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The use of transformed IMR90 cell model to identify the potential extra-telomeric effects of hTERT in cell migration and DNA damage response.

Cao X, Kong CM, Mathi KM, Lim YP, Cacheux-Rataboul V, Wang X - BMC Biochem. (2014)

Bottom Line: The RSH-transformed cells acquired hallmarks of cancer, such as they can grow under anchorage independent conditions; self-sufficient in growth signals; attenuated response to apoptosis; and possessed recurrent chromosomal abnormalities.This notion was further supported by our microarray analysis.In addition, we found that Ku70 were exclusively upregulated in both RSH-transformed IMR90 cells and hTERT-overexpressing IMR90 cells, suggesting the potential role of hTERT in DNA damage response (DDR).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 1, 5 Science Drive 2, Singapore 117545, Singapore. bchwxy@nus.edu.sg.

ABSTRACT

Background: Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomesase, is responsible for telomere maintenance and its reactivation is implicated in almost 90% human cancers. Recent evidences show that hTERT is essential for neoplastic transformation independent of its canonical function. However, the roles of hTERT in the process remain elusive. In the current work, we explore the extra-telomeric role of hTERT in the neoplastic transformation of fibroblast IMR90.

Results: Here we established transformed IMR90 cells by co-expression of three oncogenic factors, namely, H-Ras, SV40 Large-T antigen and hTERT (RSH). The RSH-transformed cells acquired hallmarks of cancer, such as they can grow under anchorage independent conditions; self-sufficient in growth signals; attenuated response to apoptosis; and possessed recurrent chromosomal abnormalities. Furthermore, the RSH-transformed cells showed enhanced migration capability which was also observed in IMR90 cells expressing hTERT alone, indicating that hTERT plays a role in cell migration, and thus possibly contribute to their metastatic potential during tumor transformation. This notion was further supported by our microarray analysis. In addition, we found that Ku70 were exclusively upregulated in both RSH-transformed IMR90 cells and hTERT-overexpressing IMR90 cells, suggesting the potential role of hTERT in DNA damage response (DDR).

Conclusions: Collectively, our study revealed the extra-telomeric effects of hTERT in cell migration and DDR during neoplastic transformation.

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Ku 70 expression in IMR90-RSH and IMR90 hTERT cells. (A) Coomassie blue staining of the protein expression in RSH transformed cells and control fibroblasts, showing an augmented ~70 kDa (indicated by the black arrow). (B) Semi-quantitative analysis of Ku70 by RT-PCR in IMR90 control, IMR90 hTERT and IMR90 RSH cells. Ku70 expression showed an increase in IMR90 RSH and IMR90 hTERT cells compared to IMR90 control cells. (C) Ku70 protein expression in IMR90 control, IMR90 hTERT and IMR90 RSH cells. Ku70 protein expression showed an increase in IMR90 RSH and IMR90 hTERT cells compared to IMR90 control cells.
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Figure 3: Ku 70 expression in IMR90-RSH and IMR90 hTERT cells. (A) Coomassie blue staining of the protein expression in RSH transformed cells and control fibroblasts, showing an augmented ~70 kDa (indicated by the black arrow). (B) Semi-quantitative analysis of Ku70 by RT-PCR in IMR90 control, IMR90 hTERT and IMR90 RSH cells. Ku70 expression showed an increase in IMR90 RSH and IMR90 hTERT cells compared to IMR90 control cells. (C) Ku70 protein expression in IMR90 control, IMR90 hTERT and IMR90 RSH cells. Ku70 protein expression showed an increase in IMR90 RSH and IMR90 hTERT cells compared to IMR90 control cells.

Mentions: To delineate the roles of hTERT in neoplastic transformation, we performed mass spectrometry analysis on the protein levels of IMR90 RSH cells. Surprisingly, analysis by mass spectrometry revealed that protein Ku70 was exclusively found in IMR90 RSH cells, but not in IMR90 control cells (Figure 3A; Additional file 2: Table S2). Moreover, both RT-PCR and immunoblotting results confirmed our observation where Ku70 expression was augmented in IMR90 RSH as well as IMR90 hTERT cells, but not in IMR90 control cells (Figure 3B,C). This could be indicative of an activated DNA damage responses (DDR) in both IMR90-RSH and IMR90-hTERT since Ku70 is a DDR sensor and is implicated in the non homologous end joining (NHEJ) pathway [29]. Ku70 forms a heterodimeric complex with Ku80 [30]. However, the protein expression of Ku80 remained unchanged (Figure 3C). Presence of Ku70 in IMR90 RSH cells and IMR90 hTERT cells prompted us to assess whether other DDR-associated proteins could be regulated by hTERT as well. Our microarray data revealed that several DDR-associated genes were upregulated in both RSH-transformed IMR90 and hTERT-overexpressing IMR90 cells (fold change ≥ 2; p < 0.05) (Additional file 3: Table S3). Taken together, these results suggest that hTERT may play an important role in DDR pathways, via Ku70 and other DDR-associated proteins.


