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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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Related in: MedlinePlus

Transformed IMR90 cells show characteristics of a cancer cell. (A) Western blot confirming the expression of the three genetic factors Ras, hTERT and SV 40 Large T in the transformed IMR90 primary human cells. The expression of hTERT on the western blot was detected using anti-FLAG antibody. (B) Changes in cellular morphology after RSH transformation. Transformation of IMR90 cells and resulted in shorter and rounder cells. Left bottom corners show the enlarged pictures. (C) Soft agar assay determining the anchorage independence of the transformed RSH cells in vitro. MCF-7 cells were used as the positive control. The experiment was carried out in triplicates. Representative image of one well is shown. (D) Survival of IMR90 control and IMR90 RSH cells in serum-free medium. (E) Cells of IMR90 control and IMR90 RSH were treated with 1 μM, 3 μM and 5 μM of doxorubicin separately for up to 48 hours. (F) G-band Karyotype analysis of IMR90 control cells infected with control vector and IMR90 RSH cells. Arrows indicate the presence of genetic aberrations. (G) Metaphase spreads of IMR90 RSH cells. White arrows indicate the presence of genetic aberrations. (H) A representative spectral karyotype of a metaphase from IMR90 RSH cells. Recurrent abnormality is defined as at least 3 metaphase cells having the abnormality at the same region of chromosomal location. Chromosomal abnormality can be observed at chromosomes 4, 18, 20.
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Figure 1: Transformed IMR90 cells show characteristics of a cancer cell. (A) Western blot confirming the expression of the three genetic factors Ras, hTERT and SV 40 Large T in the transformed IMR90 primary human cells. The expression of hTERT on the western blot was detected using anti-FLAG antibody. (B) Changes in cellular morphology after RSH transformation. Transformation of IMR90 cells and resulted in shorter and rounder cells. Left bottom corners show the enlarged pictures. (C) Soft agar assay determining the anchorage independence of the transformed RSH cells in vitro. MCF-7 cells were used as the positive control. The experiment was carried out in triplicates. Representative image of one well is shown. (D) Survival of IMR90 control and IMR90 RSH cells in serum-free medium. (E) Cells of IMR90 control and IMR90 RSH were treated with 1 μM, 3 μM and 5 μM of doxorubicin separately for up to 48 hours. (F) G-band Karyotype analysis of IMR90 control cells infected with control vector and IMR90 RSH cells. Arrows indicate the presence of genetic aberrations. (G) Metaphase spreads of IMR90 RSH cells. White arrows indicate the presence of genetic aberrations. (H) A representative spectral karyotype of a metaphase from IMR90 RSH cells. Recurrent abnormality is defined as at least 3 metaphase cells having the abnormality at the same region of chromosomal location. Chromosomal abnormality can be observed at chromosomes 4, 18, 20.

Mentions: Primary human fibroblast cells IMR90 were successfully co-transfected with Ras, SV40 Large-T, and hTERT and their protein expressions were confirmed by western blotting (Figure 1A). Morphologically, IMR90 RSH fibroblasts appeared to be shorter and rounder compared to the infection control (Figure 1B). This observation is consistent with the findings of Mason and colleagues in IMR90 cells transformed with E1a/Ras [17], suggesting that these changes are the unique characteristics of cellular transformation. Moreover, late passages of IMR90 control cells underwent significant increase in cell sizes, indicating their senescent status. However, this was not observed in IMR90 RSH cells even after several passages (data not shown).


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)

Transformed IMR90 cells show characteristics of a cancer cell. (A) Western blot confirming the expression of the three genetic factors Ras, hTERT and SV 40 Large T in the transformed IMR90 primary human cells. The expression of hTERT on the western blot was detected using anti-FLAG antibody. (B) Changes in cellular morphology after RSH transformation. Transformation of IMR90 cells and resulted in shorter and rounder cells. Left bottom corners show the enlarged pictures. (C) Soft agar assay determining the anchorage independence of the transformed RSH cells in vitro. MCF-7 cells were used as the positive control. The experiment was carried out in triplicates. Representative image of one well is shown. (D) Survival of IMR90 control and IMR90 RSH cells in serum-free medium. (E) Cells of IMR90 control and IMR90 RSH were treated with 1 μM, 3 μM and 5 μM of doxorubicin separately for up to 48 hours. (F) G-band Karyotype analysis of IMR90 control cells infected with control vector and IMR90 RSH cells. Arrows indicate the presence of genetic aberrations. (G) Metaphase spreads of IMR90 RSH cells. White arrows indicate the presence of genetic aberrations. (H) A representative spectral karyotype of a metaphase from IMR90 RSH cells. Recurrent abnormality is defined as at least 3 metaphase cells having the abnormality at the same region of chromosomal location. Chromosomal abnormality can be observed at chromosomes 4, 18, 20.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Transformed IMR90 cells show characteristics of a cancer cell. (A) Western blot confirming the expression of the three genetic factors Ras, hTERT and SV 40 Large T in the transformed IMR90 primary human cells. The expression of hTERT on the western blot was detected using anti-FLAG antibody. (B) Changes in cellular morphology after RSH transformation. Transformation of IMR90 cells and resulted in shorter and rounder cells. Left bottom corners show the enlarged pictures. (C) Soft agar assay determining the anchorage independence of the transformed RSH cells in vitro. MCF-7 cells were used as the positive control. The experiment was carried out in triplicates. Representative image of one well is shown. (D) Survival of IMR90 control and IMR90 RSH cells in serum-free medium. (E) Cells of IMR90 control and IMR90 RSH were treated with 1 μM, 3 μM and 5 μM of doxorubicin separately for up to 48 hours. (F) G-band Karyotype analysis of IMR90 control cells infected with control vector and IMR90 RSH cells. Arrows indicate the presence of genetic aberrations. (G) Metaphase spreads of IMR90 RSH cells. White arrows indicate the presence of genetic aberrations. (H) A representative spectral karyotype of a metaphase from IMR90 RSH cells. Recurrent abnormality is defined as at least 3 metaphase cells having the abnormality at the same region of chromosomal location. Chromosomal abnormality can be observed at chromosomes 4, 18, 20.
Mentions: Primary human fibroblast cells IMR90 were successfully co-transfected with Ras, SV40 Large-T, and hTERT and their protein expressions were confirmed by western blotting (Figure 1A). Morphologically, IMR90 RSH fibroblasts appeared to be shorter and rounder compared to the infection control (Figure 1B). This observation is consistent with the findings of Mason and colleagues in IMR90 cells transformed with E1a/Ras [17], suggesting that these changes are the unique characteristics of cellular transformation. Moreover, late passages of IMR90 control cells underwent significant increase in cell sizes, indicating their senescent status. However, this was not observed in IMR90 RSH cells even after several passages (data not shown).

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