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A gene expression signature classifying telomerase and ALT immortalization reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT.

Lafferty-Whyte K, Cairney CJ, Will MB, Serakinci N, Daidone MG, Zaffaroni N, Bilsland A, Keith WN - Oncogene (2009)

Bottom Line: We have shown earlier that active repression of telomerase gene expression by chromatin remodelling of the promoters is one mechanism of regulation; however, other genes and signalling networks are likely to be required to regulate telomerase and maintain the ALT phenotype.This network expands on our existing knowledge of hTERT regulation and provides a platform to understand differential regulation of hTERT in different tumour types and normal tissues.We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues.

View Article: PubMed Central - PubMed

Affiliation: Centre for Oncology and Applied Pharmacology, University of Glasgow, Cancer Research UK Beatson Laboratories, Bearsden, Glasgow G61 1BD, UK.

ABSTRACT
Telomere length is maintained by two known mechanisms, the activation of telomerase or alternative lengthening of telomeres (ALT). The molecular mechanisms regulating the ALT phenotype are poorly understood and it is unknown how the decision of which pathway to activate is made at the cellular level. We have shown earlier that active repression of telomerase gene expression by chromatin remodelling of the promoters is one mechanism of regulation; however, other genes and signalling networks are likely to be required to regulate telomerase and maintain the ALT phenotype. Using gene expression profiling, we have uncovered a signature of 1305 genes to distinguish telomerase-positive and ALT cell lines. By combining this with the gene expression profiles of liposarcoma tissue samples, we refined this signature to 297 genes. A network analysis of known interactions between genes within this signature revealed a regulatory signalling network consistent with a model of human telomerase reverse transcriptase (hTERT) repression in ALT cell lines and liposarcomas. This network expands on our existing knowledge of hTERT regulation and provides a platform to understand differential regulation of hTERT in different tumour types and normal tissues. We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues.

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The TERT regulatory network is shown at the protein level and predicted c-MYC activity is confirmed as significantly lower in ALT.(a) Western blotting shows protein level differences in 3 molecules of the 297 gene network. 15 μg of cell extracts were run on NuPAGE 4-12% Bis-Tris gels, transferred to Millipore nitrocellulose membrane and probed with appropriate antibodies. Blots were then stripped and reprobed with ERK1 loading control. Panels shown are representative panels of 2 separate blots.(b) c-Myc activity ELISA shows significantly lower activity in ALT cells. Interval plot shows the average of 6 ALT cell lines (WI38-SV40, KMST6, SKLU, SUSM1, SAOS and U2OS) and 4 telomerase cell lines (A2780, C33a, HT1080 and 5637) on 3 separate occasions with 4 replicates of each cell line. Crosshairs show mean expression for each group and error bars show 95% confidence intervals of the mean. T-test of the results were T-Value = −2.51 P-Value = 0.015 DF = 51.
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Figure 5: The TERT regulatory network is shown at the protein level and predicted c-MYC activity is confirmed as significantly lower in ALT.(a) Western blotting shows protein level differences in 3 molecules of the 297 gene network. 15 μg of cell extracts were run on NuPAGE 4-12% Bis-Tris gels, transferred to Millipore nitrocellulose membrane and probed with appropriate antibodies. Blots were then stripped and reprobed with ERK1 loading control. Panels shown are representative panels of 2 separate blots.(b) c-Myc activity ELISA shows significantly lower activity in ALT cells. Interval plot shows the average of 6 ALT cell lines (WI38-SV40, KMST6, SKLU, SUSM1, SAOS and U2OS) and 4 telomerase cell lines (A2780, C33a, HT1080 and 5637) on 3 separate occasions with 4 replicates of each cell line. Crosshairs show mean expression for each group and error bars show 95% confidence intervals of the mean. T-test of the results were T-Value = −2.51 P-Value = 0.015 DF = 51.

Mentions: Given the ability of the refined 297 gene signature to separate liposarcomas by TMM we hypothesised that the genes within the signature may comprise of functional regulatory networks involved in aspects of TMM. In order to explore this we performed network modelling using Metacore from GeneGo, allowing us to build a candidate network indicating possible interactions between genes from the 297 signature mined from published data. A regulatory network involving hTERT and telomeric DNA was revealed by this analysis (figure 4d). Expression data from the 297 gene signature was converted to fold change in ALT over telomerase positive, uploaded into Metacore analytical suite and overlaid on the direct interactions network. As can be seen from Figure 4d, by combining interactions between known signalling pathways and experimentally defined levels of expression for regulatory genes this approach allows for predictions relating to hTERT regulation and repression in ALT cells. hTERT expression is reduced in ALT cells and tumours in relation to telomerase positive samples. Consistent with this, expression of E2F1 a known repressor of hTERT, is up-regulated in ALT samples, whereas chromatin modifying enzymes with roles in gene activation such as GCN5 are down-regulated in ALT, in agreement with the decreased association of acetylated histones and low hTERT expression in ALT cell lines as we have previously shown. Western Blotting of HDAC5 PKCa and GCN5 seen in Figure 5a shows that the expression differences highlighted in this network are also seen at the protein level.


A gene expression signature classifying telomerase and ALT immortalization reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT.

