Lack of Telomerase Activity in Lung Carcinoids Is Dependent on Human Telomerase Reverse Transcriptase Transcription and Alternative Splicing and Is Associated with Long Telomeres

Zaffaroni, Nadia; Villa, Raffaella; Pastorino, Ugo; Cirincione, Rosalia; Incarbone, Matteo; Alloisio, Marco; Curto, María Ángeles; Pilotti, Silvana; Daidone, Maria Grazia

doi:10.1158/1078-0432.ccr-04-1293

Cited by 33 publications

(13 citation statements)

References 55 publications

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“…TA-DMPM did not express any hTERT transcript or, alternatively, displayed the presence of the h -splice variant alone or different combinations of full-length and deletion transcripts, with a generally higher level of alternatively spliced variants compared with that of the complete hTERT transcript. These data would suggest that the presence of the full-length hTERT transcript is necessary but not sufficient to activate the enzyme in DMPM and that not only hTERT transcription but also alternative splicing can contribute to the regulation of TA in this disease, as already reported for melanomas and lung carcinoids (41,42). In this context, we previously showed that forced accumulation of alternatively spliced transcripts with a concomitant decrease of the full-length hTERT, accomplished through the use of 2 ¶-Omethyl-RNA phosphorothioate oligonucleotides targeting the splicing site in the hTERT pre-mRNA, induced a decline of TA in prostate cancer cells (43).…”

Section: Discussionsupporting

confidence: 55%

Multiple Mechanisms of Telomere Maintenance Exist and Differentially Affect Clinical Outcome in Diffuse Malignant Peritoneal Mesothelioma

Villa¹,

Daidone²,

Motta³

et al. 2008

Clinical Cancer Research

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Purpose: This study aims to investigate the prevalence of the two known telomere maintenance mechanisms, telomerase activity (TA) and alternative lengthening of telomeres (ALT), and to assess their prognostic relevance in diffuse malignant peritoneal mesothelioma (DMPM). Experimental Design: In 44 DMPM specimens obtained from 38 patients, TA was determined using the telomeric repeat amplification protocol and ALT was detected by assaying ALT-associated promyelocytic leukemia nuclear bodies. The prognostic significance of telomere maintenance mechanisms was analyzed by Cox regression in the overall series and in a subset of 29 patients who underwent a uniform treatment regimen consisting of cytoreductive surgery and hyperthermic i.p. chemotherapy. Results: Telomere maintenance mechanisms were detectable in 86.4% of DMPM: ALT or TA alone was found in 18.2% or 63.6% of lesions, respectively, whereas two cases (4.6%) were ALT+/TA+. TA and ALT proved to be inversely associated (P = 0.002). In the overall series, TA was prognostic for 4-year relapse (TA+ versus TA-, hazard ratio, 3.30; 95% confidence interval, 1.23-8.86; P = 0.018) and cancer-related death (TA+ versusTA-, hazard ratio, 3.56; 95% confidence interval, 1.03-12.51; P = 0.045), whereas ALT failed to significantly affect clinical outcome. These results held true also in the subset of patients submitted to uniform treatment with cytoreductive surgery and hyperthermic i.p. chemotherapy. Conclusions: Our results indicate that both known telomere maintenance mechanisms,TA and ALT, are present in DMPM and differentially affect patient prognosis.

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Section: Discussionsupporting

confidence: 55%

Multiple Mechanisms of Telomere Maintenance Exist and Differentially Affect Clinical Outcome in Diffuse Malignant Peritoneal Mesothelioma

Villa¹,

Daidone²,

Motta³

et al. 2008

Clinical Cancer Research

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“…This indicates the presence of an active hTERT promoter also in VL-7 cells and suggests that alternative splicing plays a significant role in TA activation of NSCLC cells. Comparably, expression of alternatively spliced hTERT mRNA molecules has been very recently described for TA-and ALT-negative lung carcinoids (53). However, in contrast to our VL-7 cells, these carcinoid cells underwent crisis during in vitro culture, suggesting them as mortal pre-M2 cells comparable with VL-9 NSCLC cells.…”

