Human Diploid Skin Fibroblasts Research Articles

Characterization of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts

The interaction of human platelets with one another and with the blood vessel wall is thought to be regulated in part by a balance between two arachidonic acid metabolites: thromboxane A 2, synthesized by platelets, and prostacyclin (PGI 2), synthesized by the vessel wall. We have studied the ability of cultured human vascular cells to synthesize PGI 2 from arachidonic acid. Four strains of human arterial smooth muscle cells synthesized a mean of 1.36 ng PGI 2 per 10 5 cells, with a range of 0.2–5.3 ng PGI 2 per 10 5 cells among the different strains. Human umbilical vein endothelial cells synthesized a mean of 7.16 ng PGI 2 per 10 5 cells with a range of 2.3–14.0 ng per 10 5 cells. In contrast, cultured human diploid skin fibroblasts synthesized only 0.27 ng PGI 2 per 10 5 cells with a range of 0.05–0.6 ng per 10 5 cells. When cultured cells were mixed with platelets, PGI 2 synthesis from added arachidonate was reduced rather than stimulated. Thus the major precursor cyclic endoperoxides utilized for PGI 2 synthesis are formed within the cells and not from endoperoxides synthesized by platelet cyclooxygenase. Aspirin has been proposed as an anti-thrombotic agent. Aspirin could be ineffective, however, if it inhibited not only platelet cyclooxygenase but that of vessel wall cells as well. Measurement of the rate constant or potency for aspirin inhibition of PGI 2 synthesis in cultured cells indicates that the cyclooxygenase in both cell types of the blood vessel wall is 14–44 fold less sensitive to aspirin inactivation than that in platelets, and appropriate levels of aspirin can selectively block human platelet thromboxane A 2 synthesis without compromising the capacity of the vasculature to produce PGI 2.

Development of a liquid-holding technique for the study of DNA-repair in human diploid fibroblasts

Liquid-holding conditions can be obtained for human diploid skin fibroblasts by keeping confluent cultures stationary over periods of 7 days or longer by means of conditioned medium. Under this condition recovery of radiation damage induced by ultraviolet light of X-rays is observed as an increase in cloning efficiency. The amount of recovery when expressed in a dose-modifying-factor appears higher than in bacteria and yeast. The repair-deficient human cells strains XP25Ro and XP7Be (xeroderma pigmentosum from complementation groups A and D respectively) exhibit less but still discernible recovery after UV-irradiation and the same was observed for AT6Bi (ataxia telangiectasia) after X-irradiation. Experiments on mutation induction indicated that the repair which takes place during liquid holding of UV-irradiated XP7Be cells reduces the mutant frequency considerably while after liquid holding of UV-irradiated wild-type cells the same or lower mutant frequencies were found for the lower exposures and the same or higher mutant frequencies for the higher exposures.

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

During serial subcultures 50 micrograms per ml gentamicin and penicillin (100 U per ml)-streptomycin (100 micrograms per ml) depressed cell growth signficantly 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.

Comparison of polypeptides from cultured human fibroblasts and sarcoma cells

The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000–180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [ 35S]methionine and [ 3H]mannose incorporation experiments but not using the surface labeling method.

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin febroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [ 14C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [ 14C]adenine or [ 14C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.

Comparative effects of human platelet growth factor on the growth and morphology of human fetal and adult diploid fibroblasts

Human platelet growth factor (HPGF) caused marked growth and morphological responses in two lines of fetal and two lines of adult human diploid skin fibroblasts. Dose-response studies demonstrated differences between these two cell types. Adult cells required ten times more HPGF than fetal cells for optimal growth. In the presence of HPGF both cell lines decreased three- to four-fold in size, and their morphology in stationary culture changed to an extremely long, thin fibroblastic shape; reduction in size was accompanied by a corresponding decrease in total protein and fatty acid-containing lipid. These decreases were directly related to the concentration of HPGF in the growth medium. The results suggest that human cells in different stages of cellular development have different requirements for growth-promoting agents (HPGF) and that the use of HPGF on human diploid fibroblasts provides an excellent model system to study growth-associated changes in intermediary metabolism.

