Cultures Of Human Diploid Fibroblasts Research Articles

The Impact of Three Genospecies of Borrelia on Expression of Genes Associated with Chemokines and Their Receptors in Normal Human Dermal Fibroblastsin Vitro

An important role in pathomechanism of Lyme disease is played by the ability of spirochetes to spread within tissues, and to adhere (to platelets, erythrocytes and vascular epithelium). The principal factors regulating that process are chemokines, cytokines and adhesion particles. The aim of this study was to select genes related to the chemokines and their receptors, differentiating the type of infection in the system model, i.e. a culture of normal human diploid fibroblasts infected with three different spirochete genospecies: B. afzelii, B. garinii and B. burgdorferii sensu stricto, by comparing the infected fibroblast culture with that of the control fibroblast. The differences in the expression of genes selected on the basis of a scientific database Affymetrix were analysed by comparing transcriptomes from the four cultures of fibroblasts, using the oligonucleotide microarrays HG_U133A. In the result of infection of fibroblast cultivation with a specific Borrelia genospecies, a variable expression of the chemokines and their receptors, specific for one genospecies was observed. The fibroblast infected with B. afzelii expressed CCL4, CCL1, CCL2 and CCR10; with B. garinii - CXCL12, IL6, CCR3 and CXCR5; and with B. burgorferii sensu stricto - CCL5, CCR1, CCL3, CCL16, CXCR6, IL8, CXCR7 and CXCR3.

Expression of senescence-associated microRNAs and target genes in cellular aging and modulation by tocotrienol-rich fraction.

Emerging evidences highlight the implication of microRNAs as a posttranscriptional regulator in aging. Several senescence-associated microRNAs (SA-miRNAs) are found to be differentially expressed during cellular senescence. However, the role of dietary compounds on SA-miRNAs remains elusive. This study aimed to elucidate the modulatory role of tocotrienol-rich fraction (TRF) on SA-miRNAs (miR-20a, miR-24, miR-34a, miR-106a, and miR-449a) and established target genes of miR-34a (CCND1, CDK4, and SIRT1) during replicative senescence of human diploid fibroblasts (HDFs). Primary cultures of HDFs at young and senescent were incubated with TRF at 0.5 mg/mL. Taqman microRNA assay showed significant upregulation of miR-24 and miR-34a and downregulation of miR-20a and miR-449a in senescent HDFs (P < 0.05). TRF reduced miR-34a expression in senescent HDFs and increased miR-20a expression in young HDFs and increased miR-449a expression in both young and senescent HDFs. Our results also demonstrated that ectopic expression of miR-34a reduced the expression of CDK4 significantly (P < 0.05). TRF inhibited miR-34a expression thus relieved its inhibition on CDK4 gene expression. No significant change was observed on the expression of CCND1, SIRT1, and miR-34a upstream transcriptional regulator, TP53. In conclusion tocotrienol-rich fraction prevented cellular senescence of human diploid fibroblasts via modulation of SA-miRNAs and target genes expression.

Expression of Genes Associated with H Factor in Fibroblasts Infected with Borrelia Spirochaetes

Infection with Borrelia spirochaetes leads to the launch of specific and non-specific immunological response in humans. Activation of the complement system is one of the first defence mechanisms against penetrating pathogens. The aim of this study was to select genes related to the alternative pathway of the complement system [including complement factor H (CFH)], differentiating the type of infection in the system model, that is, a culture of normal human diploid fibroblasts infected with three different spirochaete genospecies: Borrelia afzelii, Borrelia garinii and Borrelia burgdorferii sensu stricto, by comparing the infected fibroblast culture with the control fibroblast one. With the use of oligonucleotide microarrays HGU-133A, the differences in the expression of genes selected on the basis of a scientific database Affymetrix were studied by comparing transcriptomes from the four cultures of fibroblasts. In the result of infection of fibroblast cultivation with a specific Borrelia genospecies, a variable expression of certain CFH and complement system-associated genes, specific for one genospecies only, B. afzelii- C1QBP, CD59, C2, CD46 and FHL1; B. garinii - C1S and CLU; Borrelia burgeforii- CFB, A2M and VSIG4, was observed. CFH differentiates infections induced by B. afzelii and B. garinii from infections induced by B. burgdorferii sensu stricto.

