Dataset Information

Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death.

ABSTRACT: 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.

SUBMITTER: Fratz EJ

PROVIDER: S-EPMC3981678 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death.

Fratz Erica J EJ Hunter Gregory A GA Ferreira Gloria C GC

PloS one 20140409 4

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 ca ...[more]

PMID: 24718052

Similar Datasets

Project description:SignificanceThe quantification of protoporphyrin IX (PpIX) in skin can be used to study photodynamic therapy (PDT) treatments, understand porphyrin mechanisms, and enhance preoperative mapping of non-melanoma skin cancers.AimWe aim to develop a smartphone-based imager for performing simultaneous radiometric fluorescence (FL) and white light (WL) imaging to study the baseline levels, accumulation, and photobleaching of PpIX in skin.ApproachA smartphone-based dual FL and WL imager (sDUO) is introduced alongside new radiometric calibration methods for providing SI-units of measurements in both pre-clinical and clinical settings. These radiometric measurements and corresponding PpIX concentration estimations are applied to clinical measurements to understand mechanistic differences between PDT treatments, accumulation differences between normal tissue and actinic keratosis lesions, and the correlation of photosensitizer concentrations to treatment outcomes.ResultsThe sDUO alongside the developed methods provided radiometric FL measurements (nW/cm2) with a demonstrated sub nanomolar PpIX sensitivity in 1% intralipid phantoms. Patients undergoing PDT treatment of actinic keratosis (AK) lesions were imaged, capturing the increase and subsequent decrease in FL associated with the incubation and irradiation timepoints of lamp-based PDT. Furthermore, the clinical measurements showed mechanistic differences in new daylight-based treatment modalities alongside the selective accumulation of PpIX within AK lesions. The use of the radiometric calibration enabled the reporting of detected PpIX FL in units of nW/cm2 with the use of liquid phantom measurements allowing for the estimation of in-vivo molar concentrations of skin PpIX.ConclusionsThe phantom, pre-clinical, and clinical measurements demonstrated the capability of the sDUO to provide quantitative measurements of PpIX FL. The results demonstrate the use of the sDUO for the quantification of PpIX accumulation and photobleaching in a clinical setting, with implications for improving the diagnosis and treatment of various skin conditions.

Dataset Information

Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death.

Publications

Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets