Fluorescence Monitoring of the Oxidative Repair of DNA Alkylation Damage by ALKBH3, a Prostate Cancer Marker

Abstract

The 2-oxoglutarate-dependent iron enzyme ALKBH3 is an antitumor target and a potential diagnostic marker for several tumor types, including prostate cancer. However, there is at present no simple way to measure this enzyme's activity. Here we describe a fluorogenic probe design (MAQ) that is directly responsive to ALKBH3 repair activity. It makes use of the fluorescence-quenching properties of 1-methyladenine; removal of the alkyl group results in a >10-fold light-up signal. The probe is specific for ALKBH3 over its related homologue ALKBH2 and can be used to identify and measure the effectiveness of enzyme inhibitors. Measurements of the enzyme substrate parameters show that MAQ displays Km and kcat values essentially the same as those of the native substrate. Finally, we show that the probe functions efficiently in cells, allowing imaging and quantitation of ALKBH3 activity by microscopy and flow cytometry. We expect that MAQ probes will be broadly useful in the study of the basic biology of ALKBH3 and in clinical cancer applications as well.

Figure 1

Demethylation of 1-methyladenine (m1A) by ALKBH3 in a MAQ probe that employs an adjacent fluorescent deoxynucleoside (Y) that is selectively quenched by the cationic alkylated base.

Figure 2

Fluorescence responses of MAQ probe to ALKBH3 and to an inhibitor. (A) Response of MAQ probe (1 μM) to ALKBH3. (B) Plot showing inhibition curve generated by MAQ probe for a recently reported inhibitor of ALKBH3.^, Y = α-pyrene deoxynucleoside.

Figure 3

Fluorescence responses of nuclease-protected probe PMAQ (2 μM, shown above) to variable levels of ALKBH3 expression in prostate cancer cell lines PC3 (A, high expression) and U2OS (B, low expression).

Figure 4

Quantitation of ALKBH3 signals by flow cytometry. Data are obtained with 2 μM probe in wild-type mouse embryonic fibroblasts and in cells with ALKBH3 knocked out. See SI for methods.