Figure 4. DNA damage by APOBEC3 family members and expression in breast cancer cell-lines.
(A) Effect of enforced APOBEC expression on cell viability. (A1) Stable transfectants of KBM7 cells that inducibly express APOBEC proteins were incubated with inducer (doxycyclin) and viability was monitored after 72 hr. (A2) Expression of FLAG-tagged APOBECs after 24 hr doxycyclin treatment. (B) Enforced expression of APOBEC3A and APOBEC3B leads to induction of histone γH2AX and of 53BP1 foci. The percentage of cells (B1) positive for histone γH2AX expression quantified by flow cytometry and (B2) exhibiting punctate rather than diffuse 53BP1 staining quantified by immunofluorescence microscopy, with the foci number per cell indicated. (C) Expression of APOBEC family members in six human breast cancer cell-lines as well as in HEK293 cells was analysed by qRT-PCR of total cellular RNA. Expression is shown relative to the average of housekeeping genes HPRT and HMBS. The effect of phorbol ester (PMA) and interferon alpha (INFα) treatment on APOBEC3A and APOBEC3B levels is also shown. In all cases, * indicates p<0.1, ** indicates p<0.0001 compared to control (unpaired t-test).