Loss of p21 Sdi1 expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21 Sdi1 gene promoter

To answer what is a critical event for higher incidence of tumor development in old than young individuals, primary culture of human diploid fibroblasts were employed and DNA damage was induced by doxorubicin or X-ray irradiation. Response to the damage was different between young and old cells; loss of p21 sdi1 expression in spite of p53 S15 activation in old cells along with [ 3H]thymidine and BrdU incorporation, but not in young cells. The phenomenon was confirmed by other tissue fibroblasts obtained from different donor ages. Induction of miR-93 expression and reduced p53 binding to p21 gene promoter account for loss of p21 sdi1 expression in senescent cells after DNA damage, suggesting a mechanism of in vivo carcinogenesis in aged tissue without repair arrest.

Tocotrienol‐Rich Fraction Prevents Cell Cycle Arrest and Elongates Telomere Length in Senescent Human Diploid Fibroblasts

This study determined the molecular mechanisms of tocotrienol-rich fraction (TRF) in preventing cellular senescence of human diploid fibroblasts (HDFs). Primary culture of HDFs at various passages were incubated with 0.5 mg/mL TRF for 24 h. Telomere shortening with decreased telomerase activity was observed in senescent HDFs while the levels of damaged DNA and number of cells in G0/G1 phase were increased and S phase cells were decreased. Incubation with TRF reversed the morphology of senescent HDFs to resemble that of young cells with decreased activity of SA-β-gal, damaged DNA, and cells in G0/G1 phase while cells in the S phase were increased. Elongated telomere length and restoration of telomerase activity were observed in TRF-treated senescent HDFs. These findings confirmed the ability of tocotrienol-rich fraction in preventing HDFs cellular ageing by restoring telomere length and telomerase activity, reducing damaged DNA, and reversing cell cycle arrest associated with senescence.

Production of the human β-interferon recombinant protein in avian cell culture

An avian recombinant adenovirus serotype 1 (CELO) expressing human beta-interferon (IB) was obtained. An expression cassette containing the IB gene was placed at the right end of the CELO genome under the ontrol of hybrid promoter hEF-1/HTLV. The resulting recombinant adenovirus CELO-IB transduced the avian cell culture LMH. The level of production of recombinant IB was 0.15 μg/ml. The IB protein yield after affine chromatography purification using Ni-NTA agarose was 50%. The biological activity of the purified IB was fairly high (7.8 × 108 per μg of protein). The purified IB inhibited replication of murine encephalomyocarditis virus (VMEC) in cell culture of human diploid fibroblasts (HDF). Thus, the expression system based on the avian cell culture is an effective system for producing biologically active protein of human interferon beta.

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles : Part II: Human cell lines

Principal aims of this study were at first, to find a relevant human derived cell line to investigate the genotoxic potential of PAH-containing complex mixtures and second, to use this cell system for the analysis of DNA adduct forming activity of organic compounds bound onto PM10 particles. Particles were collected by high volume air samplers during summer and winter periods in three European cities (Prague, Kosice, and Sofia), representing different levels of air pollution. The genotoxic potential of extractable organic matter (EOM) was compared with the genotoxic potential of individual carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as well as their artificial mixtures. Metabolically competent human hepatoma HepG2 cells, confluent cultures of human diploid lung fibroblasts (HEL), and the human monocytic leukemia cell line THP-1 were used as models. DNA adducts were analyzed by 32P-postlabeling. The total DNA adduct levels induced in HepG2 cells after exposure to EOMs were higher than in HEL cells treated under the same conditions (15–190 versus 2–15 adducts/10 8 nucleotides, in HepG2 and HEL cells, respectively). THP-1 cells exhibited the lowest DNA adduct forming activity induced by EOMs (1.5–3.7 adducts/10 8 nucleotides). A direct correlation between total DNA adduct levels and c-PAH content in EOM was found for all EOMs in HepG2 cells incubated with 50 μg EOM/ml ( R = 0.88; p = 0.0192). This correlation was even slightly stronger when B[ a]P content in EOMs and B[ a]P-like adduct spots were analyzed ( R = 0.90; p = 0.016). As THP-1 cells possess a limited metabolic capacity for most c-PAHs to form DNA reactive intermediates and are also more susceptible to toxic effects of PAHs and various EOM components, this cell line seemed to be an inappropriate system for genotoxicity studies of PAH-containing complex mixtures. The seasonal variability of genotoxic potential of extracts was stronger than variability among the three localities studied. In HepG2 cells, the highest DNA adduct levels were induced by EOM collected in Prague in the winter period, followed by Sofia and Kosice. However, in the summer sampling period, the order was quite opposite: Kosice > Sofia > Prague. When the EOM content per m 3 of air was taken into consideration in order to compare real exposures of humans to genotoxic compounds in all three localities, extracts from respirable dust particles collected in Sofia exhibited the highest genotoxicity regardless of the sampling period. The results indicate that most of DNA adducts detected in cells incubated with EOMs have their origin in low concentrations of c-PAHs representing 0.03–0.17% of EOM total mass. Finally, our results suggest that HepG2 cells have a metabolic capacity for PAHs similar to human hepatocytes and represent therefore the best in vitro model for investigating the genotoxic potential of complex mixtures containing PAHs among the three cell lines tested in this study.

Evidence for the Direct Binding of Phosphorylated p53 to Sites of DNA Breaks In vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.

Antiviral Activity of Liposomal Preparations of Antibiotic Geliomycin

A liposomal preparation with maximally possible content of incorporated geliomycin was obtained. Its cytotoxicity was studied in a culture of human embryonal diploid fibroblasts. Antiviral activity was studied on a model of cytomegaloviral infection in vitro by the capacity to plaque formation. Liposomal geliomycin was 10-fold less toxic for human cells than the solution of the antibiotic in DMSO and exhibited antiviral activity towards cytomegaloviral infection at a concentration of 0.042 microg/ml.

Apoptosis-based drug screening and detection of selective toxicity to cancer cells.

The goal of our study was to determine whether an apoptosis assay used after short-term drug exposure could predict selective toxicity to cancer cells. To this end we compared the effect of eight anticancer drugs and 10 toxic compounds without known antitumor activity in cultures of human breast cancer cells and normal diploid fibroblasts by Apoptosis ELISA and growth inhibition assays. There was an overlap in concentration values of drugs and toxins inhibiting proliferation in cancer cells. In contrast, Apoptosis ELISA clearly distinguished between the two groups of compounds. Anticancer drugs induced apoptosis in cancer cells at 0.0015-0.5 microM, while toxins were effective at much higher concentrations of 8.0-50.0 microM. Moreover, six out of the 10 toxins did not induce apoptosis in cancer cells. The normal:cancer cell (N:C) ratio for growth inhibiting concentrations was in a similar range for anticancer drugs and toxins. The N:C ratio for apoptosis inducing concentrations was 33-200 for anticancer drugs and 1.3-3.0 for toxins. Our data indicate that apoptosis assays could be used to detect selective toxicity of anticancer drugs by determining apoptosis induction in cancer cells or through a comparison of apoptosis-inducing concentrations in normal and cancer cells.

Growth kinetics rather than stress accelerate telomere shortening in cultures of human diploid fibroblasts in oxidative stress-induced premature senescence

WI-38 human diploid fibroblasts underwent accelerated telomere shortening (490 bp/stress) and growth arrest after exposure to four subcytotoxic 100 μM tert-butylhydroperoxide (t-BHP) stresses, with a stress at every two population doublings (PD). After subcytotoxic 160 μM H 2O 2 stress or five repeated 30 μM t-BHP stresses along the same PD, respectively a 322±55 and 380±129 bp telomere shortening was observed only during the first PD after stress. The percentage of cells resuming proliferation after stress suggests this telomere shortening is due to the number of cell divisions accomplished to reach confluence during the first PD after stress.

Effect of cytomegalovirus on cell cycle progression and formation of pathological mitoses in cultured human diploid fibroblasts

The effect of cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G2, or M); at the same time, at least part of infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained 3H-thymidine label above chromosomes. This fact suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of cytomegalovirus.

Effect of the Cytomegalovirus on Cell Cycle Progression and Pathological Mitoses in Cultured Human Diploid Fibroblasts

The effect of the cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G 2, or M); at the same time, at least part of the infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained 3H-thymidine label above chromosomes. This suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of the cytomegalovirus.

Malignant transformation of human diploid fibroblasts and suppression of their anchorage independence by introduction of chromosome 13.