Genetics of the connective tissue proteins: Assignment of the gene for human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids

Somatic cell hybrids between mouse and human cell lines have been used to identify the specific chromosome that governs the synthesis of type I procollagen. Fourteen hybrid clones and subclones were derived independently from crosses between mouse parents [LM (thymidine kinase-negative) or A9 (hypoxanthine phosphoribosyltransferase-negative)] and human cells (human diploid lung fibroblasts WI-38 or diploid skin fibroblasts GM5, GM17, and GM9). The cultures were labeled with [(3)H]proline in modified Eagle's medium without serum. Radioactive procollagens were purified from the medium by the method of Church et al. [(1974) J. Mol. Biol. 86, 785-799]. DEAE-cellulose chromatography was used to separate collagen and type I and type III procollagen. Human type I procollagen was assayed by double immunodiffusion analysis with type I procollagen antibodies prepared by immunizing rabbits with purified human type I procollagen. These analyses combined with karyology and isozyme analyses of each hybrid line have produced evidence for the assignment of the gene for human type I procollagen to chromosome 17. A human microcell-mouse hybrid cell line containing only human chromosome 17 was positive for human type I procollagen, lending further support to the assignment of the human type I procollagen gene to chromosome 17. Finally, by using a hybrid line containing only the long arm of human chromosome 17 translocated onto a mouse chromosome, the type I procollagen gene can be assigned more specifically to the long arm of chromosome 17.

Transport of hypoxanthine by human diploid skin fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase

Summary Transport of purine bases and nucleosides by a variety of mammalian cell lines is generally accomplished by facilitated diffusion, a non-concentrative, saturable process. However, previous investigators have been unable to detect a saturable component for the transport of hypoxanthine by human fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase, implying that in normal cells the enzyme actively participates in transport. In the present study we have used the phenomenon of countertransport to demonstrate the existence of a saturable transport mechanism in hypoxanthine-guanine phosphoribosyltransferase-deficient human diploid skin fibroblasts.

Outgrowth of human fibroblast aggregates on a substratum triggers a wide variety of morphogenetic properties in the cells

Twenty-four-hour-old aggregates of human diploid skin fibroblasts are allowed to attach to a glass or plastic substratum. As a result of this attachment the cells in the aggregate demonstrate rapid and generalized changes in cell shape, cell surface and cytoplasm ultrastructure and in their ability to incorporate [3H]thymidine. Within 24 h they grow out on the substratum to attain the regular monolayer configuration. During the process of leaving the aggregate for the substratum a great number of different morphogenetic properties are displayed by the cells, resembling the properties of embryonic or epithelial cells. The simultaneous occurrence of this great variety of cell shape and cell surface changes, many of them unusual for fibroblasts, as well as the concurrent formation of organized cytoplasmic structures - microfilaments, microtubules - at localized areas of the cells, makes this system a potentially useful tool in the study of cell behaviour.

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts

The cytotoxicity of the “K-region” epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several “K-region” epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ration of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions. To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by “K-region” epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the “K-region” epoxide of benzo(a)pyrene (BP), 7,12-dimethylbenz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.

Aggregation-induced alterations in fibroblast morphology. An ultrastructural study.

The different stages during aggregation of diploid human skin fibroblasts have been examined by transmission and scanning electron microscopy. As a result of aggregation, fibroblasts form a complex tissue configuration. Numerous intercellular junctions can be observed, while the cells remain polygonal and do not develop an organised intracellular cytoskeleton. Cell division occurs only rarely. After aggregation, signs of progressive auto-digestion develop. Adhesion to a substrate results in outgrowth of the cells and monolayer formation, even when extensive cell damage had occurred. The morphology of fibroblasts in aggregates and in the monolayers, from which they were derived is compared and the contribution of the aggregate system to the study of fibroblast behavior is discussed.

