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A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants
Published on: October 10, 2022
Similar deamination activities but different phenotypic outcomes induced by APOBEC3 enzymes in breast epithelial
Milaid Granadillo Rodríguez1, Lai Wong1, Linda Chelico1
1Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.
Abstract:
APOBEC3 (A3) enzymes deaminate cytosine to uracil in viral single-stranded DNA as a mutagenic barrier for some viruses. A3-induced deaminations can also occur in human genomes resulting in an endogenous source of somatic mutations in multiple cancers. However, the roles of each A3 are unclear since few studies have assessed these enzymes in parallel. Thus, we developed stable cell lines expressing A3A, A3B, or A3H Hap I using non-tumorigenic MCF10A and tumorigenic MCF7 breast epithelial cells to assess their mutagenic potential and cancer phenotypes in breast cells. The activity of these enzymes was characterized by γH2AX foci formation and in vitro deamination. Cell migration and soft agar colony formation assays assessed cellular transformation potential. We found that all three A3 enzymes had similar γH2AX foci formation, despite different deamination activities in vitro. Notably, in nuclear lysates, the in vitro deaminase activity of A3A, A3B, and A3H did not require digestion of cellular RNA, in contrast to that of A3B and A3H in whole-cell lysates. Their similar activities in cells, nonetheless, resulted in distinct phenotypes where A3A decreased colony formation in soft agar, A3B decreased colony formation in soft agar after hydroxyurea treatment, and A3H Hap I promoted cell migration. Overall, we show that in vitro deamination data do not always reflect cell DNA damage, all three A3s induce DNA damage, and the impact of each is different.
Insights
APOBEC3 (A3) enzymes cause DNA damage in breast cells, but their specific roles in cancer are unclear. This study reveals distinct cellular effects for A3A, A3B, and A3H, highlighting varied impacts on cancer phenotypes.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- APOBEC3 (A3) enzymes are crucial for innate immunity against viruses by deaminating cytosine in viral DNA.
- A3-induced mutations also occur endogenously in human genomes, contributing to somatic mutations in various cancers.
- The specific roles and mutagenic potential of individual A3 enzymes in cancer development remain largely uncharacterized.
Purpose of the Study:
- To investigate and compare the mutagenic potential and cancer-associated phenotypes of A3A, A3B, and A3H Hap I in breast epithelial cells.
- To determine if in vitro deamination activity correlates with cellular DNA damage and functional outcomes.
- To elucidate the distinct cellular impacts of each A3 enzyme in both non-tumorigenic and tumorigenic breast cell contexts.
Main Methods:
- Development of stable cell lines expressing A3A, A3B, or A3H Hap I in MCF10A and MCF7 cells.
- Assessment of DNA damage via γH2AX foci formation and in vitro deamination assays.
- Evaluation of cellular transformation using cell migration and soft agar colony formation assays.
Main Results:
- All three A3 enzymes induced similar levels of DNA damage (γH2AX foci), despite variations in in vitro deamination activity.
- In vitro deaminase activity of A3A, A3B, and A3H in nuclear lysates did not require RNA digestion, unlike in whole-cell lysates.
- Distinct cellular phenotypes were observed: A3A inhibited soft agar colony formation, A3B did so after hydroxyurea treatment, and A3H promoted cell migration.
Conclusions:
- In vitro deamination data do not always accurately predict cellular DNA damage or functional outcomes.
- All three studied APOBEC3 enzymes induce DNA damage in breast cells.
- Each A3 enzyme exerts unique effects on cellular phenotypes, suggesting differential roles in breast cancer development and progression.
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