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Wilms' tumour 1 can suppress hTERT gene expression and telomerase activity in clear cell renal cell carcinoma via multiple pathways.

Sitaram RT, Degerman S, Ljungberg B, Andersson E, Oji Y, Sugiyama H, Roos G, Li A - Br. J. Cancer (2010)

Bottom Line: WT1 demonstrated lower RNA expression in ccRCC compared with renal cortical tissue, whereas hTERT was increased, showing a negative correlation between WT1 and hTERT (P=0.005).These findings were experimentally confirmed in vitro.The WT1 generated effect on hTERT promoter activity seemed complex, as several negative regulators of hTERT transcription, such as SMAD3, JUN (AP-1) and ETS1, were activated by WT1 overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biosciences and Pathology, Umeå University, Umeå, Sweden.

ABSTRACT

Background: Wilms' tumour 1 (WT1) gene was discovered as a tumour suppressor gene. Later findings have suggested that WT1 also can be oncogenic. This complexity is partly explained by the fact that WT1 has a number of target genes.

Method: WT1 and its target gene human telomerase reverse transcriptase (hTERT) were analysed in clear cell renal cell carcinoma (ccRCC). In vitro experiments were performed to examine the functional link between WT1 and hTERT by overexpression of WT1 isoforms in the ccRCC cell line, TK-10.

Results: WT1 demonstrated lower RNA expression in ccRCC compared with renal cortical tissue, whereas hTERT was increased, showing a negative correlation between WT1 and hTERT (P=0.005). These findings were experimentally confirmed in vitro. The WT1 generated effect on hTERT promoter activity seemed complex, as several negative regulators of hTERT transcription, such as SMAD3, JUN (AP-1) and ETS1, were activated by WT1 overexpression. Downregulation of potential positive hTERT regulators, such as cMyc, AP-2α, AP-2γ, IRF1, NFX1 and GM-CSF, were also observed. Chromatin immunoprecipitation analysis verified WT1 binding to the hTERT, cMyc and SMAD3 promoters.

Conclusion: The collected data strongly indicate multiple pathways for hTERT regulation by WT1 in ccRCC.

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Related in: MedlinePlus

Validation of gene expression of SMAD3 (A), ETS1 (B) and AP-2α (C) by quantitative real-time PCR (qRT–PCR).
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fig4: Validation of gene expression of SMAD3 (A), ETS1 (B) and AP-2α (C) by quantitative real-time PCR (qRT–PCR).

Mentions: Gene expression alterations induced by WT1 transfection in TK-10 cells were detected using whole genome expression array analysis and several genes reported to be WT1 targets were found to be affected (Figure 3). Further analysis was focused on hTERT-regulating genes identifying an increased expression of several repressors. Fold changes, based on the array data, in expression of hTERT transcriptional regulators after WT1 transfection are summarised in Table 1. Two negative hTERT regulators, SMAD3 and JUN, were significantly upregulated. Moreover, the GM-CSF, ETS1 and IRF1 genes also with suggested negative effects on hTERT transcription demonstrated increased expression. In contrast, AP-2α, AP-2γ and NFX1, all with potential positive effects on hTERT expression, were downregulated. The altered expression of SMAD3, ETS1 and AP-2α was validated by qRT–PCR, showing a good correlation with the array data (Figure 4).


Wilms' tumour 1 can suppress hTERT gene expression and telomerase activity in clear cell renal cell carcinoma via multiple pathways.

Sitaram RT, Degerman S, Ljungberg B, Andersson E, Oji Y, Sugiyama H, Roos G, Li A - Br. J. Cancer (2010)

Validation of gene expression of SMAD3 (A), ETS1 (B) and AP-2α (C) by quantitative real-time PCR (qRT–PCR).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2967054&req=5

fig4: Validation of gene expression of SMAD3 (A), ETS1 (B) and AP-2α (C) by quantitative real-time PCR (qRT–PCR).
Mentions: Gene expression alterations induced by WT1 transfection in TK-10 cells were detected using whole genome expression array analysis and several genes reported to be WT1 targets were found to be affected (Figure 3). Further analysis was focused on hTERT-regulating genes identifying an increased expression of several repressors. Fold changes, based on the array data, in expression of hTERT transcriptional regulators after WT1 transfection are summarised in Table 1. Two negative hTERT regulators, SMAD3 and JUN, were significantly upregulated. Moreover, the GM-CSF, ETS1 and IRF1 genes also with suggested negative effects on hTERT transcription demonstrated increased expression. In contrast, AP-2α, AP-2γ and NFX1, all with potential positive effects on hTERT expression, were downregulated. The altered expression of SMAD3, ETS1 and AP-2α was validated by qRT–PCR, showing a good correlation with the array data (Figure 4).

Bottom Line: WT1 demonstrated lower RNA expression in ccRCC compared with renal cortical tissue, whereas hTERT was increased, showing a negative correlation between WT1 and hTERT (P=0.005).These findings were experimentally confirmed in vitro.The WT1 generated effect on hTERT promoter activity seemed complex, as several negative regulators of hTERT transcription, such as SMAD3, JUN (AP-1) and ETS1, were activated by WT1 overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biosciences and Pathology, Umeå University, Umeå, Sweden.

ABSTRACT

Background: Wilms' tumour 1 (WT1) gene was discovered as a tumour suppressor gene. Later findings have suggested that WT1 also can be oncogenic. This complexity is partly explained by the fact that WT1 has a number of target genes.

Method: WT1 and its target gene human telomerase reverse transcriptase (hTERT) were analysed in clear cell renal cell carcinoma (ccRCC). In vitro experiments were performed to examine the functional link between WT1 and hTERT by overexpression of WT1 isoforms in the ccRCC cell line, TK-10.

Results: WT1 demonstrated lower RNA expression in ccRCC compared with renal cortical tissue, whereas hTERT was increased, showing a negative correlation between WT1 and hTERT (P=0.005). These findings were experimentally confirmed in vitro. The WT1 generated effect on hTERT promoter activity seemed complex, as several negative regulators of hTERT transcription, such as SMAD3, JUN (AP-1) and ETS1, were activated by WT1 overexpression. Downregulation of potential positive hTERT regulators, such as cMyc, AP-2α, AP-2γ, IRF1, NFX1 and GM-CSF, were also observed. Chromatin immunoprecipitation analysis verified WT1 binding to the hTERT, cMyc and SMAD3 promoters.

Conclusion: The collected data strongly indicate multiple pathways for hTERT regulation by WT1 in ccRCC.

Show MeSH
Related in: MedlinePlus