Skin Fibroblast Strains Research Articles

Transforming growth factor-beta reverses a posttranscriptional defect in elastin synthesis in a cutis laxa skin fibroblast strain.

Skin fibroblasts from two cases of autosomal recessive cutis laxa (CL), having insignificant elastin production and mRNA levels, were challenged with transforming growth factor beta-1 (TGF-beta 1). Elastin production was brought from undetectable values to amounts typical of normal human skin fibroblasts in a dose-dependent fashion. Basic fibroblast growth factor (100 ng/ml) alone or in combination with TGF-beta 1 reduced elastin production and mRNA expression in CL skin fibroblasts more extensively than in normal cells. In situ hybridization showed that these effects were at the transcript level. One of the CL strains was examined in detail. Transcription rates for elastin were similar in normal and CL and unchanged by TGF-beta 1 or TGF-beta 2 (10 ng/ml), while in CL elastin mRNA half-life was increased > 10-fold by TGF-beta 2 and reduced 6-fold after TGF-beta 2 withdrawal, as compared with a control strain. Cycloheximide partially reversed elastin mRNA instability. These data are consistent with a defect in elastin mRNA stability that requires synthesis of labile factors or intact translational machinery, resulting in an extremely low steady state level of mRNA present in this strain of CL. Furthermore, TGF-beta can relieve elastin mRNA instability in at least one CL strain and elastin production defects in both CL strains.

Complementation studies in Niemann-Pick disease type C indicate the existence of a second group.

Niemann-Pick disease type C is a clinically heterogeneous storage disorder with an unknown primary metabolic defect. We have undertaken somatic cell hybridisation experiments using skin fibroblast strains from 12 patients representing a wide clinical spectrum. Preliminary experiments using filipin staining of free cholesterol as a marker for complementation indicated the existence of one major group (group alpha) and one minor group (group beta) represented by one mutant strain. Subsequent experiments in which sphingomyelinase activity was measured as a marker for complementation using five mutant strains showing activity consistently < 40% control levels confirmed the existence of the second group.

Spontaneous chromosomal aberrations in cell cultures from patients with neurofibromatosis 1

Neurofibroma-derived cell cultures, skin fibroblast strains from NF1 patients, melanocyte cultures from café-au-lait spots and melanocytes from skin overlying neurofibroma were investigated with regard to the frequencies of spontaneous chromosomal aberrations. A 3.4-fold increased rate of stable and unstable chromosome aberrations in 34 neurofibroma cultures of 18 NF1 patients was noticed in comparison to 17 skin biopsy-derived cultures of healthy probands. Fibroblast strains from the unaffected skin of nine NF1 patients revealed a 2.6-fold higher rate of chromosome breakage compared with the control cultures. Likewise, an increase of spontaneous chromosomal instability by a factor of 13.5 was found in cultured melanocytes from café-au-lait spots and by a factor of 11.9 in the skin-melanocyte cultures of six NF1 patients in comparison to foreskin-derived melanocyte strains of five unaffected persons. Analyses of the distribution of chromatid and chromosome breaks along the chromosomes revealed a significant clustering of these events in the centromeric regions specifically in the four kinds of NF1-derived cultures.

Effect of cell-cell and cell-matrix interactions on the response of fibroblasts to epidermal growth factor in vitro. Expression of collagen type I, collagenase, stromelysin and tissue inhibitor of metalloproteinases.

