Related Experiment Video
Updated: Aug 7, 2026
Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Published on: October 21, 2012
Differential gene expression in apoptosis: identification of ribosomal protein 23K, a cell proliferation inhibitor
F W Chen1, J P Davies, Y A Ioannou
1Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, USA.
Abstract:
Gene expression during the camptothecin-induced apoptotic death of human leukemic U937 cells and mouse T-cell hybridoma QW4.1 cells was studied by the mRNA differential display technique. Ten clones were confirmed to be differentially expressed, nine of which encoded novel sequences. One clone, U3.2, was induced approximately 10-fold in camptothecin-treated cells and was found to be identical to a highly basic 23-kDa human protein which contains basic leucine zipper-like motifs and has recently been identified as the human homologue of the rat ribosomal protein L13a. Northern blot analysis revealed a major mRNA of approximately 0.9 kb and a minor mRNA of approximately 1.3 kb. Overexpression of a full-length 23K cDNA, tagged with a FLAG sequence, in COS-7 cells revealed a predominantly nucleolar localization and the absence of any 23K protein from the cytoplasm. Subsequent transfection studies, using antisense phosphorothioate-modified oligonucleotides, revealed that inhibition of 23K expression results in an increased cell proliferation and greater sensitivity of U937 cells to the effects of camptothecin-induced cell death. Upregulation of 23K expression using a cDNA construct resulted in a decrease in cell proliferation and growth arrest, suggesting a role for 23K protein as a proliferation checkpoint following a cellular insult.
Insights
Camptothecin induces apoptosis in cancer cells by altering gene expression. A novel protein, 23K, acts as a proliferation checkpoint, regulating cell death and growth following cellular damage.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Apoptosis, or programmed cell death, is crucial for development and disease.
- Understanding gene expression changes during apoptosis is key to cancer therapy.
- Camptothecin is a potent anti-cancer agent that induces apoptosis.
Purpose of the Study:
- To investigate gene expression changes during camptothecin-induced apoptosis.
- To identify novel genes involved in the apoptotic pathway.
- To elucidate the function of a newly identified gene, 23K, in cell proliferation and death.
Main Methods:
- mRNA differential display technique to identify differentially expressed genes.
- Northern blot analysis to confirm mRNA expression levels.
- cDNA overexpression and antisense oligonucleotide transfection to study protein function.
- Subcellular localization studies using tagged proteins.
Main Results:
- Differential display identified ten differentially expressed clones, including a novel sequence, U3.2.
- U3.2 was identified as the human homologue of rat ribosomal protein L13a (23K protein).
- 23K protein localized to the nucleolus and inhibited cell proliferation.
- Inhibition of 23K expression increased proliferation and sensitivity to camptothecin-induced cell death.
Conclusions:
- 23K protein plays a significant role in regulating cell proliferation.
- 23K acts as a proliferation checkpoint, influencing cell fate after cellular insult.
- Modulating 23K expression could be a therapeutic strategy for cancer treatment.
Related Concept Videos
01:23Negative Regulator Molecules
02:24Ribosome Profiling
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...
02:23Abnormal Proliferation
01:31The Intrinsic Apoptotic Pathway