Similar deamination activities but different phenotypic outcomes induced by APOBEC3 enzymes in breast epithelial cells

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.

Protein interaction map of APOBEC3 enzyme family reveals deamination-independent role in cellular function

“…The PFD5 levels remained constant. To confirm that this relationship was dependent on the interaction between A3B and PFD5, we conducted experiments in an MCF7 cell line that expresses doxycycline (dox) inducible A3B-FLAG and transfected in siRNA against PFD5 or a scrambled siRNA ( Figure 4H-I ) (40). The siRNA resulted in a 2-fold decrease in PFD5 (Figure 4H-I ).…”

“…The doxycycline (dox) inducible A3B-FLAG expressing stable MCF7 cells have been previously reported (40). Silencer TM siRNAs were ordered against PFD5 and a scramble of the siRNA (ThermoFisher Scientific).…”

“…siRNA knockdown of PFD5 in A3B-FLAG expressing MCF7 cells. The doxycycline (dox) inducible A3B-FLAG expressing stable MCF7 cells have been previously reported (40).…”