Summary As an initial approach to optimize 8-aminolaevulinic acid (6-ALA)-induced photosensitization of tumours, we examined the response of three enzymes of the haem biosynthetic pathway: 8-ALA dehydratase, porphobilinogen deaminase (PBGD) and ferrochelatase. Only PBGD activity displayed a time-and dose-related increase in tumours after intravenous administration of 300 mg kg-1 6-ALA. The time course for porphyrin fluorescence changes, reflecting increased production of the penultimate porphyrin, protoporphyrin IX (PPIX), showed a similar pattern to PBGD. This apparent correlation between PBGD activity and porphyrin fluorescence was also observed in four cultured tumour cell lines exposed to 0.1-2.0 mm 6-ALA in vitro. The increase in PBGD activity and PPIX fluorescence was prevented by the protein synthesis inhibitor cycloheximide. As the apparent Km for PBGD was similar before and after 6-ALA, the increase in PBGD activity was attributed to induction of enzyme de novo. These observations of an associated response of PBGD and PPIX imply that PBGD may be a ratelimiting determinant for the efficacy of 6-ALA-induced photosensitization when used in photodynamic therapy.Keywords: 6-aminolaevulinic acid: photosensitization; porphobilinogen deaminase: haem biosynthesis; porphyrin fluorescence Haem is an essential prosthetic group in many critical cellular proteins such as haemoglobin, cytochrome P450 and cyclooxygenase (Abraham, 1991). Eight enzymes are involved in the biosynthesis of haem, a process that occurs in two subcellular compartments: the mitochondria and the cytosol. The first enzyme in the haem pathway, mitochondrial 8-aminolaevulinic acid synthase (8-ALA-S), forms 6-ALA from glycine and succinyl CoA and is a target for the regulation of haem biosynthesis. Feedback inhibition of 8-ALA-S occurs when intracellular haem is present in excess (Ade, 1990).The last step in the haem biosynthetic pathway, the insertion of iron into PPIX to form haem, is catalysed by the mitochondrial enzyme ferrochelatase (FC). In unperturbed systems, FC is regulated by the availability of iron and/or PPIX. Two of the metabolic events that occur between 8-ALA-S and FC are catalysed by the cytosolic enzymes 8-ALA dehydratase (8-ALA-D) and porphobilinogen deaminase (PBGD). The dehydratase enzyme catalyses the condensation of two 8-ALA molecules to form porphobilinogen (PBG). It is the first enzyme that will metabolize the administered 8-ALA. The next enzyme in haem biosynthesis, PBGD, forms the tetrapyrole ring from four porphobilinogen molecules (Abraham, 1991).By providing the 8-ALA-S product, 8-ALA, the initial feedback step has been circumvented and this approach has been exploited for use in photodynamic therapy (PDT) of cancer (Kennedy and Pottier, 1992;Grant et al, 1993; Cairnduff et al, 1994;Regula et al, 1995). Traditional PDT regimens consist of the systemic adminis- Accepted 13 June 1997 Correspondence to: R Hilf tration of a photosensitizer followed by an appropriate interval to allow for its maximal accumulation in ma...

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Photodynamic therapy (PDT), a novel treatment for a variety of human malignancies, usually consists of visible light irradiation of lesions following the systemic administration of a photosensitizer. Induction of the endogenous photosensitizer protoporphyrin IX by the systemic or topical administration of delta-aminolevulinic acid (delta-ALA) is being investigated for use in PDT. We have determined that the incubation of two human and two rodent tumor cell lines in culture with delta-ALA over a 24 h period results in an increase in the accumulation of fluorescent porphyrins in all of these cell lines. However, the two human cell lines produce fluorescent porphyrin at different rates from those seen in the rodent cell lines. The uptake of 14C-delta-ALA was concentration dependent, similar for all the cell lines studied and rapidly reached an intra/extracellular equilibrium after delta-ALA was added to the culture medium. The increase in intracellular fluorescent porphyrin was dependent on the level of delta-ALA in the medium and the incubation time and was directly related to the phototoxicity observed upon exposure of cultured monolayers to light. The data demonstrate that equivalent levels of phototoxicity can be attained by exposing cells to 0.04 mM delta-ALA for 24 h or to 0.5 mM delta-ALA for 2 h. These findings may have implications for optimization of PDT treatment regimens that use delta-ALA.