Lafferty-Whyte K, Cairney CJ, Will MB, Serakinci N, Daidone MG, Zaffaroni N, Bilsland A, Keith WN - Oncogene (2009)

The TERT regulatory network is shown at the protein level and predicted c-MYC activity is confirmed as significantly lower in ALT.(a) Western blotting shows protein level differences in 3 molecules of the 297 gene network. 15 μg of cell extracts were run on NuPAGE 4-12% Bis-Tris gels, transferred to Millipore nitrocellulose membrane and probed with appropriate antibodies. Blots were then stripped and reprobed with ERK1 loading control. Panels shown are representative panels of 2 separate blots.(b) c-Myc activity ELISA shows significantly lower activity in ALT cells. Interval plot shows the average of 6 ALT cell lines (WI38-SV40, KMST6, SKLU, SUSM1, SAOS and U2OS) and 4 telomerase cell lines (A2780, C33a, HT1080 and 5637) on 3 separate occasions with 4 replicates of each cell line. Crosshairs show mean expression for each group and error bars show 95% confidence intervals of the mean. T-test of the results were T-Value = −2.51 P-Value = 0.015 DF = 51.
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Related In: Results  -  Collection

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Figure 5: The TERT regulatory network is shown at the protein level and predicted c-MYC activity is confirmed as significantly lower in ALT.(a) Western blotting shows protein level differences in 3 molecules of the 297 gene network. 15 μg of cell extracts were run on NuPAGE 4-12% Bis-Tris gels, transferred to Millipore nitrocellulose membrane and probed with appropriate antibodies. Blots were then stripped and reprobed with ERK1 loading control. Panels shown are representative panels of 2 separate blots.(b) c-Myc activity ELISA shows significantly lower activity in ALT cells. Interval plot shows the average of 6 ALT cell lines (WI38-SV40, KMST6, SKLU, SUSM1, SAOS and U2OS) and 4 telomerase cell lines (A2780, C33a, HT1080 and 5637) on 3 separate occasions with 4 replicates of each cell line. Crosshairs show mean expression for each group and error bars show 95% confidence intervals of the mean. T-test of the results were T-Value = −2.51 P-Value = 0.015 DF = 51.
Mentions: Given the ability of the refined 297 gene signature to separate liposarcomas by TMM we hypothesised that the genes within the signature may comprise of functional regulatory networks involved in aspects of TMM. In order to explore this we performed network modelling using Metacore from GeneGo, allowing us to build a candidate network indicating possible interactions between genes from the 297 signature mined from published data. A regulatory network involving hTERT and telomeric DNA was revealed by this analysis (figure 4d). Expression data from the 297 gene signature was converted to fold change in ALT over telomerase positive, uploaded into Metacore analytical suite and overlaid on the direct interactions network. As can be seen from Figure 4d, by combining interactions between known signalling pathways and experimentally defined levels of expression for regulatory genes this approach allows for predictions relating to hTERT regulation and repression in ALT cells. hTERT expression is reduced in ALT cells and tumours in relation to telomerase positive samples. Consistent with this, expression of E2F1 a known repressor of hTERT, is up-regulated in ALT samples, whereas chromatin modifying enzymes with roles in gene activation such as GCN5 are down-regulated in ALT, in agreement with the decreased association of acetylated histones and low hTERT expression in ALT cell lines as we have previously shown. Western Blotting of HDAC5 PKCa and GCN5 seen in Figure 5a shows that the expression differences highlighted in this network are also seen at the protein level.

Bottom Line: We have shown earlier that active repression of telomerase gene expression by chromatin remodelling of the promoters is one mechanism of regulation; however, other genes and signalling networks are likely to be required to regulate telomerase and maintain the ALT phenotype.This network expands on our existing knowledge of hTERT regulation and provides a platform to understand differential regulation of hTERT in different tumour types and normal tissues.We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues.

View Article: PubMed Central - PubMed

Affiliation: Centre for Oncology and Applied Pharmacology, University of Glasgow, Cancer Research UK Beatson Laboratories, Bearsden, Glasgow G61 1BD, UK.

ABSTRACT
Telomere length is maintained by two known mechanisms, the activation of telomerase or alternative lengthening of telomeres (ALT). The molecular mechanisms regulating the ALT phenotype are poorly understood and it is unknown how the decision of which pathway to activate is made at the cellular level. We have shown earlier that active repression of telomerase gene expression by chromatin remodelling of the promoters is one mechanism of regulation; however, other genes and signalling networks are likely to be required to regulate telomerase and maintain the ALT phenotype. Using gene expression profiling, we have uncovered a signature of 1305 genes to distinguish telomerase-positive and ALT cell lines. By combining this with the gene expression profiles of liposarcoma tissue samples, we refined this signature to 297 genes. A network analysis of known interactions between genes within this signature revealed a regulatory signalling network consistent with a model of human telomerase reverse transcriptase (hTERT) repression in ALT cell lines and liposarcomas. This network expands on our existing knowledge of hTERT regulation and provides a platform to understand differential regulation of hTERT in different tumour types and normal tissues. We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues.

Show MeSH
Related in: MedlinePlus