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PubMed2002

Efficient and tightly regulated expression systems for the human cytomegalovirus major transactivator protein IE2p86 in permissive cells.

The 86-kDa IE2 protein (IE2p86) of human cytomegalovirus is a pleiotropic regulatory polypeptide that is essential for activation of viral early promoters and thus, for the entire viral replication cycle. Moreover, this protein modulates cellular gene expression and contributes to the pathogenic features of HCMV. The full spectrum of IE2p86 mediated effects on cellular gene expression has not been defined yet, since efficient expression systems for this protein in HCMV permissive cells are, so far, limited. Here, we report the establishment of two efficient model systems that allow a tightly regulated expression of IE2p86 in various permissive cell types including primary human fibroblasts, primary endothelial cells and U373MG cells. Firstly, we generated a tetracycline-regulated U373MG cell line, which expresses high levels of IE2p86 upon tetracycline removal from the culture medium. Secondly, a recombinant baculovirus was constructed, which expresses IE2p86 under the control of the HCMV major immediate early enhancer/promoter upon transduction of various cell types. Importantly, IE2p86 was functional in both systems, since strong transactivation of luciferase promoter constructs could be measured. Furthermore, a cell cycle arrest was detectable after infection of primary human fibroblasts with IE2p86-expressing baculoviruses. Both expression systems represent useful tools to fully define the effects of this pleiotropic transactivator on cellular gene expression and to establish screening systems for novel antiviral drugs targeting this critical immediate early protein of HCMV.

Animals↗

PubMed2004

Nuclear localization signal and cell synchrony enhance gene targeting efficiency in primary fetal fibroblasts.

The use of primary somatic cells in nuclear transfer procedure has opened a new opportunity to manipulate domestic animal genomes via homologous recombination. To date, while a few loci have been targeted in somatic cells using similar enrichment strategies as those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells within a colony and difficulties in cloning the cell after targeting. Utilizing the hypoxanthine guanine phosphoribosyl transferase (HPRT) as a test locus, it was determined that while no targeted colonies were identified using a conventional targeting construct, an average of 1 per million targeted cells were identified when a nuclear localization signal (nls) was added to the construct. When the nls was combined with cell synchronization using a thymidine block, targeting efficiency increased 7-fold. Moreover, the number of random integrants decreased by over 54-fold resulting in a 1:3 targeted to random integration ratio. This method should facilitate the application of homologous recombination to primary somatic cells.

Animals↗

PubMed1991

Effect of 9,10-dimethyl-1,2-benzanthracene on the growth of hamster dermal fibroblasts in primary culture.

Newborn Syrian hamster dermal fibroblasts in primary culture have high plating efficiency, manifest as homogeneous contact-sensitive monolayer in mass cultures and above all possesses a short life-span of 15-20 days in culture. These cells are quite sensitive to mutagenic carcinogens. The DMBA treatment induced multi-layer and foci formation and growth in soft-agar suggesting that immortal variants can be obtained by treatment with carcinogens.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2006

Microarray analysis of radiation response genes in primary human fibroblasts.

PURPOSE: To identify radiation-induced early transcriptional responses in primary human fibroblasts and understand cellular pathways leading to damage correction. METHODS AND MATERIALS: Primary human fibroblast cell lines were irradiated with 2 Gy gamma-radiation and RNA isolated 2 h later. Radiation-induced transcriptional alterations were investigated with microarrays covering the entire human genome. Time- and dose dependent radiation responses were studied by quantitative real-time polymerase chain reaction (RT-PCR). RESULTS: About 200 genes responded to ionizing radiation on the transcriptional level in primary human fibroblasts. The expression profile depended on individual genetic backgrounds. Thirty genes (28 up- and 2 down-regulated) responded to radiation in identical manner in all investigated cells. Twenty of these consensus radiation response genes were functionally categorized: most of them belong to the DNA damage response (GADD45A, BTG2, PCNA, IER5), regulation of cell cycle and cell proliferation (CDKN1A, PPM1D, SERTAD1, PLK2, PLK3, CYR61), programmed cell death (BBC3, TP53INP1) and signaling (SH2D2A, SLIC1, GDF15, THSD1) pathways. Four genes (SEL10, FDXR, CYP26B1, OR11A1) were annotated to other functional groups. Many of the consensus radiation response genes are regulated by, or regulate p53. Time- and dose-dependent expression profiles of selected consensus genes (CDKN1A, GADD45A, IER5, PLK3, CYR61) were investigated by quantitative RT-PCR. Transcriptional alterations depended on the applied dose, and on the time after irradiation. CONCLUSIONS: The data presented here could help in the better understanding of early radiation responses and the development of biomarkers to identify radiation susceptible individuals.

