Differentiation-specific increase in ALA-induced protoporphyrin IX accumulation in primary mouse keratinocytes.

B Ortel,E Maytin,T Hasan,N Chen,Gp Dotto,J Brissette

doi:10.1038/bjc.1998.292

Abstract

A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT). Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes. Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA. These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells. The differentiation-dependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media. Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged. From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium- and differentiation specific. Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation. Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation. Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable selective targeting of several tissue types.

Highlights

We investigated the effect of differentiation upon the ability of keratinocytes to form and accumulate protoporphyrin IX (PPIX) in response to exogenous amino-laevulinic acid (ALA) exposure, and upon their subsequent response to photosensitization
Earlier reports showed a positive correlation between the proliferation rates of several primary cells and cell lines in vitro and the amounts of PPIX produced from exogenous ALA (Iinuma et al, 1994; Rittenhouse-Diakun et al, 1995). In contrast to these earlier findings, the present study shows an increase in ALA-induced PPIX production in differentiating cells, elucidates some of the mechanisms involved in the differentiationdependent increase in ALA-induced PPIX formation; the relevance of this increase for photodynamic sensitization is discussed
Intracellular PPIX accumulation increased with time after inducing differentiation andI growth arrest as determined by a decrease in [3H]Td incorporation rrate

Summary

Methods

ALA from DUSA Pharmaceuticals, Ontario, Canada, was prepared as a 0.1 M stock solution in 0.1 M HCl. The stock was kept at 4°C and final concentrations were reached by direct dilution of the stock into the media. 8-[4-14C]Aminolaevulenic acid hydrochloride ([14C]ALA) (51.3 mCi mmol-1) and [methyl-3H]thymidine ([3H]Td) (20.0 Ci mmol-') were obtained from Dupont/NEN, Wilmington, DE, USA. DMSO, 12-o-Tetradecanoylphorbol-13-acetate (TPA), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MIF), succinyl acetone, PPIX and salmon sperm DNA were all purchased from Sigma, St Louis, MO, USA. Epidermal growth factor (EGF) was purchased from Collaborative Research, Bedford, MA, USA. Calcimycin (A23187) was purchased from Molecular Probes, Eugene, OR, USA. Transfonrning growth factor beta 1 (TGF-[I) was obtained from R&D Systems, Minneapolis, MN, USA, and nerve growth factor (NGF) from Boehringer Mannheim, Indianapolis, IN, USA. Stock solutions of TPA and PPIX were made in DMSO, TGF-

Results

Discussion

Conclusion