Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death.
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.
Department of Molecular Medicine, Morsani College of Medicine, Tampa, Florida, United States of America.
Erica J
EJ
Fratz
Department of Molecular Medicine, Morsani College of Medicine, Tampa, Florida, United States of America.
Gregory A
GA
Hunter
Department of Molecular Medicine, Morsani College of Medicine, Tampa, Florida, United States of America; Department of Chemistry, University of South Florida, Tampa, Florida, United States of America.
Gloria C
GC
Ferreira
D000624:5-Aminolevulinate Synthetase / Q000378:metabolism*; D000818:Animals; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D003470:Culture Media; D003676:Deferoxamine / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D008027:Light; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011524:Protoporphyrins / Q000378:metabolism*; D014162:Transfection
D003470:Culture Media; D050505:Mutant Proteins; D011524:Protoporphyrins; D049452:Green Fluorescent Proteins; C028025:protoporphyrin IX; D000624:5-Aminolevulinate Synthetase; C588337:ALAS2 protein, mouse; D003676:Deferoxamine; D017239:Paclitaxel; D005998:Glycine