Aged↗

PubMed2000

Role of Ras isoforms in the stimulated proliferation of human renal fibroblasts in primary culture.

The proliferation of renal fibroblasts is implicated in the pathophysiologic processes of renal fibrosis. Many of the growth factors involved in proliferation are known to activate intracellular signaling pathways that converge on Ras monomeric GTPases. Although three ras family genes exist, their functional specificity is not yet known. Using antisense oligonucleotides, a role for Kirsten (Ki)-Ras in the stimulated proliferation of a primate renal fibroblast cell line was previously demonstrated. This study examines Ras in primary cultures of adult human renal fibroblasts. Using reverse transcription-PCR, mRNA for Harvey (Ha)-ras, Ki(4B)-ras, and neural (N)-ras, but not Ki(4A)-ras, were detected. Antisense oligonucleotides targeting Ha-, Ki-, and N-ras mRNA, which were used for liposomal transfection at 100 to 200 nM, were demonstrated to be active and isoform-specific in quantitative reverse transcription-PCR assays. Cellular Ras protein levels, as estimated using isoform-specific monoclonal antibodies, indicated that Ki-Ras was the predominantly expressed isoform (>95% of total Ras protein) under both serum-containing and serum-free conditions, with N- and Ha-Ras being detected in small amounts. Consistent with this finding, the antisense oligonucleotide directed against Ki-Ras reduced total cellular Ras levels by >70%, whereas Ha-Ras, N-Ras, and control oligonucleotides had no significant effect. Proliferation of oligonucleotide-transfected cells was measured using epidermal growth factor (EGF) and serum stimulation. The Ki-Ras oligonucleotide at 100 nM reduced serum-stimulated proliferation by >50% and EGF-stimulated proliferation by 25%, compared with data obtained with the control oligonucleotide (P: < 0. 01). The N-Ras oligonucleotide was not active, compared with the control oligonucleotide. The Ha-Ras oligonucleotide was not significantly active at 100 nM but reduced serum-stimulated proliferation by 13% and EGF-stimulated growth by 40% at 200 nM (P: < 0.01). These results demonstrate that Ki-Ras(4B) is the predominantly expressed Ras isoform in human renal fibroblasts in primary culture and is important for both serum- and EGF-stimulated proliferation. Ha-Ras appears to be expressed at low levels but may also play a distinct role in stimulated proliferation.

Antibodies, Monoclonal↗

PubMed2000

Effect of spermine synthase deficiency on polyamine biosynthesis and content in mice and embryonic fibroblasts, and the sensitivity of fibroblasts to 1,3-bis-(2-chloroethyl)-N-nitrosourea.

Mutant Gy male mice, which have previously been described as having disruption of the phosphate-regulating Phex gene and a spermine synthase gene [Meyer, Henley, Meyer, Morgan, McDonald, Mills and Price (1998) Genomics, 48, 289-295; Lorenz, Francis, Gempel, Böddrich, Josten, Schmahl and Schmidt (1998) Hum. Mol. Genet. 7, 541-547], as well as mutant Hyp male mice, which have disruption of the Phex gene only, were examined along with their respective normal male littermates. Biochemical analyses of extracts of brains, hearts and livers of 5-week-old mice showed that Gy males lacked any significant spermine synthase activity as well as spermine content. Organs of Gy males had a higher spermidine content. This was caused not only by the lack of conversion of spermidine into spermine, but also because of compensatory increases in the activities of other polyamine biosynthetic enzymes. Gy males were half the body weight of their normal male littermates at weaning age. Hyp males, however, were no different in size when compared with their controls. High mortality of Gy males occurs by weaning age and this mortality was shown to be largely post-natal. Embryonic fibroblasts were isolated from Gy males and their normal male littermates and were similarly shown to lack any significant spermine synthase activity as well as spermine content. The lack of spermine, however, had no significant effect on the growth of immortalized fibroblasts or of primary fibroblast cultures. Similarly, there was no difference in the time of senescence of primary fibroblast cultures from Gy males compared with cultures derived from normal male littermates. However, the lack of spermine did increase the sensitivity of immortalized fibroblasts to killing by the chloroethylating agent 1, 3-bis(2-chloroethyl)-N-nitrosourea. Therefore both the Gy male mice and derived embryonic fibroblasts provide valuable models to study the importance of spermine and spermine synthase, without the use of inhibitors which may have additional side effects.

