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Published on: October 4, 2019
Identification of genes downregulated in tumor cells expressing antisense glutaminase mRNA by differential display
Mercedes Martín-Rufián1, Juan A Segura, Carolina Lobo
1Departamento de Biología Molecular y Bioquímica, Laboratorio de Química de Proteínas, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain.
Abstract:
Ehrlich ascites tumor cells (EATC) is a highly proliferative malignant cell line derived from mouse mammary epithelia, whereas their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, show a less transformed phenotype and loss of their tumorigenic capacity in vivo correlated with an inhibition of glutaminase expression. The mRNA differential display technique was applied to these two cell lines for the identification and isolation of genes whose transcription was altered. Side-by-side comparisons of cDNA patterns among relevant RNA samples revealed four genes significantly downregulated in 0.28AS-2 cells: high-mobility group Hmga2 protein, Fmnl3 or formin-like protein 3, Nedd-4 ubiquitin-protein ligase, and ubiquitin carboxyl-terminal hydrolase Usp-15. These positives were confirmed by Northern analysis. The four targeted genes have relevant functions in cell growth and proliferation. Our results show the validity of mRNA differential display technique to get insights into the molecular mechanisms underlying the acquisition of a more differentiated phenotype by tumor cells after inhibition of glutaminase expression.
Insights
Researchers identified four genes downregulated in less tumorigenic cells derived from Ehrlich ascites tumor cells (EATC) after glutaminase inhibition. This finding offers insights into tumor cell differentiation mechanisms.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Ehrlich ascites tumor cells (EATC) are highly proliferative malignant cells.
- 0.28AS-2 cells, derived from EATC, exhibit reduced transformation and tumorigenicity due to inhibited glutaminase expression.
Purpose of the Study:
- To identify genes with altered transcription in 0.28AS-2 cells compared to EATC.
- To understand the molecular mechanisms behind the differentiated phenotype in tumor cells following glutaminase inhibition.
Main Methods:
- mRNA differential display technique was employed for gene identification.
- Northern analysis was used to confirm the downregulation of identified genes.
Main Results:
- Four genes were found to be significantly downregulated in 0.28AS-2 cells: high-mobility group Hmga2 protein, formin-like protein 3 (Fmnl3), Nedd-4 ubiquitin-protein ligase, and ubiquitin carboxyl-terminal hydrolase Usp-15.
- These downregulated genes are involved in critical cellular processes such as growth and proliferation.
Conclusions:
- The study validates the mRNA differential display technique for uncovering molecular mechanisms in cancer research.
- Inhibition of glutaminase expression in tumor cells leads to a more differentiated phenotype, associated with the downregulation of specific genes involved in proliferation.
