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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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Migration capability analysis of IMR90 RSH and IMR90 hTERT cells. (A) Boyden assay comparing the migration capability of IMR90 control and IMR90 RSH cells after 10 hours. (B) Wound healing assay comparing the migration of IMR90 control and IMR90 RSH cells after 9 hours of incubation. Images at 0 hour and at 9 hours, representative of triplicate experiments for IMR90 control and IMR90 RSH cells, are shown. White arrows indicate individual cells that have migrated. (C) The ‘wound closure’ areas are visualized under an inverted microscope and bar graphs show the distance travelled by IMR90 control and IMR90 RSH cells in the wound healing assay. Results are indicated as the mean ± standard deviation (SD) (n = 2). *p < 0.05; **p < 0.01; ***p <0.001. (D) Western blot confirming the overexpression of hTERT in IMR90 primary human cells. (E) Wound healing assay comparing the migration of IMR90 control and IMR90 hTERT cells after 32 hours of incubation. Images at 0 hour and at 32 hours, representative of triplicate experiments for IMR90 control and IMR90 hTERT cells, are shown. White arrows indicate individual cells that have migrated.(F) Bar graphs showing the distance travelled by IMR90 control and IMR90 hTERT cells in the wound healing assay. Results are indicated as the mean ± standard deviation (SD) (n = 2). *p <0.05; **p < 0.01; ***p <0.001.
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Figure 2: Migration capability analysis of IMR90 RSH and IMR90 hTERT cells. (A) Boyden assay comparing the migration capability of IMR90 control and IMR90 RSH cells after 10 hours. (B) Wound healing assay comparing the migration of IMR90 control and IMR90 RSH cells after 9 hours of incubation. Images at 0 hour and at 9 hours, representative of triplicate experiments for IMR90 control and IMR90 RSH cells, are shown. White arrows indicate individual cells that have migrated. (C) The ‘wound closure’ areas are visualized under an inverted microscope and bar graphs show the distance travelled by IMR90 control and IMR90 RSH cells in the wound healing assay. Results are indicated as the mean ± standard deviation (SD) (n = 2). *p < 0.05; **p < 0.01; ***p <0.001. (D) Western blot confirming the overexpression of hTERT in IMR90 primary human cells. (E) Wound healing assay comparing the migration of IMR90 control and IMR90 hTERT cells after 32 hours of incubation. Images at 0 hour and at 32 hours, representative of triplicate experiments for IMR90 control and IMR90 hTERT cells, are shown. White arrows indicate individual cells that have migrated.(F) Bar graphs showing the distance travelled by IMR90 control and IMR90 hTERT cells in the wound healing assay. Results are indicated as the mean ± standard deviation (SD) (n = 2). *p <0.05; **p < 0.01; ***p <0.001.

Mentions: Metastasis is often correlated to two attributes: migration followed by invasion [21]. Metastatic tumors are believed to occur in the late stages of cancer [22]. However, in numerous early-stage cancers, recurrence can be observed even after the removal of non-invasive, benign tumors, suggesting the possibility of the cancer cells undergoing metastasis at a much earlier stage [23]. We questioned if IMR90 RSH cells possess migration capability similar to that of a cancer cell. Our Boyden assay results showed that IMR90 RSH cells had a greater migration capability as compared to IMR90 control cells (Figure 2A). This result was further validated with wound healing assay, in which the gap was reduced to 40% for IMR90 RSH cells compared to 84% in IMR90 control cells (p < 0.01) (Figure 2B,C). Moreover, we observed that IMR90 RSH cells migrated faster and in a more individualistic pattern compared to IMR90 control cells, implying some degree of autonomy (Figure 2B).


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)

Migration capability analysis of IMR90 RSH and IMR90 hTERT cells. (A) Boyden assay comparing the migration capability of IMR90 control and IMR90 RSH cells after 10 hours. (B) Wound healing assay comparing the migration of IMR90 control and IMR90 RSH cells after 9 hours of incubation. Images at 0 hour and at 9 hours, representative of triplicate experiments for IMR90 control and IMR90 RSH cells, are shown. White arrows indicate individual cells that have migrated. (C) The ‘wound closure’ areas are visualized under an inverted microscope and bar graphs show the distance travelled by IMR90 control and IMR90 RSH cells in the wound healing assay. Results are indicated as the mean ± standard deviation (SD) (n = 2). *p < 0.05; **p < 0.01; ***p <0.001. (D) Western blot confirming the overexpression of hTERT in IMR90 primary human cells. (E) Wound healing assay comparing the migration of IMR90 control and IMR90 hTERT cells after 32 hours of incubation. Images at 0 hour and at 32 hours, representative of triplicate experiments for IMR90 control and IMR90 hTERT cells, are shown. White arrows indicate individual cells that have migrated.(F) Bar graphs showing the distance travelled by IMR90 control and IMR90 hTERT cells in the wound healing assay. Results are indicated as the mean ± standard deviation (SD) (n = 2). *p <0.05; **p < 0.01; ***p <0.001.
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Figure 2: Migration capability analysis of IMR90 RSH and IMR90 hTERT cells. (A) Boyden assay comparing the migration capability of IMR90 control and IMR90 RSH cells after 10 hours. (B) Wound healing assay comparing the migration of IMR90 control and IMR90 RSH cells after 9 hours of incubation. Images at 0 hour and at 9 hours, representative of triplicate experiments for IMR90 control and IMR90 RSH cells, are shown. White arrows indicate individual cells that have migrated. (C) The ‘wound closure’ areas are visualized under an inverted microscope and bar graphs show the distance travelled by IMR90 control and IMR90 RSH cells in the wound healing assay. Results are indicated as the mean ± standard deviation (SD) (n = 2). *p < 0.05; **p < 0.01; ***p <0.001. (D) Western blot confirming the overexpression of hTERT in IMR90 primary human cells. (E) Wound healing assay comparing the migration of IMR90 control and IMR90 hTERT cells after 32 hours of incubation. Images at 0 hour and at 32 hours, representative of triplicate experiments for IMR90 control and IMR90 hTERT cells, are shown. White arrows indicate individual cells that have migrated.(F) Bar graphs showing the distance travelled by IMR90 control and IMR90 hTERT cells in the wound healing assay. Results are indicated as the mean ± standard deviation (SD) (n = 2). *p <0.05; **p < 0.01; ***p <0.001.
Mentions: Metastasis is often correlated to two attributes: migration followed by invasion [21]. Metastatic tumors are believed to occur in the late stages of cancer [22]. However, in numerous early-stage cancers, recurrence can be observed even after the removal of non-invasive, benign tumors, suggesting the possibility of the cancer cells undergoing metastasis at a much earlier stage [23]. We questioned if IMR90 RSH cells possess migration capability similar to that of a cancer cell. Our Boyden assay results showed that IMR90 RSH cells had a greater migration capability as compared to IMR90 control cells (Figure 2A). This result was further validated with wound healing assay, in which the gap was reduced to 40% for IMR90 RSH cells compared to 84% in IMR90 control cells (p < 0.01) (Figure 2B,C). Moreover, we observed that IMR90 RSH cells migrated faster and in a more individualistic pattern compared to IMR90 control cells, implying some degree of autonomy (Figure 2B).

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