Animals↗

PubMed1993

Subcellular localization of protein kinase CK-2 alpha- and beta-subunits in synchronized cells from primary human fibroblasts and established cell lines.

Casein kinase 2 (CK-2) subunits were sublocalized by indirect immunofluorescence in synchronized primary human fibroblasts, human HT1080, and mouse 3T3 cells. The antibodies used were directed against the CK-2 alpha- and beta-subunits. In human primary fibroblasts and the fibrosarcoma (HT1080) both the alpha and beta-subunits were predominantly localized in the nucleus, irrespective of the stage of the cell cycle. By contrast, in murine 3T3 cells, CK-2 alpha-subunit was found in the cytoplasm whereas the beta-subunit was mostly localized in the nucleus. In parallel, CK-2 activity was measured throughout the cell cycle. Apart from the well-known differences between cell lines with their high proliferation rate and higher CK-2 activity, and the slower growing primary cell cultures with lower CK-2 activity, no significant activity changes were measured. Densitometric analysis of CK-2 alpha- vs. beta-subunits after immunoblot detection revealed a 10:1 ratio for primary human skin fibroblasts at quiescent state which is significantly more than at later stages. Such an asymmetrical subunit distribution at G0 was not observed with the established cell line 3T3 where the alpha/beta ratio was 0.7-1 throughout the cell cycle.

3T3 Cells↗

PubMed2001

Chromosome nondisjunction and loss induced by protons and X rays in primary human fibroblasts: role of centromeres in aneuploidy.

To study the origin of micronuclei induced in human primary fibroblasts by low-energy protons (7.7 and 28.5 keV/microm) and X rays, we have developed a combined antikinetochore-antibody (CREST) and FISH staining with pancentromeric probes. This technique allowed us to analyze the integrity of the kinetochore and centromeric DNA structures and to assess their role in induced aneuploidy. The effect of LET on radiation-induced chromosome nondisjunction was studied in binucleated cells with centromeric-specific DNA probes for chromosomes 7 and 11. Our results indicate that, though more than 90% of radiation-induced micronuclei were CREST(-)/FISH(-), 28.5 keV/microm protons and X rays were also able to induce statistically significant increases in the number of micronuclei that were CREST(-)/FISH(+) and CREST(+)/FISH(+), respectively. One interpretation of these results could be that the protons induced chromosome loss by kinetochore detachment or by breakage in the centromeric DNA region, whereas X rays induced aneuploidy through a non-DNA damage mechanism. Nondisjunction appears to be a far more important mechanism leading to radiation-induced aneuploidy. Irrespective of the higher frequency of micronuclei induced by 28.5 keV/microm protons, the frequency of chromosome loss was markedly higher for X rays than for 28.5 keV/microm protons, strengthening the hypothesis that non-DNA targets, such as components of the mitotic spindle apparatus, may be involved in aberrations in chromosome segregation after X irradiation.

Aneuploidy↗

PubMed2002

Primary skin fibroblasts as human model system for proteome analysis.

Elucidation of cellular processes and their changes at the level of protein expression and post-translational modification patterns may allow identification of novel proteins and thereby mechanisms involved in the pathogenesis of multigenic diseases. The aim of this study was to test cultured, nontransformed primary fibroblasts derived from human skin biopsies as a suitable model system for proteome analysis. Therefore soluble protein fractions were separated on several overlapping ultrazoom gels covering the pH range from 3.5-9. Correlation analysis of gel-pairs revealed a highly reproducible protein expression pattern within (intra-assay) and between (inter-assay) independent experiments of a single fibroblast cell line (intra-cell line comparison). Spot intensity variations were less than a factor of two for more than 80% of identical spots. In addition, inter-cell line comparison exhibits no significant variations in spot intensities. To achieve further improvements in reproducibility we generated master gels for each pH range by combining averaged spot information derived from two different cell lines each analysed by two independent experiments using the raw master gel algorithm of the Z3 image analysis software. The resulting reference images of primary human fibroblasts provided a basis for investigating regulation by extracellular stimuli and drugs as well as their alterations in patients with different diseases.

Cells, Cultured↗

PubMed1981

Variability in collagen and fibronectin synthesis by scleroderma fibroblasts in primary culture.