Investigations of the effect of epidermal growth factor (EGF) on the expression of four genes involved in the turnover of the extracellular matrix, collagen type I, collagenase, stromelysin and tissue inhibitor of metalloproteinases (TIMP) were performed on four strains of skin fibroblasts in vitro. Addition of EGF to subconfluent cultures for increasing periods of time up to 5 days induced an inhibition of procollagen alpha 1(I) mRNA and a strong stimulation of collagenase (100-fold) and stromelysin (1000-fold) mRNAs, whereas the mRNA of TIMP was increased to a lesser extent (5-fold). After a 40 h pulse with EGF, these effects persisted for 24-48 h after withdrawal of the growth factor and slowly diminished thereafter to attain control values after several days. By culturing fibroblasts for increasing periods of time, different levels of confluence were obtained allowing for the deposition of an extracellular biomatrix. The steady-state level of collagenase and stromelysin mRNAs were profoundly depressed in confluent as against non-confluent cultures, whereas no major change for TIMP and procollagen alpha 1(I) mRNAs was observed. Upon treatment of these cultures with EGF for 48h, the steady-state level of collagenase, stromelysin and TIMP increased, whereas procollagen alpha 1(I) mRNA was slightly reduced. These modifications were, at least in part, dependent upon a regulation of the transcription rate, as suggested from run-off experiments. Similar states of confluence were obtained by seeding cells at increasing densities in short-term cultures in which cell-cell contact predominated. In such culture conditions, the collagenase and stromelysin mRNAs were enhanced in high as compared to low density cultures. The response to EGF was progressively decreased for collagenase, stromelysin and, to a lesser extent, TIMP mRNAs at most densities and a complete lack of response to EGF at the highest cell density was observed. Under all culture conditions the modulation of collagenase mRNA was paralleled by similar modifications of enzyme activity. These results emphasize the importance of the cell-cell contacts and cell-matrix interactions in the expression of the genes coding for metalloproteinases or their inhibitor and their modulation by growth factors.

Induction and superinduction of messenger ribonucleic acid specific for aromatase cytochrome P-450 in cultured human skin fibroblasts.

Aromatase cytochrome P-450 (cytochrome P-450AROM) catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. Several studies indicate that cytochrome P-450AROM activity is induced by glucocorticoids such as dexamethasone (DEX) and superinduced by DEX plus cycloheximide (CHX). We have used cultured human skin fibroblasts as a model system to investigate the regulation of aromatase gene expression. Whereas Northern blot analysis of total cellular RNA or poly (A)+ RNA from untreated strains of normal human skin fibroblasts failed to demonstrate any hybridization with a specific human placental cytochrome P-450AROM complementary DNA, analysis of RNA from cells treated with DEX demonstrated hybridization of the cytochrome P-450AROM complementary DNA to two transcripts of about 2.5 and 3.0 kilobases. Incubation of cells with DEX plus CHX resulted in a further increase in levels of cytochrome P-450AROM messenger RNA (mRNA) when compared to cells treated with DEX alone, suggesting that inhibition of protein synthesis superinduces transcription of the cytochrome P-450AROM gene. By contrast, levels of beta-actin mRNA were not affected by treatment with DEX and CHX. Treatment of cells with CHX alone did not produce a change in either aromatase activity or levels of cytochrome P-450AROM mRNA transcripts. These results indicate that aromatase activity is regulated by changes in the concentration of cytochrome P-450AROM mRNA, and imply that control of cytochrome P-450AROM gene expression is at the level of gene transcription. We conclude that the cytochrome P-450AROM gene is regulated by a complex mechanism that includes both positive and negative transcription factors.

Transformation and immortalization of diploid xeroderma pigmentosum fibroblasts

Diploid xeroderma pigmentosum (XP) skin fibroblast strains from various XP-complementation groups (B, C, G, and H) were transformed with an origin-defective SV40 early region or with the pSV3gpt plasmid. In the latter case, transfected cells were selected for their ability to express the dominant xgpt gene. Immortalized cell lines were obtained from XP-complementation groups C (8CA, 3MA, and 20MA; XP3MA and XP20MA were formerly considered to belong to complementation group I), G (2BI and 3BR), and H (2CS). No immortalized cells could be isolated from complementation group B (11BE). The immortalization frequency of wild-type diploid fibroblasts and diploid cultures from XP patients was not significantly increased by cotransfection with the SV40 early region plus several selected viral and cellular oncogenes. In fact, co-transfection with some of the oncogenes caused a marked decrease of the transformation frequency. The observed immortalization occurred at a frequency of approximately 5 × 10 −8.

