5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) catalyzes the first committed step of
heme biosynthesis in animals. The erythroid-specific ALAS
isozyme (ALAS2) is negatively regulated by
heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of
protoporphyrin IX (
PPIX), the precursor for
heme. Importantly, generation of
PPIX is a crucial component in the widely used
photodynamic therapies (
PDT) of
cancer and other dysplasias. ALAS2 variants that cause high levels of
PPIX accumulation provide a new means of targeted, and potentially enhanced,
photosensitization. In order to assess the prospective utility of ALAS2 variants in
PPIX production for
PDT, K562 human
erythroleukemia cells and HeLa human cervical
carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type
enzyme. The levels of accumulated
PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21-22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of
PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to
aminolevulinic acid (ALA) treatment producing a similar amount of
PPIX. The delivery of stable and highly active ALAS2 variants has the potential to expand and improve upon current
PDT regimes.