Skin Fibroblast Strains Research Articles

Gene Expression Profiles Reveal Extracellular Matrix and Inflammatory Signaling in Radiation-Induced Premature Differentiation of Human Fibroblast in vitro.

PurposeFibroblasts are considered to play a major role in the development of fibrotic reaction after radiotherapy and premature radiation-induced differentiation has been proposed as a cellular basis. The purpose was to relate gene expression profiles to radiation-induced phenotypic changes of human skin fibroblasts relevant for radiogenic fibrosis.Materials and MethodsExponentially growing or confluent human skin fibroblast strains were irradiated in vitro with 1–3 fractions of 4 Gy X-rays. The differentiated phenotype was detected by cytomorphological scoring and immunofluorescence microscopy. Microarray analysis was performed on Human Genome U133 plus2.0 microarrays (Affymetrix) with JMP Genomics software, and pathway analysis with Reactome R-package. The expression levels and kinetics of selected genes were validated with quantitative real-time PCR (qPCR) and Western blotting.ResultsIrradiation of exponentially growing fibroblast with 1 × 4 Gy resulted in phenotypic differentiation over a 5-day period. This was accompanied by downregulation of cell cycle-related genes and upregulation of collagen and other extracellular matrix (ECM)-related genes. Pathway analysis confirmed inactivation of proliferation and upregulation of ECM- and glycosaminoglycan (GAG)-related pathways. Furthermore, pathways related to inflammatory reactions were upregulated, and potential induction and signaling mechanisms were identified. Fractionated irradiation (3 × 4 Gy) of confluent cultures according to a previously published protocol for predicting the risk of fibrosis after radiotherapy showed similar downregulation but differences in upregulated genes and pathways.ConclusionGene expression profiles after irradiation of exponentially growing cells were related to radiation-induced differentiation and inflammatory reactions, and potential signaling mechanisms. Upregulated pathways by different irradiation protocols may reflect different aspects of the fibrogenic process thus providing a model system for further hypothesis-based studies of radiation-induced fibrogenesis.

Cadaverine effect on microtubule polymerization

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.

Apolipoprotein D (APOD) is a putative biomarker of androgen receptor function in androgen insensitivity syndrome

Androgen insensitivity syndrome (AIS) is the most common cause of disorders of sex development usually caused by mutations in the androgen receptor (AR) gene. AIS is characterized by a poor genotype-phenotype correlation, and many patients with clinically presumed AIS do not seem to have mutations in the AR gene. We therefore aimed at identifying a biomarker enabling the assessment of the cellular function of the AR as a transcriptional activator. In the first step, we used complementary DNA (cDNA) microarrays for a genome-wide screen for androgen-regulated genes in two normal male primary scrotal skin fibroblast strains compared to two labia majora fibroblast strains from 46,XY females with complete AIS (CAIS). Apolipoprotein D (APOD) and two further transcripts were significantly upregulated by dihydrotestosterone (DHT) in scrotum fibroblasts, while CAIS labia majora cells were unresponsive. Microarray data were well correlated with quantitative real-time polymerase chain reaction (qRT-PCR; R = 0.93). Subsequently, we used qRT-PCR in independent new cell cultures and confirmed the significant DHT-dependent upregulation of APOD in five normal scrotum strains [13.5 +/- 8.2 (SD)-fold] compared with three CAIS strains (1.2 +/- 0.7-fold, p = 0.028; t test) and six partial androgen insensitivity syndrome strains (2 +/- 1.3-fold, p = 0.034; t test). Moreover, two different 17ss-hydroxysteroid dehydrogenase III deficiency labia majora strains showed APOD induction in the range of normal scrotum (9.96 +/- 1.4-fold), supporting AR specificity. Therefore, qRT-PCR of APOD messenger RNA transcription in primary cultures of labioscrotal skin fibroblasts is a promising tool for assessing AR function, potentially allowing a function-based diagnostic evaluation of AIS in the future.

Rescue of Migratory Defects of Ehlers–Danlos Syndrome Fibroblasts In Vitro by Type V Collagen but not Insulin-Like Binding Protein-1

Mutations in the genes encoding for type V collagen have been found in the classical type of Ehlers-Danlos syndrome (EDS); the most common mutations lead to a non-functional COL5A1 allele. We characterized three skin fibroblast strains derived from patients affected by classical EDS caused by COL5A1 haploinsufficiency. As a typical clinical hallmark of EDS is the impaired wound healing, we analyzed the repair capability of fibroblasts in a monolayer wounding assay. The mutant fibroblast strains were unable to move into the scraped area showing then a marked delay in wound repair. In all the EDS strains, type V collagen was absent in the extracellular space, also leading to the lack of fibronectin fibrillar network and impairing the expression of alpha(2)beta(1) and alpha(5)beta(1) integrins. The abnormal integrin pattern inhibited the positive effect of insulin-like growth factor-binding protein-1 on cell migration, whereas the migratory capability remarkably improved in the presence of exogenous type V collagen.

