Telomerase Alone Extends the Replicative Life Span of Human Skeletal Muscle Cells Without Compromising Genomic Stability

Wootton, Martha; Steeghs, Karen; Watt, Diana J.; Munro, June; Gordon, Katrina E.; Ireland, H. Elyse; Morrison, Vivienne; Behan, W. M. H.; Parkinson, Eric Kenneth

doi:10.1089/104303403769211682

Cited by 27 publications

(23 citation statements)

References 49 publications

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“…Some other cell types including retinal pigment epithelial cells (Bodnar et al, 1998), oesophageal keratinocytes (Harada et al, 2003), skeletal muscle cells (Wootton et al, 2003), mesothelial cells (Dickson et al, 2000) and endothelial cells (Yang et al, 1999) are immortalised by transduction with hTERT alone. However, in other cell types, loss of p16 in addition to expression of hTERT is necessary for immortalisation (Dickson et al, 2000;Farwell et al, 2000;Rheinwald et al, 2002;Muntoni et al, 2003) probably to bypass a culture stress-induced premature senescence.…”

Section: Discussionmentioning

confidence: 99%

Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway

et al. 2006

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The CDKN2A locus is frequently inactivated in urothelial cell carcinoma (UCC), yet how this alteration contributes to bladder tumorigenesis is not known. Although most UCC express telomerase, inactivation of the p16/Rb pathway is generally required for in vitro immortalisation. This and the involvement of p16 in senescence of normal human urothelial cells (NHUC) suggest that CDKN2A deletion may aid bypass of senescence and allow immortalisation. CDKN2A encodes p16 and p14 ARF and therefore inactivation of this locus can disrupt both the Rb and p53 tumour suppressor pathways. Retrovirusmediated transduction was used to specifically modulate the p16/Rb and/or p53 tumour suppressor pathways in NHUC and to express human telomerase reverse transcriptase (hTERT). Expression of hTERT bypassed Rb and p53 pathway-dependent barriers to proliferation and immortalised NHUC. TERT-NHUC had normal karyotypes, were non-tumorigenic and unexpectedly retained CDKN2A. Thus, the phenotypic significance of inactivation of CDKN2A in UCC may not be solely related to bypass of senescence. Phenotypic assays in human urothelial cells have relied on cell strains derived from invasive tumours or NHUC immortalised by expression of SV40-large T. The production of genetically normal but immortal NHUC lines now provides a valuable platform for experiments to examine the timing and combination of events necessary for UCC tumorigenesis.

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Section: Discussionmentioning

confidence: 99%

Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway

et al. 2006

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“…Although cellular immortalization in the vast majority of human tumors relies on derepression of the native cellular hTERT gene, telomerase is itself not widely believed to be an oncogene that is independently capable of inducing tumor formation (35). The overexpression of telomerase activity in cultured cells has failed to result in malignant change in a number of cell types, including fibroblasts (12,33), endothelial cells (15,33), skeletal muscle cells (36), and esophageal squamous cells (31).…”

Section: Discussionmentioning

confidence: 99%

Relevance and safety of telomerase for human tissue engineering

Klinger

Blum

Hearn

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Tissue engineering holds the promise of replacing damaged or diseased tissues and organs. The use of autologous donor cells is often not feasible because of the limited replicative lifespan of cells, particularly those derived from elderly patients. Proliferative arrest can be overcome by the ectopic expression of telomerase via human telomerase reverse transcriptase (hTERT) gene transfection. To study the efficacy and safety of this potentially valuable technology, we used differentiated vascular smooth muscle cells (SMC) and vascular tissue engineering as a model system. Although we previously demonstrated that vessels engineered with telomerase-expressing SMC had improved mechanics over those grown with control cells, it is critical to assess the phenotypic impact of telomerase expression in donor cells, because telomerase upregulation is observed in >95% of human malignancies. To study the impact of telomerase in tissue engineering, expression of hTERT was retrovirally induced in SMC from eight elderly patients and one young donor. In hTERT SMC, significant lifespan extension beyond that of control was achieved without population doubling time acceleration. Karyotype changes were seen in both control and hTERT SMC but were not clonal nor representative of cancerous change. hTERT cells also failed to show evidence of neoplastic transformation in functional assays of tumorigenicity. In addition, the impact of donor age on cellular behavior, particularly the synthetic capability of SMC, was not affected by hTERT expression. Hence, this tissue engineering model system highlights the impact of donor age on cellular synthetic function that appears to be independent of lifespan extension by hTERT.tumorigenicity ͉ smooth muscle cells ͉ vascular graft