Isolation of cell lines that display various degrees of transformed phenotypes may be very useful to clarify multistep mechanisms of oncogenesis, but malignant transformation of human diploid fibroblasts in culture is a very rare event. We attempted to isolate variously transformed cell lines from human diploid fibroblasts (RB) of a patient with hereditary retinoblastoma. The RB cells exhibited normal karyotypes with the exception of one copy of chromosome 13, which contained a large deletion at the q14-22 region, where the RB1 gene is located. By transfection with SV40 early genes and repeated passage, we succeeded in obtaining SV40-transfected mortal, immortalized, anchorage-independent, and tumorigenic RB cell lines. DNA fingerprinting showed that these cell lines were not contaminants, but derivatives of the original RB cells. The remaining RB1 allele may be wild-type even in the malignant cell lines, because the expression and the LT-binding ability were normal. Furthermore, we did not find any homozygous loss in 16 polymorphic markers located in the 13q14-22 region in the transformed cell lines. However, introduction of a copy of a normal chromosome 13 into the anchorage-independent cell line suppressed its anchorage-independent growth ability. All these data, together with the fact that the RB cells containing the deletion progressed to a tumorigenic state spontaneously, but normal fibroblasts did not, raise the possibility that a new tumor suppressor gene, located at 13q14-22, may play a critical role in neoplastic transformation. We conclude that these RB cell lines provide an excellent system for identification of genes involved in malignant transformation of human cells. Genes Chromosomes Cancer 26:47-53, 1999.

Grafting of genetically modified human fetal fibroblasts to produce human butyrylcholinesterase in mice

Human diploid fibroblast cultures were established from fetal skin tissue. Enzymic dissociation yielded cultures of higher growth capacity of fibroblasts than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. Transfer of recombinant human butyrylcholinesterase (BChE, EC 3.1.1.8) gene into primary human fibroblasts was achieved successfully using lipofection and retrovirus-mediated transfection. The analysis of drug-resistant colonies suggested the presence of the transcripted BChE mRNA in the cytoplasm of transfected cells. The secreted BChE protein in culture medium was assayed for enzyme activity using butyrylthiocholine as substrate. The genetically modified fibroblasts were mixed with rat tail collagen and transplanted subcutaneously and intraperitoneally to mice. Immunoreactive human BChE appeared in the plasma from the transplanted mice, reaching the top level at day 13. It was not present any longer in most of the mice 20 days later.

Attenuated heat shock transcriptional response in aging: molecular mechanism and implication in the biology of aging.

A characteristic feature of aging is a progressive impairment in the ability to adapt to environmental challenges. The purpose of this review is to present the experimental evidence of an attenuated heat shock transcriptional response to heat and physiological stresses in a number of aging mammalian model systems. These include the human diploid fibroblasts in culture, whole animals and animal derived cells and cell cultures, as well as peripheral blood mononuclear cells obtained from human donors. The possibility that age-dependent changes in cellular redox status, as exemplified by the increased production of reactive oxygen inter-mediates and accumulation of oxidatively-modified proteins, affects the regulation and function of the heat shock factor 1 (HSF1) and contributes to the attenuated heat shock transcriptional response in aging cells and organisms is discussed. Given the fundamentally important role of HSPs in many aspects of protein homeostasis and signal transduction, it seems likely that the inability, or compromised ability, of aging cells and organisms to produce HSPs in response to stress would contribute to the well known increase in morbidity and mortality of the aged when challenged.

Heat shock response, heat shock transcription factor and cell aging.

A characteristic feature of aging is a progressive impairment in the ability to adapt to environmental challenges. The purpose of this article is to review the evidence of an attenuated response to heat and physiological stresses in a number of mammalian aging model systems, including the human diploid fibroblasts in culture, whole animals and animal-derived cells and cell cultures, as well as peripheral blood mononuclear cells obtained from human donors. Analyses of the regulation and function of heat shock factor 1 (HSF1), a transcription factor that mediates the response to heat shock, showed that while the relative abundance of both the hsf1 transcript and the HSF1 protein did not change as a function of age, the responsiveness of HSF1 to heat-induced activation, as measured by its trimerization and ability to bind to the heat shock element consensus sequence, was inversely related to the age of the cells used. Given the fundamentally important role of heat shock proteins (HSPs) in many aspects of protein homeostasis and signal transduction it seems likely that the inability, or compromised ability, of aging cells and organisms to activate HSF1 and produce HSPs in response to stress would contribute to the well-known increase in morbidity and mortality of the aged when challenged.

Heterogeneity of bromodeoxyuridine sensitivity of cultured cells from melanoma metastases.