Aging increases adenosine and inosine release by human fibroblast cultures

The effect of in vitro age and donor age on net release of adenosine and inosine was studied in cultures of normal human fibroblasts. Confluent cultures of low- (population doubling level [PDL] 23–25) and high- (PDL 43–45) passage human lung fibroblasts derived from a 16-week-old fetal donor (IMR-90) were incubated for 30 min in physiological saline and the release of adenosine and inosine into the saline was determined by HPLC. Release of adenosine and inosine into the saline bathing low-passage human skin fibroblasts derived from a 16-week-old fetal donor (GM6111) was also determined and compared with two strains of low-passage skin fibroblasts from aged (66–67 years) donors (GM3529 and GM3524). The release of adenosine and inosine by low-passage cultures of fetal lung fibroblasts was 911 and 225 pmol/30 min per mg protein, respectively. In high-passage cultures of lung fibroblasts, release of adenosine and inosine was significantly greater at 1403 and 351 pmol/30 min per mg protein, respectively. The release of adenosine and inosine by low-passage cultures of fetal skin fibroblasts was 250 and 179 pmol/30 min per mg protein, respectively. In low-passage skin fibroblasts from aged donors, release of adenosine and inosine was significantly greater at 583 and 652 pmol/30 min per mg protein, respectively. These results indicate that the net release of adenosine and inosine by cultured human fibroblasts into their extracellular environment is enhanced by in vitro aging of lung fibroblasts and is greater in skin fibroblasts from aged donors.

Glutamine dependency of human skin fibroblasts: Modulation by hexoses

The combined effects of carbohydrates and glutamine were investigated in diploid strains of normal human skin fibroblasts cultured for 21 days under eight different culture conditions: hexose-free medium or medium containing d-glucose, d-galactose, or d-fructose, with or without added glutamine. Cell growth, hexose consumption, lactate production, intracellular glycogen content and extracellular amino acid levels were measured every third to fourth day. In the presence of glutamine, cells reached a higher saturation density in fructose medium than in glucose or galactose medium but per cell consumption of fructose and galactose was much less than that of glucose. Consumption of all three carbohydrates per unit cell growth exhibited three distinct phases: Days 1–3, 3–10, and 10–20, respectively. In the absence of glutamine the rate of cell growth was not altered in glucose or galactose medium, but slowed down considerably in fructose medium. Glutamine deprivation also led to changes in hexose consumption. In hexose-free media the cell growth rate at first was very slow, but rose after 2 or 3 weeks of culture. The levels of extracellular nonessential amino acids varied according to medium and growth phase. One of the most exciting findings was that human fibroblasts are able to maintain a slight excess of glutamine in all media not supplemented with glutamine and, more surprisingly, to synthesize it in a medium containing galactose and glutamine.

Mitomycin C-induced postmitotic fibroblasts retain the capacity to repair pyrimidine photodimers formed after UV-irradiation

The formation and excision of UV-C light-induced cyclobutane-type pyrimidine photodimers were determined in cultures of human skin fibroblasts at time zero and several weeks following treatment with mitomycin C (MMC). Characteristic morphological changes of the fibroblasts and specific shifts in the [35S]methionine polypeptide pattern of total cellular proteins support the notion that MMC accelerates the differentiation pathway from mitotic (MF) to post-mitotic fibroblasts (PMF). No discernible difference could be detected between the fluence-response curves of pyrimidine dimers for untreated and MMC-treated repair-deficient xeroderma pigmentosum cells of group A. Furthermore we investigated the removal of pyrimidine dimers in 3 normal human skin fibroblast strains frequently used in mutation, transformation and aging research. We were able to demonstrate that no significant difference exists in the rate and extent of the excision-repair response to thymine-containing pyrimidine dimers following UV-irradiation shortly after MMC treatment of fibroblasts and in the MMC-induced PMF stage of these cells.

Deficient activation by a human cell strain leads to mitomycin resistance under aerobic but not hypoxic conditions

Two non-transformed human skin fibroblast strains, GM38 and 3437T, were found to be more sensitive to the bioreductive alkylating agents mitomycin C (MMC) and porfiromycin (PM) under hypoxic compared to aerobic conditions. One of these strains, 3437T, was 6-7 times more resistant to these agents under aerobic exposure conditions, but was identical in sensitivity to the normal strain, GM38, under hypoxic conditions. Aerobic 3437T cells demonstrated no increased resistance to cisplatin compared to the normal strain, arguing against enhanced ability to repair DNA interstrand cross-links as the underlying explanation for the mitomycin resistance. The aerobic resistance of 3437T was not altered by dicumarol, an inhibitor of the enzyme DT-diaphorase which is believed to be involved in aerobic activation of MMC and PM. Dicumarol did increase the resistance of GM38, but not to the same level of resistance demonstrated by 3437T. These results suggest that the aerobic MMC and PM resistance of 3437T may arise, in part, from a deficiency in DT-diaphorase activity. The identical sensitivities under hypoxic conditions indicate that drug activation pathways operative in the absence of oxygen are similar in both the normal and 3437T cells.