Understanding the regulation and control of heme/porphyrin biosynthesis is critical for the optimization of the delta-aminolevulinic-acid (ALA)-mediated photodynamic therapy of cancer, in which endogenously produced protoporphyrin IX (PPIX) is the photosensitizer. The human breast cancer cell line MCF-7, the rat mammary adenocarcinoma cell line R3230AC, the mouse mammary tumor cell line EMT-6 and the human mesothelioma cell line H-MESO-1 were used to study ALA-induced PPIX levels and their relationship to delta-aminolevulinic acid dehydratase (ALA-D) activity in vitro. Incubation of these cell lines with 0.5 mM ALA for 3 h resulted in a significant increase in PPIX accumulation, compared with control cells, but there was no significant change in ALA-D activity. Exposure of cells incubated with ALA to 30 mJ/cm2 of fluorescent light, a dose that would cause a 50% reduction in cell proliferation, did not significantly alter the activity of ALA-D. Increasing the activity of porphobilinogen deaminase (PBGD), the enzyme immediately subsequent to ALA-D, by four- to seven-fold via transfection of cells with PBGD complementary DNA did not alter the activity of ALA-D. However, incubation of cells with various concentrations of succinyl acetone, a potent inhibitor of ALA-D, caused a concomitant decline in both PPIX accumulation and ALA-D activity. These data imply that when cells are exposed to exogenous ALA, ALA-D is an important early-control step in heme/porphyrin biosynthesis and that regulation of PPIX synthesis by this dehydratase may impact the effectiveness of ALA-mediated photosensitization.

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.

Summary Recently, considerable interest has been given to photodynamic therapy of cancer using δ-aminolaevulinic acid to induce protoporphyrin IX as the cell photosensitizer. One advantage of this modality is that protoporphyrin IX is cleared from tissue within 24 h after δ-aminolaevulinic acid administration. This could allow for multiple treatment regimens because of little concern regarding the accumulation of the photosensitizer in normal tissues. However, the haem biosynthetic pathway would have to be fully functional after the first course of therapy to allow for subsequent treatments. Photosensitization of cultured R3230AC rat mammary adenocarcinoma cells with δ-aminolaevulinic acid-induced protoporphyrin IX resulted in the inhibition of porphobilinogen deaminase, an enzyme in the haem biosynthetic pathway, and a concomitant decrease in protoporphyrin IX levels. Cultured R3230AC cells exposed to 0.5 mM δ-aminolaevulinic acid for 27 h accumulated 6.07 × 10 -16 mol of protoporphyrin IX per cell and had a porphobilinogen deaminase activity of 0.046 fmol uroporphyrin per 30 min per cell. Cells cultured under the same incubation conditions but exposed to 30 mJ cm -2 irradiation after a 3-h incubation with δ-aminolaevulinic acid showed a significant reduction in protoporphyrin IX, 2.28 × 10 -16 mol per cell, and an 80% reduction in porphobilinogen deaminase activity to 0.0088 fmol uroporphyrin per 30 min per cell. Similar effects were evident in irradiated cells incubated with δ-aminolaevulinic acid immediately after, or following a 24 h interval, post-irradiation. There was little gain in efficacy from a second treatment regimen applied within 24 h of the initial treatment, probably a result of initial metabolic damage leading to reduced levels of protoporphyrin IX. These findings suggest that a correlation may exist between the δ-aminolaevulinic acid induction of porphobilinogen deaminase activity and the increase in intracellular protoporphyrin IX accumulation.

Understanding the regulation and control of heme/porphyrin biosynthesis is critical for the optimization of the ␦-aminolevulinic-acid (ALA)-mediated photodynamic therapy of cancer, in which endogenously produced protoporphyrin IX (PPIX) is the photosensitizer. The human breast cancer cell line MCF-7, the rat mammary adenocarcinoma cell line R3230AC, the mouse mammary tumor cell line EMT-6 and the human mesothelioma cell line H-MESO-1 were used to study ALA-induced PPIX levels and their relationship to ␦-aminolevulinic acid dehydratase (ALA-D) activity in vitro. Incubation of these cell lines with 0.5 mM ALA for 3 h resulted in a significant increase in PPIX accumulation, compared with control cells, but there was no significant change in ALA-D activity. Exposure of cells incubated with ALA to 30 mJ/ cm 2 of fluorescent light, a dose that would cause a 50% reduction in cell proliferation, did not significantly alter the activity of ALA-D. Increasing the activity of porphobilinogen deaminase (PBGD), the enzyme immediately subsequent to ALA-D, by four-to seven-fold via transfection of cells with PBGD complementary DNA did not alter the activity of ALA-D. However, incubation of cells with various concentrations of succinyl acetone, a potent inhibitor of ALA-D, caused a concomitant decline in both PPIX accumulation and ALA-D activity. These data imply that when cells are exposed to exogenous ALA, ALA-D is an important early-control step in heme/porphyrin biosynthesis and that regulation of PPIX synthesis by this dehydratase may impact the effectiveness of ALA-mediated photosensitization.

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