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Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene.

Kapanadze B, Morris E, Smith E, Trojanowska M - J. Cell. Mol. Med. (2009)

Bottom Line: A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed.This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture.The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Medical University of South Carolina, Charleston, SC 29425, USA.

ABSTRACT
Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-betaRI indicative of altered TGF-beta signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.

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hTERT clones maintain telomerase activity after repeated passaging. hTERT activity was assayed in each clone using the TRAPeze?Telomerase Detection Kit (Chemicon) by PCR amplification of the telomerase added repeats with primers specific for these telomeres. The resulting PCR product was resolved in an acrylamide gel. The presence of 6bp DNA steps demonstrates hTERT activity. Several NS/SSc clones show persistent hTERT activity after repeated passaging in culture. Representative hTERT clones NS342–3, SSc345–1 and SSc 345–3 are shown in lanes 1, 2 and 3. Lane 4 is a non-infected normal dermal fibroblast for a negative control.
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fig01: hTERT clones maintain telomerase activity after repeated passaging. hTERT activity was assayed in each clone using the TRAPeze?Telomerase Detection Kit (Chemicon) by PCR amplification of the telomerase added repeats with primers specific for these telomeres. The resulting PCR product was resolved in an acrylamide gel. The presence of 6bp DNA steps demonstrates hTERT activity. Several NS/SSc clones show persistent hTERT activity after repeated passaging in culture. Representative hTERT clones NS342–3, SSc345–1 and SSc 345–3 are shown in lanes 1, 2 and 3. Lane 4 is a non-infected normal dermal fibroblast for a negative control.

Mentions: Fibroblasts were isolated and cultured from SSc and NS biopsies and transduced with hTERT DNA in passage zero. The hTERT gene was introduced into each culture using a lentivirus construct containing zeocin resistance. Several zeocin-resistant colonies were selected from each cell population for further characterization. We established 35 fibroblast clones that showed recombinant hTERT expression, 19 normal and 16 SSc, which may suggest that normal fibroblasts are easier to immortalize by this method. To confirm that hTERT overexpression was stably incorporated into the cellular DNA, hTERT activity was measured in several clones using the TRAP. This assay is not sensitive enough to detect levels of endogenous hTERT. The presence of six base pair DNA repeats indicates that hTERT is overexpressed in the clonal cell line. The PCR product of a representative clone is shown in Figure 1.


Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene.

Kapanadze B, Morris E, Smith E, Trojanowska M - J. Cell. Mol. Med. (2009)

hTERT clones maintain telomerase activity after repeated passaging. hTERT activity was assayed in each clone using the TRAPeze?Telomerase Detection Kit (Chemicon) by PCR amplification of the telomerase added repeats with primers specific for these telomeres. The resulting PCR product was resolved in an acrylamide gel. The presence of 6bp DNA steps demonstrates hTERT activity. Several NS/SSc clones show persistent hTERT activity after repeated passaging in culture. Representative hTERT clones NS342–3, SSc345–1 and SSc 345–3 are shown in lanes 1, 2 and 3. Lane 4 is a non-infected normal dermal fibroblast for a negative control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822752&req=5

fig01: hTERT clones maintain telomerase activity after repeated passaging. hTERT activity was assayed in each clone using the TRAPeze?Telomerase Detection Kit (Chemicon) by PCR amplification of the telomerase added repeats with primers specific for these telomeres. The resulting PCR product was resolved in an acrylamide gel. The presence of 6bp DNA steps demonstrates hTERT activity. Several NS/SSc clones show persistent hTERT activity after repeated passaging in culture. Representative hTERT clones NS342–3, SSc345–1 and SSc 345–3 are shown in lanes 1, 2 and 3. Lane 4 is a non-infected normal dermal fibroblast for a negative control.
Mentions: Fibroblasts were isolated and cultured from SSc and NS biopsies and transduced with hTERT DNA in passage zero. The hTERT gene was introduced into each culture using a lentivirus construct containing zeocin resistance. Several zeocin-resistant colonies were selected from each cell population for further characterization. We established 35 fibroblast clones that showed recombinant hTERT expression, 19 normal and 16 SSc, which may suggest that normal fibroblasts are easier to immortalize by this method. To confirm that hTERT overexpression was stably incorporated into the cellular DNA, hTERT activity was measured in several clones using the TRAP. This assay is not sensitive enough to detect levels of endogenous hTERT. The presence of six base pair DNA repeats indicates that hTERT is overexpressed in the clonal cell line. The PCR product of a representative clone is shown in Figure 1.

Bottom Line: A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed.This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture.The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Medical University of South Carolina, Charleston, SC 29425, USA.

ABSTRACT
Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-betaRI indicative of altered TGF-beta signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.

Show MeSH
Related in: MedlinePlus