Effect of cellular aging on collagen biosynthesis: II. Collagen synthesis and deposition by a human skin fibroblast strain over 25 passages

Collagen synthesis and accumulation were studied by serial cultures of human dermal fibroblasts. A freshly seeded strain of cells was compared to cryopreserved cells from the same donor. Up to 25 passages there was no clear sign of reaching phase III, the decline of the culture. This was ascertained by the count of the dead cells floating on top of the culture fluid, the time to reach saturation density and the number of cells at confluence, counted after trypsinization. Although collagen synthesis oscillated to some extent, the average value of collagen deposited by the cells did not show any clear sign of decrease of collagen synthesis, on the contrary, there was an increase between the 15th and 25th passages. A review of the literature revealed that recent experiments on the age-dependent variation of maximal passage number did not confirm previous results showing a progressive decline, which was much delayed in recent studies as compared to previous records. The same appears to happen with collagen synthesis, found by earlier investigators to decline with increasing passage numbers. This is not confirmed, at least up to passage 25 in the present experiments. As skin tissue is progressively lost with age, our results are more in favor of increasing matrix degradation with age as an important factor of the age-dependent loss of skin tissue, more than decrease of matrix synthesis.

The gene expression profile of psoralen plus UVA-induced premature senescence in skin fibroblasts resembles a combined DNA-damage and stress-induced cellular senescence response phenotype

After a finite number of population doublings, normal human cells undergo replicative senescence accompanied by growth arrest. We previously described a model of stress-induced premature senescence by treatment of dermal fibroblasts with psoralen plus UVA, a common photodermatological therapy. Psoralen photoactivation has long been used as a therapy for hyperproliferative skin disorders. The repetitive therapeutical treatment is accompanied by premature aging of the skin. Treatment of fibroblasts in vitro with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation results in growth arrest with morphological and functional changes reminiscent of replicative senescence. For gene expression profiling in two strains of human skin fibroblasts after PUVA treatment, we used a low-density DNA array representing 240 genes involved in senescence and stress response. Twenty-nine genes were differentially expressed after PUVA treatment in the two strains of human skin fibroblasts. These genes are involved in growth arrest, stress response, modification of the extracellular matrix and senescence. This study contributes further to the elucidation of the PUVA model and its validation as a useful stress-induced premature senescence model aiming to characterize the premature senescence of fibroblasts and to identify biomarkers that could be applied in vivo.

The Elastin Receptor Complex Transduces Signals through the Catalytic Activity of Its Neu-1 Subunit

The binding of elastin peptides on the elastin receptor complex leads to the formation of intracellular signals but how this is achieved remains totally unknown. Using pharmacological inhibitors of the enzymatic activities of its subunits, we show here that the elastin peptide-driven ERK1/2 activation and subsequent pro-MMP-1 production, observed in skin fibroblasts when they are cultured in the presence of these peptides, rely on a membrane-bound sialidase activity. As lactose blocked this effect, the elastin receptor sialidase subunit, Neu-1, seemed to be involved. The use of a catalytically inactive form of Neu-1 and the small interfering RNA-mediated decrease of Neu-1 expression strongly support this view. Finally, we report that N-acetyl neuraminic acid can reproduce the effects of elastin peptides on both ERK1/2 activation and pro-MMP-1 production. Altogether, our results indicate that the enzymatic activity of the Neu-1 subunit of the elastin receptor complex is responsible for its signal transduction, presumably through sialic acid generation from undetermined substrates.

Fibroblast heterogeneity in collagenolytic response to colchicine

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in various physiological and pathological conditions, including those that involve homeostasis of collagen. Drug induced regulation of MMP-1, other MMPs and TIMPs is critical in treatment of various diseases, e.g. the use of the plant alkaloid, colchicine. One possible factor that might explain the failure in colchicine-treatment of some patients is interindividual variability on the cellular level. To investigate the possible individual heterogeneity in response to colchicine, we studied the effect of colchicine-induced synthesis of collagenase from 32 different human skin fibroblast strains derived from both healthy individuals as well as individuals with different skin diseases. We showed that colchicine induced an increased synthesis of collagenase in 22 of 32 cases. This heterogeneity occurred in fibroblasts from healthy as well as diseased individuals. To determine if colchicine also affected the fibroblast synthesis of gelatinase, stromelysin and tissue inhibitors of MMPs, we investigated several individuals from a single family. The results showed that both colchicine responsive and non-responsive fibroblasts with respect to collagenase synthesis responded to colchicine by an increased stromelysin synthesis, while the synthesis of gelatinase and TIMP-1 were unaffected. As a whole, our results indicate that individual heterogeneity in collagenase response to colchicine treatment may partly explain some of the controversial results obtained with colchicine as a drug.