Section: Discussionsupporting

confidence: 57%

Telomerase- and Alternative Telomere Lengthening–Independent Telomere Stabilization in a Metastasis-Derived Human Non–Small Cell Lung Cancer Cell Line: Effect of Ectopic hTERT

et al. 2006

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In the majority of human malignancies, maintenance of telomeres is achieved by reactivation of telomerase, whereas a smaller fraction uses an alternative telomere lengthening (ALT) mechanism. Here, we used 16 non-small cell lung cancer (NSCLC) cell lines to investigate telomere stabilization mechanisms and their effect on tumor aggressiveness. Three of 16 NSCLC cell lines (VL-9, SK-LU-1, and VL-7) lacked telomerase activity, correlating with significantly reduced tumorigenicity in vitro and in vivo. Of the three telomerasenegative cell lines, only SK-LU-1 displayed characteristics of an ALT mechanism (i.e., highly heterogeneous telomeres and ALT-associated promyelocytic leukemia bodies). VL-9 cells gained telomerase during in vitro propagation, indicating incomplete immortalization in vivo. In contrast, NSCLC metastasis-derived VL-7 cells remained telomerase and ALT negative up to high passage numbers and following transplantation in severe combined immunodeficient mice. Telomeres of VL-7 cells were homogenously short, and chromosomal instability (CIN) was comparable with most telomerase-positive cell lines. This indicates the presence of an efficient telomere stabilization mechanism different from telomerase and ALT in VL-7 cells. To test the effect of ectopic telomerase reverse transcriptase (hTERT) in these unique ALT-and telomerase-negative tumor backgrounds, hTERT was transfected into VL-7 cells. The activation of telomerase led to an excessively rapid gain of telomeric sequences resulting in very long (f14 kb), uniform telomeres. Additionally, hTERT expression induced a more aggressive growth behavior in vitro and in vivo without altering the level of CIN. These data provide further evidence for a direct oncogenic activity of hTERT not based on the inhibition of CIN. (Cancer Res 2006; 66(7): 3584-92)

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“…Total RNA (0.5 Ag) from each sample was reverse-transcribed by using the reverse transcription-PCR (RT-PCR) Core kit (Applied Biosystems) with random hexamers, and the resultant cDNA was then amplified with the same kit. Amplification of full-length and alternatively spliced human telomerase reverse transcriptase (hTERT) cDNA was obtained as previously described (26).…”

Section: Methodsmentioning

confidence: 99%

Human Bone Marrow–Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-termIn vitroCulture and Do Not Exhibit Telomere Maintenance Mechanisms

et al. 2007

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Significant improvement in the understanding of mesenchymal stem cell (MSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that MSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different in vitro culture time points. MSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. MSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of MSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping, and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. MSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages. A huge variability in terms of proliferative capacity and MSCs life span was noted between donors. In eight of 10 donors, MSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two MSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that MSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the MSCs tested. BM-derived MSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches.

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Lack of Telomerase Activity in Lung Carcinoids Is Dependent on Human Telomerase Reverse Transcriptase Transcription and Alternative Splicing and Is Associated with Long Telomeres

Cited by 33 publications

References 55 publications

Multiple Mechanisms of Telomere Maintenance Exist and Differentially Affect Clinical Outcome in Diffuse Malignant Peritoneal Mesothelioma

Multiple Mechanisms of Telomere Maintenance Exist and Differentially Affect Clinical Outcome in Diffuse Malignant Peritoneal Mesothelioma

Telomerase- and Alternative Telomere Lengthening–Independent Telomere Stabilization in a Metastasis-Derived Human Non–Small Cell Lung Cancer Cell Line: Effect of Ectopic hTERT

Human Bone Marrow–Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-termIn vitroCulture and Do Not Exhibit Telomere Maintenance Mechanisms

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