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An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry
Published on: May 13, 2019
Expulsion of micronuclei containing amplified genes contributes to a decrease in double minute chromosomes from
1Laboratory of Medical Genetics, Harbin Medical University, Harbin, People's Republic of China.
Abstract:
Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM-carried genes remains to be clarified. The human colorectal cancer cell line NCI-H716 and human malignant primitive neuroectodermal tumor cell line SK-PN-DW are known to contain many DMs. To examine the amplification of DM-carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI-H716 and SK-PN-DW tumor cells. We identified the amplification regions and the DM-carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM-carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM-carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM-carried genes led to a decrease in the number of DMs and reduced the amplification of the DM-carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM-carried genes resulted in a decrease in cell proliferation and invasion ability.
Insights
Suppressing double minute chromosome (DM)-carried genes reduces DM formation and amplification in tumor cells. This process involves DNA damage and leads to decreased cell proliferation and invasion.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Double minute chromosomes (DMs) are associated with gene amplification in cancer.
- The precise relationship between DM formation and the amplification of genes they carry is not fully understood.
Purpose of the Study:
- To investigate the role of DM-carried genes in DM formation and amplification.
- To explore the impact of downregulating DM-carried genes on tumor cell behavior.
Main Methods:
- Analysis of gene amplification in NCI-H716 and SK-PN-DW cell lines using Affymetrix SNP Array 6.0.
- RNA interference to downregulate seven specific DM-carried genes.
- Assessment of DM formation, gene amplification, DNA damage (γH2AX foci), cell proliferation, and invasion.
Main Results:
- Downregulation of DM-carried genes decreased DM numbers and their amplification via micronuclear expulsion.
- Increased γH2AX foci indicated a link between DNA damage and DM loss.
- Reduced DM amplification and expression of DM-carried genes correlated with decreased cell proliferation and invasion.
Conclusions:
- DM-carried genes play a crucial role in the maintenance and amplification of DMs.
- Targeting these genes can lead to DM loss, DNA damage, and impaired tumor cell growth and metastasis.
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