Establishment and Culture of Human Skin Fibroblasts

The establishment of skin fibroblast strains provides a vehicle by which biochemical and genetic studies may be applied. In addition to providing genetic material to study the basis of disease, fibroblast cell lines established from skin biopsies may provide the investigator with a model in which to study specific disease states or normal physiology. This unit provides methods for the biopsy, establishment, and culture maintenance of skin fibroblasts to be used in further scientific study.

Decreased Expression of IGF-II and Its Binding Protein, IGF-Binding Protein-2, in Genital Skin Fibroblasts of Patients with Complete Androgen Insensitivity Syndrome Compared with Normally Virilized Males

The action of androgen by way of the AR is required for the development of male gonads and external genitalia. The interplay between androgens and the somatotropic axis, in particular the IGFs in sexual development, is currently under thorough investigation. The IGF system is thought to mediate the androgen action in androgen-responsive cells. To investigate the interaction of androgens with the IGF system, we compared the expression of IGFs and IGF-binding proteins in cultured genital skin fibroblasts from nine patients with the syndrome of complete androgen insensitivity with that in genital skin fibroblasts from 10 normally virilized males. Mutations in the AR gene and/or abnormalities of the AR protein in the immunoblot were detected in all complete androgen insensitivity genital skin fibroblast strains. They caused a complete failure of DHT binding. RIA and RT-PCR demonstrated that the genital skin fibroblast strains expressed IGF-II, IGF-binding protein-2, and IGF-binding protein-3, but no IGF-I. Most strikingly, complete androgen insensitivity genital skin fibroblast strains produced significantly lower IGF-II (P < 0.001; 42.2 ± 9.7 vs. 106.9 ± 11.8 ng/mg protein) and IGF-II mRNA (P < 0.01, by RT-PCR) than control genital skin fibroblast strains. The production of IGF-binding protein-2 was also decreased (P < 0.03) in complete androgen insensitivity genital skin fibroblasts, whereas that of IGF-binding protein-3 did not differ. Furthermore, high levels of IGF-binding protein-5 mRNA were detected in all genital skin fibroblast strains, whereby the 28-kDa band in the ligand blot, probably representing IGF-binding protein-5, was more abundant in complete androgen insensitivity genital skin fibroblasts. Exposure of the genital skin fibroblasts to T (5 × 10−8m) had only weak effects on the expression of IGFs and IGF-binding proteins. In conclusion, although the mechanism underlying these differences requires further study, it is conceivable that in addition to the endocrine actions of IGF-I, IGF-II and IGF-binding protein-2, as local growth factors, are involved in the mediation of androgen action and growth of genital tissues.

Assessment of the relationship between genotypic status of a DT-diaphorase point mutation and enzymatic activity.

DT-diaphorase, a cytosolic reductase, has been implicated as an activator of chemotherapeutic prodrugs and a detoxifier of certain potentially carcinogenic xenobiotics. A common C to T nucleotide 609 substitution in DT-diaphorase cDNA has been associated with protein instability and reduced catalytic activity. The degree to which the allelic status of the substitution correlates with enzymatic activity was assessed in 45 normal human skin fibroblast strains using a PCR-RFLP assay. Included in this study was the 3437T strain, which is unique in that it is heterozygous for the polymorphism yet contains undetectable enzymatic activity. An allele-specific RT-PCR-RFLP technique attributed this phenomenon to exclusive DT-diaphorase mRNA expression from the variant allele. Overlap in activities was observed between individual strains homozygous for the wild-type allele and heterozygotes, but the former group displayed enzymatic activity that was on average 2-fold higher. Western blot analysis of the two strains in this panel that are homozygous for the variant allele revealed that they express relatively low amounts of DT-diaphorase protein, consistent with the role of the substitution in protein instability. This work confirms that genotypic status is a reliable initial estimate of DT-diaphorase activity. © 2000 Cancer Research Campaign

Occurrence and expansion of trisomy 7 in a fibroblast strain from a centenarian individual.