Analysis of cell surface glycoconjugates in fibroblasts from patients with cystic fibrosis

Although the basic biochemical defect in cystic fibrosis (CF) is unknown, previous studies have indicated that errors in protein glycosylation may be involved in the pathogenesis of the disease. Utilizing human skin fibroblasts, the present study was designed to quantitatively analyze glycosylation of cell surface glycoconjugates in CF and normal cells. Cell surface glycoconjugates were analyzed using 125I-concanavilin A (Con A), 125I-WGA, and Con A-ferritin conjugates. Under our binding conditions, Con A was used as a probe for mannose residues and WGA was used as a probe for N-acetylglucosamine residues. Saturable binding of both probes was observed and appropriate sugar controls confirmed the specificity of each lectin. When compared on a DNA basis, iodinated lectin binding studies indicated that no consistent differences existed between CF and normal strains of human skin fibroblasts. Ultrastructural quantitative morphometric analysis of Con A-ferritin conjugate binding indicated that neither proteolysis of cell surface glycoconjugates or internalization of lectin probes was occurring at saturable binding concentrations. In summary, our results indicated that no consistent differences in cell surface mannose and N-acetylglucosamine residues could be detected between the normal and CF strains of human skin fibroblasts used in these studies.

Cell-culture studies on neurofibromatosis (von Recklinghausen's disease). III. Experiments on X-ray sensitivity.

The X-ray sensitivity of strains of fibroblast-like cells derived from peripheral neurofibromas of ten patients with neurofibromatosis was compared with that of 12 strains of skin fibroblasts derived from healthy donors. Quantitative parameters of the dose-dependent reduction in colony-forming ability did not differ significantly between these two groups of strains. The cloning efficiencies of nonirradiated controls varied within the same range in strains derived from patients and from healthy donors.

Abnormal fibroblast aging and DNA replication in the Werner syndrome.

Cell and DNA replicative potentials were studied in 10 strains of skin fibroblasts from unrelated patients with the Werner syndrome (WS) and in a progeric CRL 1277 strain. The lifespans of all WS strains and of CRL 1277 cells are greatly abbreviated in vitro, due to large fractions of non-cycling cells and the basic process of progressive clonal attenuation during Phase II. In some WS strains, the population-doubling rate per day and the cloning efficiency fluctuated concurrently in random fashion during the cellular aging process, indicating alternating successions of adaptively well and poorly growing clones, probably resulting from various chromosome translocations. No detectable defect in excision repair was found in WS or CRL 1277 cells. However, the rate of increase in the molecular weight of pulse-chased DNA involving overall rates of chain elongation and replicon fusion was retarded in WS and CRL 1277 cells. Also, pulse-labelled DNA in WS fibroblasts was less enriched in the nuclear matrix and was more slowly chased out than in normal cells. These results led us to postulate a misfiring or delayed initiation due to the sticky attachment of replicating DNA to the nuclear matrix in the replisomes of WS fibroblasts. A suggested model for abnormal DNA replication is presented and discussed to explain the loss of DNA and the chromosome abnormalities in WS cells. The abnormal DNA-synthetic profiles so derived appeared to be normalized in SV40-transformed PSV811 (WS) cells as were in gamma ray-transformed wild-type WI38CT-1 cells.

Fibroblast androgen receptors in patients with genitourinary anomalies.