The use of transformed IMR90 cell model to identify the potential extra-telomeric effects of hTERT in cell migration and DNA damage response.

Cao X, Kong CM, Mathi KM, Lim YP, Cacheux-Rataboul V, Wang X - BMC Biochem. (2014)

Ku 70 expression in IMR90-RSH and IMR90 hTERT cells. (A) Coomassie blue staining of the protein expression in RSH transformed cells and control fibroblasts, showing an augmented ~70 kDa (indicated by the black arrow). (B) Semi-quantitative analysis of Ku70 by RT-PCR in IMR90 control, IMR90 hTERT and IMR90 RSH cells. Ku70 expression showed an increase in IMR90 RSH and IMR90 hTERT cells compared to IMR90 control cells. (C) Ku70 protein expression in IMR90 control, IMR90 hTERT and IMR90 RSH cells. Ku70 protein expression showed an increase in IMR90 RSH and IMR90 hTERT cells compared to IMR90 control cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4126993&req=5

Figure 3: Ku 70 expression in IMR90-RSH and IMR90 hTERT cells. (A) Coomassie blue staining of the protein expression in RSH transformed cells and control fibroblasts, showing an augmented ~70 kDa (indicated by the black arrow). (B) Semi-quantitative analysis of Ku70 by RT-PCR in IMR90 control, IMR90 hTERT and IMR90 RSH cells. Ku70 expression showed an increase in IMR90 RSH and IMR90 hTERT cells compared to IMR90 control cells. (C) Ku70 protein expression in IMR90 control, IMR90 hTERT and IMR90 RSH cells. Ku70 protein expression showed an increase in IMR90 RSH and IMR90 hTERT cells compared to IMR90 control cells.
Mentions: To delineate the roles of hTERT in neoplastic transformation, we performed mass spectrometry analysis on the protein levels of IMR90 RSH cells. Surprisingly, analysis by mass spectrometry revealed that protein Ku70 was exclusively found in IMR90 RSH cells, but not in IMR90 control cells (Figure 3A; Additional file 2: Table S2). Moreover, both RT-PCR and immunoblotting results confirmed our observation where Ku70 expression was augmented in IMR90 RSH as well as IMR90 hTERT cells, but not in IMR90 control cells (Figure 3B,C). This could be indicative of an activated DNA damage responses (DDR) in both IMR90-RSH and IMR90-hTERT since Ku70 is a DDR sensor and is implicated in the non homologous end joining (NHEJ) pathway [29]. Ku70 forms a heterodimeric complex with Ku80 [30]. However, the protein expression of Ku80 remained unchanged (Figure 3C). Presence of Ku70 in IMR90 RSH cells and IMR90 hTERT cells prompted us to assess whether other DDR-associated proteins could be regulated by hTERT as well. Our microarray data revealed that several DDR-associated genes were upregulated in both RSH-transformed IMR90 and hTERT-overexpressing IMR90 cells (fold change ≥ 2; p < 0.05) (Additional file 3: Table S3). Taken together, these results suggest that hTERT may play an important role in DDR pathways, via Ku70 and other DDR-associated proteins.

Bottom Line: The RSH-transformed cells acquired hallmarks of cancer, such as they can grow under anchorage independent conditions; self-sufficient in growth signals; attenuated response to apoptosis; and possessed recurrent chromosomal abnormalities.This notion was further supported by our microarray analysis.In addition, we found that Ku70 were exclusively upregulated in both RSH-transformed IMR90 cells and hTERT-overexpressing IMR90 cells, suggesting the potential role of hTERT in DNA damage response (DDR).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 1, 5 Science Drive 2, Singapore 117545, Singapore. bchwxy@nus.edu.sg.

ABSTRACT

Background: Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomesase, is responsible for telomere maintenance and its reactivation is implicated in almost 90% human cancers. Recent evidences show that hTERT is essential for neoplastic transformation independent of its canonical function. However, the roles of hTERT in the process remain elusive. In the current work, we explore the extra-telomeric role of hTERT in the neoplastic transformation of fibroblast IMR90.

Results: Here we established transformed IMR90 cells by co-expression of three oncogenic factors, namely, H-Ras, SV40 Large-T antigen and hTERT (RSH). The RSH-transformed cells acquired hallmarks of cancer, such as they can grow under anchorage independent conditions; self-sufficient in growth signals; attenuated response to apoptosis; and possessed recurrent chromosomal abnormalities. Furthermore, the RSH-transformed cells showed enhanced migration capability which was also observed in IMR90 cells expressing hTERT alone, indicating that hTERT plays a role in cell migration, and thus possibly contribute to their metastatic potential during tumor transformation. This notion was further supported by our microarray analysis. In addition, we found that Ku70 were exclusively upregulated in both RSH-transformed IMR90 cells and hTERT-overexpressing IMR90 cells, suggesting the potential role of hTERT in DNA damage response (DDR).

Conclusions: Collectively, our study revealed the extra-telomeric effects of hTERT in cell migration and DDR during neoplastic transformation.

Show MeSH
Related in: MedlinePlus