We describe the presence of metaphases with non-random gain of one or two chromosomes in a skin fibroblast strain derived from a centenarian individual. The extra elements were chromosomes 7, X, and 18, and, among these, the most frequent was a 7. During in vitro propagation +7 cells seemed to be stable and overrode the diploid ones. After prolonged growth in culture, the cell population displayed the typical senescence signs. Our findings confirm the proneness to aneuploidy in cells from aged individuals and indicate that, while the presence of a trisomic 7 may confer a selective advantage to cells grown in vitro, it does not seem to prevent cellular senescence.

Radiosensitivity in Ataxia Telangiectasia Fibroblasts Is Not Associated with Deregulated Apoptosis

Ataxia telangiectasia (AT) is an autosomal recessive human disorder featuring diverse clinical abnormalities including proneness to cancer and extreme sensitivity to ionizing radiation. Although cells from AT patients exhibit faulty activation of the p53 signal transduction pathway at early times after radiation exposure, it has been proposed that high levels of DNA damage persisting in AT cells may up-regulate p53 through an ATM-independent mechanism at late times after irradiation, leading to cell death by apoptosis. In this study we demonstrate that diploid skin fibroblast strains homozygous for the AT mutation fail to up-regulate p53 protein at late times (< or = 48 h) after irradiation with 60Co gamma rays. Moreover, exposure of normal and AT fibroblasts to a dose of 8 Gy does not result in a significant increase in the fraction of apoptotic cells. Since this treatment reduces the clonogenic potential of human cells by at least two orders of magnitude, we conclude that apoptosis is not the primary mechanism of cell death induced by ionizing radiation in human normal and AT fibroblast cultures. Therefore, our results are not in accordance with the current hypothesis suggesting that increased radiosensitivity of AT cells is associated with deregulated apoptosis.

The time-pattern of rises and falls in proliferation fades with senescence of mortal lines and is perpetuated in immortal rat hepatoma Fao cell line.

Immortal cells perpetuate the rises and falls of proliferation that are progressively damped in mortal long-term cultured cells. For immortal rat hepatoma Fao cells, similar waves of proliferation occurred about every 3-4 wk. Under the same conditions, embryonic human fibroblasts and transformed but not immortalized embryonic fibroblasts display similarly recurring proliferation waves that progressively decrease in amplitude until senescence of the lines. In addition, strains of diploid normal human skin fibroblasts cultured under different culture conditions display a similar time-pattern of proliferation. Although the amplitude and baseline of these fluctuations are characteristic for each cell line, a common point was marked slow down in proliferation after every sequence of about 25 population doublings for all cells. Renewed proliferation waves of Fao cells allow about 22-23 additional population doublings each. Normal embryonic fibroblast culture and its transformed counterpart accumulate about 30 and 60 population doublings, respectively, before senescence. Normal fibroblast strains accumulate about 25 population doublings over their entire life spans. This halt in proliferation after every stretch of about 25 population doublings may correspond to a structural or functional stop following attrition of telomeric DNA. This putative stop may be bypassed once in transformed embryonic cells and repetitively in immortal cells. In support of this hypothesis, we observed rapid telomere shortening, in two steps, during divisions of mortal embryonic cells, and maintenance of long telomeres in immortal Fao cells, which may indicate episodic repair of telomeres. Alternatively, such maintenance of long telomeres may reflect survival and successive clonal growth of rare cells with long telomeres. We suggest that the balance between telomere attrition and repair processes regulates the waves of proliferation.

Novel Putative Fragile Sites Observed in Feline Fibroblasts Treated with Aphidicolin and Fluorodeoxyuridine.

Fragile sites are non-randomly distributed chromosomal breaks and gaps observed in the cells cultivated under certain conditions. Feline fragile sites were analyzed using skin fibroblast strains after the treatments with aphidicolin and fluorodeoxyuridine in combination with caffeine. Three aphidicolin-induced fragile sites (A1q21, C2q13 and E1p21) as well as a folate-sensitive site (B1q14) were observed in all the 3 fibroblast strains tested for each treatment group. The loci in A1q21 and B1q14 are very close to that reported previously for peripheral blood lymphocytes and lung cells. Two chromosomal break points in C2q13 and E1p21 seem to be new fragile sites. Fifteen candidates for feline fragile sites were also assigned their locations in feline chromosomes. Both the incidence and distribution of feline fragile sites in skin fibroblasts seem to be different at least in part from those in lymphocytes.