The use of correction factors in the determination of mutant frequencies in populations of human diploid skin fibroblasts

The need and validity for using a number of correction factors in the determination of mutant frequencies in human diploid skin fibroblasts was investigated. Resistance to the purine analogue 8-azaguanine was used as selective system. It appears that, under the conditions used, there was no effect of the cell density on the cloning efficiency. Therefore observed mutant frequencies need to be corrected for cloning efficiency. The reliability of using Lesch-Nyhan cells as a prototype mutant in the estimation of the efficiency of mutant recovery was investigated and found to hold true. The relation between the efficiency of mutant recovery and cell density, which reflects the degree of inter-clone metabolic cooperation, was measured. The use of this relationship enhances the accuracy of the estimated mutant frequencies. With these correction factors the mutation rate was estimated to be 5.7 · 10 −6 per cell per generation. The influence of intra-clone metabolic cooperation was determined and found to be small if present.

Induction of DNA synthesis and cell division in human diploid skin fibroblasts by fibroblast growth factor.

Abstract The growth requirements of several normal diploid human skin fibroblasts in cell culture were investigated. The human diploid cells ceased growing predominantly due to depletion of serum factors in the medium. Quiescent cultures could be reinitiated to synthesize DNA by the addition of the bovine pituitary factor, fibroblast growth factor (FGF). FGF at saturating concentrations (50 ng/ml) can initiate DNA synthesis in quiescent cultures at approx. 40 to 50% of the efficiency compared with 30% fetal calf serum. Hydrocortisone and insulin did not potentiate the action of FGF in completely quiescent cultures. In the absence of serum FGF, insulin and hydrocortisone failed to maintain continuous cell growth suggesting the involvement of additional serum components. FGF failed to stimulate cells arrested by histidine deprivation to grow.

In vitro cellular radiosensitivity of human malignant tumors

Cells from four human tumors considered to be either clinically curable by radiation therapy (medulloblastoma) or non-radiocurable (osteogenic sarcoma, glioblastoma multiforme) were grown in tissue culture and the in vitro X-ray survival curve parameters determined. These were compared with the survival parameters for normal human diploid skin fibroblasts grown and irradiated under identical conditions. There were no significant differences in response among the tumor cell lines: the D o values ranged from 130 to 144 rads, and ñ from 1·4 to 1·9. For normal lcells, D o = 132 rads, and the ñ = 0·95. These results suggest that the wide range of clinical radiocurabilities found among various human tumors cannot be explained on the basis of the results of the in vitro survival curve parameters.

Electron microscopy of human skin fibroblasts in situ during growth in culture

We have studied the electron microscopic (EM) appearance of cultured diploid human skin fibroblasts during logarithmic and confluent stages of growth using a simple technique which permits in situ visualization of individual cells in monolayer. By comparison, cells disrupted from a monolayer and pelleted showed drastic distortion of the cell surface and appearance of organelles: mechanical scraping produced massive dilatation of the rough endoplasmic reticulum (RER); and trypsin-produced multiple blebs of the plasma membrane and cytoplasmic vacuoles. In situ, these changes in trypsinized cells disappeared within 1 h after plating. Six hours later, microtubules and microfilaments had surrounded the nucleus and were oriented in longitudinal bundles beneath the plasma membrane during rapid growth. At confluence these cytoskeletal elements seemed to extend beyond the plasma membrane at intercellular junctions. Pinocytotic vesicles were abundant at those surfaces devoid of filaments. Mitochondria, aligned with the long axis of the cell, were extremely long and narrow. During logarithmic growth there were many free ribosomes, polysomes, and some flat cisternae of RER. At confluence most ribosomes were found in spirals on dilated saccules of RER which contained electron-dense material. Lysosomes of several types were present during all phases of growth and varied in number from cell to cell. Late in culture the lysosomes tended to be larger, often occupying whole areas of cytoplasm or even extruding from the cell, and resembled those seen in lysosomal storage diseases. Understanding the in situ ultrastructure of normal human fibroblasts during growth in culture will permit systematic examination of such cells in a variety of pathological states.

Human chromosome 21 dosage: effect on the expression of the interferon induced antiviral state.