The etiology of certain disorders of sexual differentiation is unclear. The authors have examined the hypothesis that hypospadias and other disorders compatible with a defect in androgen action, such as cryptorchidism, micropenis, chordee/penile torsion, and ectopic testis, might be explained by androgen receptor abnormalities. Therefore, 25 subjects were studied who were selected only because they had one of these developmental defects, together with a predominantly male phenotype, and no readily ascertainable explanation for the defect. Four of these subjects had mixed gonadal dysgenesis with multiple genito-urinary anomalies. They were included for comparative purposes, since there is no evidence for androgen resistance in this disorder. Patients with testicular regression syndrome (gross testosterone deficiency), impaired testosterone biosynthesis (relative testosterone deficiency), 5 alpha-reductase deficiency (altered T/DHT ratio), and a family history or endocrine profile suggestive of androgen resistance, were all excluded from evaluation. Androgen receptor content (R0) and binding affinity (Kd) were measured in 26 genital or pubic skin fibroblast strains cultured from 25 affected patients using a dispersed, whole cell assay at 22 C. There was no difference in the mean androgen receptor content (approximately 10,000 sites/cell) or binding affinity (approximately 1 nM) between the patients' fibroblasts and those from 26 fibroblast strains established from 26 normal males. Moreover, there were no differences in the nuclear uptake of [3H]dihydro-testosterone into dispersed, intact fibroblasts incubated at 37 C when 11 patient and seven normal male fibroblast strains were compared.(ABSTRACT TRUNCATED AT 250 WORDS)

α&lt;sub&gt;2&lt;/sub&gt;-Macroglobulin Production by Cultured Human Melanoma Cells

alpha 2-Macroglobulin (alpha 2M) was identified in chemically defined spent medium of cultured human melanoma cells. This wide-spectrum protease inhibitor was subsequently shown to be synthesized and was secreted by 10 of 13 cultured melanoma cells but not by neoplastic cells of other histologic origins (0/5 breast, 0/2 colon, 0/2 lung, 0/2 glioblastomas, 0/1 neuroblastoma, 0/4 lymphoid). alpha 2M was also produced by one strain of fetal lung fibroblasts, as was previously shown, but not by two strains of normal fetal uveal melanocytes or two strains of fetal skin fibroblasts. Biochemical characterization of melanoma alpha 2M indicated that it has a similar tetrameric structure but is smaller in size than authentic serum alpha 2M. Preliminary studies indicated that melanoma alpha 2M can bind to protease and has possible functional capability.

Connective tissue activation

Four normal (NF) and 4 scleroderma skin fibroblast (SF) strains were compared with respect to 1) basal 14C-glucosamine and 35SO4-labeled glycosaminoglycan (GAG) synthesis, 2) responsiveness to autacoid mediators, and 3) performance following maximal stimulation. Under basal conditions, SF synthesized and secreted 2-3 times more radioactive hyaluronic acid than the NF (P less than 0.001); molecular volume by gel chromatography was similar and suggested a high molecular weight product. SF were essentially as responsive to normal lymphoid and platelet factors as were NF. No consistent qualitative or quantitative differences in sulfated GAG synthesis were noted between the 2 groups of cells. Incubation of NF and SF with a false "core protein" such as p-nitrophenyl-beta-D-xyloside suggested that synthesis of the core protein was rate limiting; SF and NF were equally facile in SO4-GAG chain synthesis in the presence of a beta-xyloside. SF appear to retain in vitro a partially activated state for many generations, at least with respect to hyaluronic acid synthesis.

Action spectra for inactivation of normal and xeroderma pigmentosum human skin fibroblasts by ultraviolet radiations.

Abstract—Action spectra for UV‐induced lethality as measured by colony forming ability were determined both for a normal human skin fibroblast strain (lBR) and for an excision deficient xeroderma pigmentosum strain (XP4LO) assigned to complementation group A using 7 monochromatic wavelengths in the range 254‐365 nm. The relative sensitivity of the XP strain compared to the normal skin fibroblasts shows a marked decrease at wavelengths longer than 313 nm. changing from a ratio of about 20 at the shorter wavelengths to just greater than 1.0 at the longer wavelengths. The action spectra thus indicate that the influence on cell inactivation of the DNA repair defect associated with XP cells is decreased and almost reaches zero at longer UV wavelengths. This would occur, for example, if the importance of pyrimidine dimers as the lethal lesion decreased with increasing wavelength. In common with other studies both in bacterial and mammalian cells, our results are consistent with pyrimidine dimers induced in DNA being the major lethal lesion in both cell strains over the wavelength range 254‐313 nm. However, it is indicated that different mechanisms of inactivation operate at wavelengths longer than 313 nm.