Primary cultures of fibroblasts obtained from the papillary, reticular and subcutaneous layer of scleroderma skin were analyzed for protein synthesis by metabolic labeling and radioimmunoassays. Several of these cultures showed a 10- to 20-fold increase in the production of total protein and collagen as well as of fibronectin and type III procollagen as compared to cells from unaffected individuals. Most of the increases were noted in the reticular and subcutaneous layers. With cells from other patients increased synthesis was found in some of the explants or for only some of the products. The heterogeneity observed here could represent heterogeneity in the disease, in the cells studied or in the state of the disease at the time the cells were obtained.

Adult↗

PubMed1984

Chemical transformation of Swiss mouse embryo fibroblasts in primary culture.

Trypsinized secondary cultures of various cell systems have been frequently used by different investigators as models to study, in vitro chemical transformation. In the present study, non-trypsinized primary cultures of fibroblasts derived from fetuses of 14 days gestational age have been used to find out the timings of in vitro chemical transformation by 20-methylcholanthrene and has been designated as PMM-14 cells. These PMM-14 cells have been compared with trypsinized cultures of the same cell systems. The criteria for neoplastic transformation considered in the study involved the appearance of morphological changes, indefinite growth in tissue culture, acquisition of tumorigenic potential in vitro as evidenced by in vivo tests and in vivo latency period for palpable tumor formation. The time span of neoplastic transformation of non-trypsinized embryo fibroblasts in culture was remarkably reduced in comparison with trypsinized cultures of same cell systems. Such discrepancy seems to be due to repeated use of trypsin which may replace many of vital cell surface molecules causing a delay in the expression of carcinogen-induced malignancy. Similarly, repeated subculture before transformation may also have a role in the delayed expression of malignancy.

Animals↗

PubMed1988

Tumour-associated antigens in primary mouse fibroblasts induced by transformation with bovine papillomavirus type 1.

Bovine papillomavirus type 1 (BPV 1) DNA was used to transform primary fibroblasts of C57BL/6J mice. Transformation frequency in these cells was much lower than in C127 cells and not associated with the appearance of morphologically distinct foci. However, continuous lines of transformed C57BL/6J cells were developed by serial subculturing of transfected cells. These transformed cell lines showed phenotypic properties associated with transformation including abnormal karyotypes. They contained variable amounts of viral DNA, but the copy number was in the same range as in six C127 transformants tested for comparison. In two cell lines monomeric viral DNA in an episomal form was detected. Slowly migrating viral sequences in these and in the third line were probably episomal concatamers, but the possibility of integration could not be excluded. There was some variation in immunogenicity, but all cell lines induced a cell-mediated immune response in syngeneic mice detected by the chromium release assay. In addition to BPV 1-transformed cell lines, the effector cells also reacted against an unidentified antigen shared by 2 cell lines transformed by SV40 and UV irradiation, respectively.

Animals↗

PubMed1988

Tumorigenicity of primary mouse fibroblasts transformed by bovine papillomavirus type 1.

Cell lines transformed by bovine papillomavirus type 1 DNA were established from transfected primary fibroblast cultures of C57BL/6 mice. Southern blot analysis revealed that most of the viral DNA in the cells was in episomal oligomeric form. These cell lines were tested for tumorigenicity in nude mice in comparison to properties associated with malignant transformation including growth in low-serum medium, focus formation in agar, and resistance to natural killer cells. Two of the 5 cell lines were highly tumorigenic, producing spindle cell sarcomas in all animals. Metastatic spreading to local lymph nodes was also observed. The same cell lines also showed anchorage-independent growth in agar, but no correlation was observed between natural killer cell resistance and tumorigenicity. Viral transcripts were detected in two nontumorigenic and in two tumorigenic cell lines.

Animals↗

PubMed1980

Isolation and characterization of hyaluronidase from cultures of chick embryo skin- and muscle-derived fibroblasts.

Primary and passaged cultures of chick embryo fibroblasts, prepared from 10-day embryonic skin and 12-day embryonic skeletal muscle, synthesize and secrete hyaluronidase at level comparable to the tissues of origin. The level of cell-associated hyaluronidase remains approximately constant during culture while the enzyme accumulates extracellularly such that within a 48-h incubation period, levels of hyaluronidase in the medium are 2 to 4 times higher than that found associated with the cell layer. The cultures continue to produce hyaluronidase at approximately the same rate and distribution through as many as nine passages. In addition to the chick fibroblasts, a variety of mammalian cells also produce hyaluronidase in culture. Both the cell- and medium-associated forms of the chick fibroblast hyaluronidase are endoglycosidases of estimated molecular weight of 62,000, which preferentially cleave hyaluronate substrate at an acidic pH optimum. Neither the cell-associated nor secreted hyaluronidase are enzymatically active at neutral pH.