Human primary skin fibroblasts trisomic for chromosome 13, 18, or 21 and diploid human skin fibroblasts were induced for an antiviral response with human interferon. The cells that were trisomnic for chromosome 21 were three to seven times more sensitive to protection by human interferon than the normal diploid or trisomic 18 or 13 fibroblasts. The differential response in trisomnic 21 cells is consistent with the known assignment of the human antiviral gene to chromosome 21.

HL-A markers on human diploid skin fibroblasts

Skin fibroblasts from 14 normal, healthy volunteers were typed in parallel by cytotoxicity and complement fixation for 25 alleles of the HL-A system. The results of comparing the typing of lymphocytes and platelets with those obtained with fibroblasts from the same donors are in good agreement. However, using both techniques, the accuracy of the results is increased. The necessity of standardization in the choice of HL-A reagents specially adapted for typing fibroblasts is underlined.

Inhibition of collagen synthesis by alpha, alpha1-dipyridyl in human skin fibroblasts in culture.

In human diploid skin fibroblasts in culture we have shown that nonhydroxylated collagen precursors remain in the cell when proline hydroxylation is inhibited by α, α′-dipyridyl, a chelator of ferrous ions. The inhibition of proline hydroxylation is reversed by addition of fresh medium containing 50 μg per ml of sodium ascorbate, whereupon nonhydroxylated collagen precursors are hydroxylated within the cell and extruded into the medium. Extrusion of collagen already formed within the cell is not appreciably affected by α, α′-dipyridyl inhibition. Under normal conditions collagen is released from the monolayer into the medium within 3 hr of a pulse ofL[14C]proline. In the presence of α, α′-dipyridyl, about 35% of theL[14C]proline incorporated into protein is released into the medium within 8 hr as a proline-rich, hydroxyproline-deficient protein; at the same time, approximately 15% of the protein-boundl-[14C]proline remains in the cell for as long as 12 hr. When proline hydroxylation is restored after 2 and 12 hr of α, α′-dipyridyl inhibition, approximately the same amount of hydroxyproline is formed after each time interval in the monolayer. Therefore, nonhydroxylated collagen precursors retained in the cell are not appreciably degraded during at least 12 hr of inhibition by α, α′-dipyridyl and are extruded into the medium only upon restoration of hydroxylation.

Reciprocal changes in the levels of functionally related folate enzymes during the culture cycle in human fibroblasts

The activities of 6 folate enzymes were measured in extracts of human diploid skin fibroblasts during the lag, log and stationary phases of the culture cycle. The levels of 4 folate enzymes involved in nucleic acid biosynthesis, viz. , folate reductase, serine hydroxymethyltransferase, thymidylate synthetase and 10-formyl-THF synthetase, increased from 2–20 fold during the log phase of growth. In contrast, the levels of 2 enzymes, viz. , methylene-THF reductase and 5-methyl-THF: homocysteine methyltransferase, involved in regulating the levels of 5-methyl-THF, the major tissue and serum folate compound, decreased 3–4 fold during log growth, returning to high levels again only after the cells had been in the stationary phase for 5 and 20 days respectively. This reciprocal pattern of change is consistent with the known or postulated functions of these folate enzymes.

Alkaline phosphatase activation and collagen synthesis in human skin fibroblasts in culture

It has been noted in regenerating wounds that alkaline phosphatase activity in fibroblasts reaches a maximum when the collagen production is greatest. Tissue culture studies were carried out to show that prednisolone phosphate, while increasing the specific activity of alkaline phosphatase in human diploid skin fibroblasts, did not affect accumulation of collagen-hydroxyproline in monolayers or media. Addition of sodium ascorbate, resulted in rapid accumulation of hydroxyproline in the culture over a 13-day interval, while alkaline phosphatase activity increased only slightly over the last 6 days. When prednisolone and ascorbate were added in combination, alkaline phosphatase activity was significantly increased: but accumulation of hydroxyproline was no greater than in cultures to which ascorbate alone was added. Activation of alkaline phosphatase induced by prednisolone phosphate does not appear to be directly related to the biosynthesis of collagen in human skin fibroblasts in tissue culture.