Effect of δ-Aminolevulinic Acid on Protoporphyrin IX Accumulation in Tumor Cells Transfected with Plasmids Containing Porphobilinogen Deaminase DNA

Abstract

Recently, we reported that the delta-aminolevulinic acid (delta-ALA)-induced increase in porphobilinogen deaminase (PBGD) activity was closely correlated with an increase in the accumulation of protoporphyrin IX (PPIX), resulting in augmented phototoxicity. In this report, we asked whether increasing the cellular expression of PBGD by use of gene transfection techniques in vitro would further enhance delta-ALA-induced PPIX accumulation and hence, phototoxicity. For these experiments we constructed plasmid vectors containing the PBGD-DNA, using a reverse transcription-polymerase chain reaction-generated cDNA fragment encoded from its published sequence. Subsequently, transfection of the human mammary tumor cell line, MCF-7, and the human mesothelioma cell line, H-MESO-1, with the PBGD-DNA-containing plasmids was shown to produce a 2.5-2.7-fold increase in enzyme activity. Twenty-four hours after completion of the transfection procedure, transfectants were exposed for 3 h to 0.5 mM delta-ALA. Exposure of either wild type or transfectants to delta-ALA led to measurable levels of PPIX. Although this produced a modest but significant increase in intracellular PPIX content in H-MESO-1 cells compared to wild-type cells incubated with delta-ALA alone, the increase above the transfection control did not reach statistical significance. Likewise, a significant increase in PPIX was not observed in transfected MCF-7 cells subsequently exposed to delta-ALA. These data demonstrate that transient transfection of cells with the cDNA of PBGD was successful in elevating enzyme activity in both tumor cell lines, but this did not result in a comparable difference in the levels of PPIX. Such an approach for the study of other enzymes in the heme pathway should provide a model to better define rate-limiting steps in the delta-ALA induction of PPIX, and ultimately, to enhance the effectiveness of photodynamic therapy.

Review of Neurosurgical Fluorescence Imaging Methodologies

“…A large body of research on this topic is compiled in a review by Collaud, et al [12]. Although much of past research points to porphobilinogen deaminase (PBGD) as the rate limiting enzyme in the ALA-PpIX pathway [13, 26], recent research suggests that PBGD may play a minor role in the accumulation of PpIX in vitro [27–29]. Additionally, it has been suggested that ALA-dehydratase, which converts ALA to porphobilinogen, may act as the rate-limiting step as its inhibition causes decreased PpIX production [30].…”

Is δ-Aminolevulinic Acid Dehydratase Rate Limiting in Heme Biosynthesis Following Exposure of Cells to δ-Aminolevulinic Acid?¶

“…We reported that PPIX levels and PBGD activity increased concomitantly over time during the exposure of tumors to ALA in vivo or tumor cells in vitro (10)(11)(12). We then transfected cultured tumor cells with plasmids containing PBGD-complementary DNA (cDNA) in order to enhance PBGD expression, and we ascertained whether this strategy would increase ALAinduced PPIX accumulation above that obtained in wild-type cells exposed to ALA (13). Curiously, although we were †Abbreviations: ALA, ␦-aminolevulinic acid; ALA-D, ␦-aminolevulinic acid dehydratase; ALA-S, ␦-aminolevulinic acid synthase; FBS, fetal bovine serum; FC, ferrochelatase; ␣-MEM, minimum essential medium; PBG, porphobilinogen; PBGD, porphobilinogen deaminase; PBS, phosphate-buffered saline; PDT, photodynamic therapy; PPIX, protoporphyrin IX; SA, succinyl acetone.…”

Nuclear distribution of porphobilinogen deaminase (PBGD) in glioma cells: a regulatory role in cancer transformation?

“…Transient transfection of cells with the cDNA of PBGD was successful in elevating enzyme activity in both the human mammary tumour cell line MCF-7 and the human mesothelioma cell line, H-MESO-1, but this did not result in a comparable difference in the levels of PpIX (Hilf et al, 1999). PBGD has always been considered exclusively as a cytoplasmic enzyme of the heme biosynthesis pathway (Sassa, 1990).…”