Animals↗

PubMed1996

Primary human fibroblasts induce diverse tumor invasiveness: involvement of HGF as an important paracrine factor.

Fibroblasts have been considered to play an important role in tumor progression. In order to evaluate the contribution of fibroblasts to tumor invasion, TE2-NS, an esophageal cancer cell line, was cultured on collagen gel containing primary fibroblasts derived from normal esophageal submucosa or cancerous tissues of seven esophageal cancer patients. TE2-NS showed diverse invasiveness into the underlying gel containing fibroblasts, but did not invade the gel not containing fibroblasts. The invasiveness of TE2-NS, which expressed hepatocyte growth factor (HGF) receptor, was well-correlated with the concentration of HGF in conditioned medium. Administration of neutralizing antibody against HGF effectively suppressed the invasion, but application of recombinant HGF without fibroblasts induced little invasion into the gel. Fibroblasts from non-cancerous tissue generally secreted a larger amount of HGF and induced tumor invasion to a greater extent than those from cancer tissue, with large diversity. Interestingly, HGF secretion of fibroblasts from non-cancerous tissue was stimulated by co-culture with TE2-NS in two lines, but not in the other four. These results indicate that HGF is an important paracrine factor which induces tumor cell invasion, and the diversity of HGF production by fibroblasts might suggest different potentiality to induce tumor invasion among patients.

Cadherins↗

PubMed2005

Specific radial positions of centromeres of human chromosomes X, 1, and 19 remain unchanged in chromatin-depleted nuclei of primary human fibroblasts: evidence for the organizing role of the nuclear matrix.

Radial positions of centromeres of human chromosomes X, 1, and 19 were determined in the nuclei of primary fibroblasts before and after removal of 60%-80% of chromatin. It has been demonstrated that the specific radial positions of these centromeres (more central for the chromosome 19 centromere and more peripheral for the centromeres of chromosomes 1 and X) remain unchanged in chromatin-depleted nuclei. Additional digestion of nuclear RNA did not influence this specific distribution. These results strongly suggest that the characteristic organization of interphase chromosomes is supported by the proteinous nuclear matrix and is not maintained by simple repulsing of negatively charged chromosomes.

Cell Nucleus↗

PubMed1997

In vitro radiosensitivity of primary human fibroblasts. Lack of correlation with acute radiation toxicity in patients with head and neck cancer.

BACKGROUND AND PURPOSE: There is a considerable hope among clinicians and radiobiologists to detect genetically radiosensitive patients prior to radiotherapy. A predictive assay would enable adjustment of the total irradiation dose to the individual at a constant risk of normal tissue complications. In this prospective study, the clonogenic survival assay for primary human fibroblasts to determine radiosensitivity in vitro was evaluated and then correlated with clinically observed acute radiation reactions. MATERIALS AND METHODS: One hundred twenty-five independent survival experiments with primary fibroblasts derived from 63 biopsies from 55 cancer and non-cancer patients were performed. RESULTS: A wide variation of cell survival between biopsies was detected. Statistical analysis revealed a highly significantly larger interindividual than intraindividual variation of SF2 values. However, a considerable scatter of SF2 values in repeated experiments was observed in individual cases. Age, gender, disease status (cancer patient, non-cancer patient) and origin of fibroblasts (skin, periodontal tissue) were demonstrated not to be statistically significant confounding factors on the intrinsic radiosensitivity in vitro. In a prospective study, no correlation of the SF2 and acute reactions in 25 patients with head and neck cancer treated with a primary accelerated radiochemotherapy was detected. CONCLUSION: Our data show that the clonogenic assay is able to distinguish between intrinsic radiosensitivities of primary human fibroblasts if a statistical approach is used but does not predict acute radiation toxicity.

Acute Disease↗

PubMed1994

Cellular radiosensitivity and DNA damage in primary human fibroblasts.

PURPOSE: To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are the more predictive of normal tissue cellular radiosensitivity. METHODS AND MATERIALS: Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. RESULTS: Following HDR irradiation, cell survival varied between SF2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). CONCLUSION: The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival.

Cell Survival↗