The mammalian metabolite, 2-methoxyestradiol, affects p53 levels and apoptosis induction in transformed cells but not in normal cells

The endogenous metabolite, 2-methoxyestradiol (2ME), is an inhibitor of tubulin polymerization and is therefore toxic to dividing fast-growing tumor cells. Transformed cells are not equally susceptible to the effects of 2ME. In this study the effects of 1–2 μM doses of 2ME on cell cycle progression, apoptosis induction and on p53 levels were evaluated using flow cytometry in cells with different p53 status. No effect of 2ME was seen in normal human skin fibroblast strain HSF43 with wild-type (wt) p53. However, in SV40 T antigen transformed HSF43 cells (line E8T4), 2ME caused a prominent G 2/M arrest, with subsequent micronuclei formation followed by apoptosis. Increased p53 levels were present in the G 2/M cells. Our results suggest that 2ME, being a microtubule poison, may release the bound p53 from T antigen, and that this p53 may enhance the apoptotic effects. Two lymphoblast cell lines derived from the same donor, TK6, expressing low levels of wt p53, and WTK1, expressing high levels of mutant p53, showed similar moderate responses to 2ME at 37°C. The effects included enhanced apoptosis and a modest G 2/M block. No increase in p53 levels was seen. However, at the permissive temperature of 30°C marked increases in apoptosis and a prominent G 2/M-phase block, similar to that seen in the E8T4 cells, were present in the WTK1 cells, indicating that the high levels of mutant p53 have now become functional, enhancing the apoptotic effects initiated by 2ME.

Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment

To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. The cell system of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10(5) fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumor fragments.

Models of initiation of replicative senescence by loss of telomeric DNA.

Situated at the ends of all eukaryotic chromosomes are telomeres, genetic elements that are essential for genomic stability. It has recently been established that telomere length shortens during replicative aging of normal human somatic cells. Although the cause of replicative senescence of somatic cells is still debated, we believe that telomere shortening plays a causal role in this process. In support of this hypothesis, mutant strains of yeast and ciliates that are incapable of maintaining telomere length during cell division eventually acquire a senescent-like phenotype wherein the cells become sickly, stop growing and die. Also, replicative capacity of cultured human skin fibroblast strains shows a strong positive correlation with telomere length. Several theories explaining how telomere shortening could lead to the induction of replicative senescence are now presented. We favor a model in which replicative senescence is caused by the shortening of telomeres below a length that is critical for the maintenance of proper telomere structure and function.

Radiation-induced differentiation of human skin fibroblasts: relationship with cell survival and collagen production.

The aim of this study was to determine whether significant inter-individual differences exist between skin fibroblast strains obtained from radiotherapy patients in both radiation-induced differentiation and collagen production in vitro, for use as potential parameters for a predictive assay for fibrosis following radiotherapy in patients. Morphological cell differentiation was determined 7 days after irradiation in seven early-passage primary human fibroblast cell strains and correlated with cell survival. Collagen production was measured in two cell strains by flow cytometry and incorporation of 3H-proline. There was a wide variation in the extent of radiation-induced differentiation for the seven cell strains, each showing a dose-related increase. The correlation between induced differentiation and cell survival was poor (r = 0.64) but statistically significant (p < 0.01). Collagen synthesis increased 7 days after irradiation for one cell strain (HF-48), as measured by incorporation of 3H-proline, but not in radiation sensitive AT-1 cells. The collagen I content of the two cell strains was assessed by flow cytometry but no significant differences were observed between the strains tested or with increasing dose. In conclusion, marked variations in radiation-induced fibroblast differentiation were observed between patients, this being an important criterion for a predictive assay.

Asynchronous DNA replication between 15q11.2q12 homologs: cytogenetic evidence for maternal imprinting and delayed replication.

DNA replication kinetics of Prader-Willi/Angelman syndrome region of 15q11.2q12 was studied without synchronization in five human amniotic cell and five skin fibroblast strains with a marker 15 chromosome, i.e., 15p+ or der(15), as cytological marker to distinguish between the two homologs. BrdU-33258 Hoechst-Giemsa techniques were used to analyze and compare the late replication patterns in the 15q11.2q12 region between the homologs. Asynchronous replication between the homologs was observed in both amniocytes and fibroblasts. From cells of a marker 15 of known parental origin, the paternal 15q11.2q12 replicated earlier than that of the maternal 15 in 92%-95% of asynchronous metaphases. The remaining 5%-8% of asynchronous metaphases displayed maternal early/paternal late replication. This mosaic pattern of replication in the 15q11.2q12 region may be due to methylation mosaicism of genomic imprinting or a relative lack of self-control of replication. These results provide cytogenetic evidence of maternal imprinting and delayed replication in the 15q11.2q12 region.