Continuously growing cell cultures, testing positive for tyrosine activity, were derived from two brain and three lymph-node metastases of five patients with malignant melanoma. These cell cultures were analyzed regarding their proliferation rate with continuous bromodeoxyuridine (BrdUrd) labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry. Melanoma cell cultures are more sensitive toward BrdUrd in comparison to human diploid fibroblast cultures: 50% growth inhibition at 360 +/- 130 microM BrdUrd (range: 130-520; n = 11) vs. 650 +/- 50 microM BrdUrd (n = 3) for fibroblasts. Moreover, BrdUrd sensitivity in melanoma cells is oxygen dependent: 50% growth inhibition at 200 +/- 55 microM (range: 65-400 microM) for 20% oxygen vs. 360 +/- 130 microM BrdUrd for 5% oxygen. The cell cycle kinetic mechanisms of BrdUrd-induced growth inhibition is accumulation of cells in the G2 phase. Cultures from a single metastasis showed up to a 3-fold variation in BrdUrd sensitivity. In one of the brain metastases two populations of different ploidy level (pseudotriploid vs. pseudotetraploid) and BrdUrd sensitivity could be resolved. Thus, continuous BrdUrd labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry is a powerful tool to detect heterogeneity in proliferative capacity and drug sensitivity of cell populations within one tumor biopsy.

7α-Hydroxylation of 25-hydroxycholesterol and 27-hydroxycholesterol in human fibroblasts

The metabolism of 27-hydroxycholesterol and 25-hydroxycholesterol was studied in cultures of human diploid fibroblasts. Both steroids underwent 7α-hydroxylation with subsequent oxidation to 7α-hydroxy-3-oxo-Δ 4 steroids. A minor fraction of the 27-hydroxysteroids was oxidized to acids. Competition experiments indicated that both hydroxycholesterols were hydroxylated by the same enzyme, different from cholesterol 7α-hydroxylase. 7x,25-Dihydroxycholesterol suppressed the activity of HMG-CoA reductase at least as effectively as 25-hydroxycholesterol whereas 7α,25-dihydroxy-4-cholesten-3-one was a less effective suppressor. The results suggest that cholesterol might be converted to 7α-hydroxylated bile acid precursors in extrahepatic tissues in vivo and that the regulation of the activity of HMG-CoA reductase by oxysterols might be modulated by 7α-hydroxylation and subsequent oxidation by 3 β-hydroxy- Δ 55-C 27-steroid dehydrogenase/isomerase.

Diminished Responsiveness of Senescent Normal Human Fibroblasts to TNF-Dependent Proliferation and Interleukin Production is Not Due to Its Effect on the Receptors or on the Activation of a Nuclear Factor NF- KB

The limited life span in culture of normal human diploid fibroblasts (HDF) has provided a model of cellular senescence. The short-term growth of these cells in culture is regulated by a number of different cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and fibroblast growth factor (FGF). However, the effect of senescence on the responsiveness of HDF to these cytokines is not known. In the present report, we examined the effects of TNF on foreskin-derived HDF at different passage levels. We compared the response of HDF cells at population doubling (PD) 23 (young) with that of cells at PD 70 (senescent). Young cells proliferated in response to TNF in a dose-dependent manner. Under these conditions TNF had no effect on senescent HDF. The decrease in TNF responsiveness was found to be dependent on PD. The lack of response of senescent HDF was not unique to TNF, since FGF and IL-1 were also ineffective. In contrast to senescent HDF, TNF-dependent proliferation of young HDF could be further potentiated by IL-1 and FGF, suggesting an independent signaling mechanism. On exposure to TNF, senescent HDF produced IL-6 and IL-8, but to a much lower degree than that produced by young HDF. The diminished responsiveness of senescent HDF to TNF does not appear to be due to the difference in either receptor number or affinity, since senescent cells had two-to threefold higher number of TNF receptors than young HDF but the same affinity. TNF induced the activation of a nuclear transcriptional factor, NF- k B, equally in both young and senescent cells, which indicates the lack of a defect in the early events of TNF signal transduction in senescent fibroblasts. Overall, our results indicate that there is an age-dependent decline in TNF-induced proliferation and in the production of interleukins by fibroblasts; this unresponsiveness appears not to be due to TNF receptors or NF- K B activation. These results may have importance in understanding the diminished immune response, inflammation, and wound healing associated with aging.