins and protein fragments were identified in both CF and normal plasma fractions, and were not detected in CF fibroblast media, it appears that none of these proteins can be identified as the CFCI. Identification of these proteins will permit further purification of the CFCI by immunochemical methods.     

Plasma membrane lipids of human diploid fibroblasts from normal individuals and patients with cystic fibrosis.

The lipid composition of isolated plasma membranes of human skin fibroblasts is described for the first time. Plasma membranes from a number of strains of fibroblasts from patients with cystic fibrosis and matched normals were isolated by a recently described procedure and analysed for major phospholipid classes, cholesterol and fatty acids. No differences in the quantities of these compounds were detected between cells of the two different origins. The fetal calf serum used to supplement the growth medium contained relatively more palmitoleate and oleate but less stearate than the membranes. There were also no consistent differences between cystic fibrosis and normal membranes in terms of the fatty acid compositions of their individual phospholipid classes. Consistent with this lack of chemical change in the lipids of membranes of cystic fibrosis cells, the degree of fluorescence polarization of diphenylhexatriene, an index of fluidity, was also unchanged.     

Human papilloma virus E6/E7 genes can expand the lifespan of human corneal fibroblasts

Summary Human corneal fibroblasts were infected with a retroviral delivery vector containing the E6 and E7 genes from human Papilloma virus type 16 in order to produce cell lines that have an expanded lifespan in culture. Morphologically, some of the trasfected corneal fibroblast lines appeared to have the normal spindle-shape morphology of diploid fibroblasts, whereas other lines appeared to have a more elongated morphology. All the cell lines were anchorage-dependent. Cells that had a normal morphology grew at a rate similar to normal diploid human corneal fibroblasts and had a population doubling time of 48 h. All E6/E7 expressing cell lines, regardless of morphology, produce types I, III, and V collagen, at levels similar to those observed in the parent corneal diploid fibroblast. These corneal fibroblast lines will be a usefulin vitro system to study collagen expression and fibril formation, as well as normal stroma development. These results also demonstrate that the use of E6/E7 genes to expand a cell’s lifespan can be a powerful tool because it does not appear to alter either the growth rate of the cell or collagen expression.     

Cell size and growth characteristics of cultured fibroblasts isolated from normal and keloid tissue.

No differences in appearance or in cell size distribution were observed between cultured fibroblasts derived from normal skin, mature scars, or keloids. Artifactual differences in cell size distributions between strains can result when populations are compared at different cell densities. Keloid derived fibroblasts remain euploid in culture, and they have the same growth rate and same degree of density-dependent growth inhibition as cultured normal human fibroblasts.     

Comparison of pulmonary endothelial cell and fibroblast proliferation using time-lapse cinematographic analysis

Time-lapse cinematography was used to study and compare the proliferation and migration activity of pulmonary endothelial cells and fibroblasts, two cell types with very different structural and functional properties. Endothelial cells were found to have a mere rapid growth rate than fibroblasts. Contributing to the shorter population doubling time of the endothelial cells were lower interdivision times and a tendency for these cells to remain in division cycle with successive generations of growth. Striking differences between endothelial cells and fibroblasts were seen in migration behaviour. Endothelial cells had lower migration rates and tended to remain within a restricted growth area, whereas fibroblasts migrated freely throughout the growth area.     

Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate

Whole blood serum is widely recognized as essential for the growth of diploid cells in culture. Dermal fibroblasts and arterial smooth muscle cells fail to proliferate in culture in the presence of serum derived from platelet-poor plasma. Platelet-poor plasma serum is capable of maintaining monkey arterial smooth muscle cells quiescent in culture at either low (1.5 x 10(3)) or high (2.0 x 10(4)) population densities. The proportion of cell traversing the cell cycle under these conditions was approximately 3%. Equal numbers of quiescent smooth muscle cells initiated DNA synthesis and cell division when treated with whole blood serum or with an equivalent quantity of platelet-poor plasma serum supplemented with a factor(s) derived from a supernate obtained after exposure of human platelets to purified thrombin in vitro.