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“…10 The HPV16 E7 [11][12][13][14][15][16][17][18][19][20] -specific human CTL clone A9 was obtained as described previously in detail. 11 CTLs were maintained in vitro in Yssel medium 25 supplemented with 1% human serum (HS; Perbio, Helsingborg, Sweden) and antibiotics (penicillin/streptomycin; Gibco, Paisley, Scotland) in 24-well plates (Nunc, Intermed, Denmark).…”

Section: Ctl Culture and Htert Transductionmentioning

confidence: 99%

“…Allophycocyanin (APC)-labeled HLA-A2.1 tetramers (T A2) presenting the HPV16 E7 [11][12][13][14][15][16][17][18][19][20] and influenza A virus MP 58-66 epitopes were prepared in house as described previously. 27 Tetramer and/or antibody staining of cells was performed in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline [PBS] supplemented with 0.1% bovine serum albumin [BSA] and 0.01% azide) for 15 minutes at 37°C and/or 20 minutes on ice, followed by washing with FACS buffer.…”

Section: Antibodies Tetramers and Flow Cytometrymentioning

confidence: 99%

“…Ectopic expression of hTERT has successfully mediated the in vitro lifespan extension of various human cell types, including fibroblasts, endothelial cells, muscle cells, and neuronal cells. [13][14][15][16] Additionally, hTERT transduction of human T lymphocytes has resulted in restoration of their replicative potential in vitro. This has allowed the successful lifespan extension of bulk and clonal cultures consisting of cytotoxic, helper, and regulatory type T lymphocytes.…”

Section: Introductionmentioning

confidence: 99%

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Genomic stability and functional activity may be lost in telomerase-transduced human CD8+ T lymphocytes

Schreurs¹,

Hermsen²,

Geltink³

et al. 2005

Blood

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To obtain the large amount of T cells required for adoptive immunotherapy in a clinical setting, T-cell lifespan extension by human telomerase reverse transcriptase (hTERT) transduction is of particular interest. However, constitutive expression of hTERT is associated with malignant transformation and thus warrants a detailed evaluation of the safety of hTERTtransduced T cells before clinical application. In view of this, we performed an extensive cytogenetic analysis of hTERTtransduced MART-1 (melanoma antigen recognized by T cell 1)-and human papillomavirus type 16 (HPV16) E7-specific human CD8 ؉ cytotoxic T lymphocytes (CTLs), reactive against melanoma and cervical carcinoma, respectively. Our results, obtained by (spectral) karyotyping and array comparative genomic hybridization, showed the development of minor chromosomal aberrations in an hTERTtransduced MART-1-specific CTL clone, whereas severe clonal aberrations were detected in an hTERT-transduced HPV16 E7-specific CTL clone. Furthermore, hTERT transduction did not protect CTLs from immunosenescence, because the HPV16 E7-specific, hTERT-transduced CTL clone showed a decreased functional activity on prolonged culture. Although the general frequency of major chromosomal aberrations in hTERTtransduced CTLs and the in vivo significance of our observations remain still unclear at this point, the currently available data suggest that clinical application of hTERT-transduced CTLs should pro- IntroductionAdoptive immunotherapy using tumor-reactive T lymphocytes represents an alternative strategy to treat malignant disease. In different animal models, adoptive transfer of specific cytotoxic T lymphocytes (CTLs) has resulted in the eradication of established tumors. [1][2][3][4] Also in patients with melanoma, adoptive CTL therapy has resulted in objective tumor regressions 5,6 and thus holds promise as a treatment modality against malignant disease in a clinical setting. 7 Given the poor immunogenicity of most tumor cells, the in vitro selection prior to adoptive transfer of high-avidity tumor reactive CTL clones could further improve the efficacy of in vivo tumor rejection. 8,9 However, the expansion for therapeutic purposes of CTL clones with considerable in vitro replicative history can be severely hampered by replicative senescence. 10,11 It has become clear that the catalytic subunit of the human telomerase complex, human telomerase reverse transcriptase (hTERT), can efficiently reverse replicative senescence (reviewed in Ducrest et al 12 ). The ability of hTERT to elongate and stabilize telomeric ends can be exploited to restore the replicative potential of presenescent cells. Ectopic expression of hTERT has successfully mediated the in vitro lifespan extension of various human cell types, including fibroblasts, endothelial cells, muscle cells, and neuronal cells. [13][14][15][16] Additionally, hTERT transduction of human T lymphocytes has resulted in restoration of their replicative potential in vitro. This has allowed the successful lifespan extension o...

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Telomerase Alone Extends the Replicative Life Span of Human Skeletal Muscle Cells Without Compromising Genomic Stability

Cited by 27 publications

References 49 publications

Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway

Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway

Relevance and safety of telomerase for human tissue engineering

Genomic stability and functional activity may be lost in telomerase-transduced human CD8+ T lymphocytes

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