Induction and repair of DNA damage in normal and ataxia-telangiectasia skin fibroblasts treated with neocarzinostatin.

Cells from patients with the hereditary multisystem disorder ataxia-telangiectasia (A-T) are hypersensitive to the cytotoxic action of DNA-breaking agents, such as X-rays, bleomycin and neocarzinostatin (NCS). A defect in the repair of a certain DNA lesion induced by all three agents may underlie this hypersensitivity. This DNA lesion may be a certain type of DNA strand break. Most of the previous experiments done with X-rays and bleomycin failed to show any retardation in the rejoining of DNA strand breaks in A-T cells. However, since both A-T homozygous and heterozygous cells are particularly hypersensitive to NCS, we studied the time course of strand breakage induction and repair in A-T skin fibroblast strains treated with NCS, using the sensitive method of alkaline or neutral elution. A linear dose response was obtained for the induction by NCS of single-strand breaks and double-strand breaks. A-T cells did not respond with a higher initial extent of strand breakage compared with normal cells. NCS is an appropriate agent for studying the kinetics of rejoining strand breaks, due to its rapid action in the cells; this action, which is completed within 2--4 min, was studied by monitoring strand break induction, inhibition of DNA synthesis and decrease in cellular survival. The time course of strand break rejoining found after NCS treatment was very similar to that found following X-irradiation: with both single- and double-strand breaks, a rapid phase of rejoining was first noticed (t 1/2 approximately 5 min for single-strand breaks and 20--25 min for double-strand breaks). This was followed by a second, slow phase that continued for several hours. No difference could be detected between normal and A-T cells either with regard to the time course of rejoining or the fraction of non-rejoined breaks remaining several hours after treatment.

Repair of potentially lethal and sublethal damage induced by neocarzinostatin in normal and ataxia-telangiectasia skin fibroblasts

Neocarzinostatin is a radiomimetic antibiotic with a potent cytotoxic effect which elicits a hypersensitive response in human cells homozygous or heterozygous for the gene for ataxia-telangiectasia. The extent and the time course of potentially lethal damage repair and sublethal damage repair following neocarzinostatin treatment were investigated in human skin fibroblast strains and were found to be remarkably similar to those obtained following X-irradiation. Ataxia-telangiectasia homozygous cells essentially lacked potentially lethal damage repair, but were able to perform some degree of sublethal damage repair following neocarzinostatin treatment. Ataxia-telangiectasia heterozygous cells which show an intermediate degree of neocarzinostatin sensitivity could perform both processes but with somewhat reduced efficiency as compared to normal cells. These observations provide further evidence for a DNA repair defect in ataxia-telangiectasia cells.

Abnormal sensitivity to UV-radiation in cultured skin fibroblasts from patients with hereditary cutaneous malignant melanoma and dysplastic nevus syndrome.

The dysplastic nevus syndrome (DNS) is a preneoplastic melanocyte abnormality which occurs in families affected by hereditary cutaneous malignant melanoma (HCMM). Although environmental exposures, especially solar UV-irradiation, have been implicated as risk factors in sporadic melanoma, the role of such exposures in the pathogenesis of HCMM is unknown. We have studied the in vitro radiation responses of six non-tumor skin fibroblast strains from HCMM/DNS patients representing five families. All six HCMM/DNS strains were found to show some degree of enhanced cell killing sensitivity, compared with normal controls, following 254 nm UV-irradiation. The abnormal survival responses appeared to relate to specific characteristics of HCMM/DNS cells since the six strains had essentially normal sensitivity to gamma-radiation. The enhanced photosensitivity was not associated with abnormal patterns in either DNA repair synthesis or UV-induced inhibition and recovery of de novo DNA synthesis. The survival results are consistent with the hypothesis that the genetically determined predisposition to malignant melanoma may directly or indirectly be the consequence of increased susceptibility to UV-induced cellular damage.