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p73 expression is associated with cellular chemosensitivity in human non-small cell lung cancer cell lines
Kaishan Liu1, Xiaomei Zhuang, Zhuoying Mai
1Department of Pathology, School of Medicine, Jinan University, Guangzhou, Guangdong 510632, P.R. China.
Abstract:
p73 is a member of the p53 tumor suppressor protein family and induces apoptosis in tumor cells that lack functional p53. It has been demonstrated that methylation of CpG islands in the promoter and exon 1 region may result in silencing of the p73 gene. The aim of this study was to investigate the correlation between p73 gene expression and chemosensitivity in non-small cell lung cancer (NSCLC) cell lines. The expression of the p73 transcript in six NSCLC cell lines was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The methylation status in these cell lines was determined by methylation-specific PCR (MSP) analysis. An in vitro demethylation assay was conducted using the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-dC). Restored expression of p73 in the human lung squamous cell carcinoma cell line C57, both at the mRNA and protein level, was investigated by RT-PCR and immunohistochemistry, respectively. A colony formation assay was used to measure the surviving fraction of the C57 cell line. Transcript silencing of the p73 gene in the six NSCLC cell lines was observed and related to aberrant methylation. The expression of the p73 transcript and protein in the C57 cell line was restored by 5-aza-dC. The surviving fraction for colony formation in C57 cells pre-treated with 5-aza-dC was 0.059±0.006, which was significantly different from that of the control group (0.12±0.008; P<0.05). Our data demonstrated a significant correlation between expression of p73 and cellular chemosensitivity in NSCLC.
Insights
Aberrant methylation silences the p73 gene in non-small cell lung cancer (NSCLC). Restoring p73 expression via demethylation enhances chemosensitivity in NSCLC cells, suggesting p73 as a therapeutic target.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- p73, a p53 family member, induces apoptosis in tumor cells lacking functional p53.
- CpG island methylation in the p73 promoter and exon 1 can lead to gene silencing.
- Aberrant gene methylation is a known mechanism in cancer development and progression.
Purpose of the Study:
- To investigate the correlation between p73 gene expression and chemosensitivity in non-small cell lung cancer (NSCLC) cell lines.
- To determine if p73 gene silencing in NSCLC is associated with aberrant methylation.
- To assess the impact of restoring p73 expression on chemosensitivity in NSCLC cells.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) to assess p73 transcript levels.
- Methylation-specific PCR (MSP) to determine methylation status of the p73 gene.
- In vitro demethylation assay using 5-aza-2-deoxycytidine (5-aza-dC) to restore gene expression.
- Immunohistochemistry to confirm protein level restoration.
- Colony formation assay to evaluate cellular chemosensitivity.
Main Results:
- Transcript silencing of the p73 gene was observed in six NSCLC cell lines and correlated with aberrant methylation.
- Treatment with 5-aza-dC restored p73 transcript and protein expression in the C57 NSCLC cell line.
- Pre-treatment with 5-aza-dC significantly reduced the surviving fraction of C57 cells in a colony formation assay (0.059±0.006 vs. 0.12±0.008, P<0.05).
Conclusions:
- Aberrant methylation silences the p73 gene in NSCLC.
- Restoration of p73 expression through demethylation enhances chemosensitivity in NSCLC cells.
- p73 expression levels are significantly correlated with cellular chemosensitivity in NSCLC, indicating its potential as a therapeutic target.
