Figure 2. Cell type-specificity and dose-dependence of PpIX accumulation following MTX pre-conditioning, and correlation with changes in PpIX-metabolic enzymes.

(A) Quantification of PpIX signal in the different cell lines using spectrofluorimetry. Cells were seeded in replicate 25 cm² flasks at ~60% confluence and induced with MTX (0.01 mg/L) for 72 h. Following incubation with ALA, cell numbers were determined by photography and manual counting; then cells were lysed and the lysates measured in a spectrofluorimeter (see Methods). PpIX levels per cell are shown relative to untreated controls. Each bar represents mean ± SD of 3 separate flasks; differences (MTX versus no MTX) are significant at the p < 0.005 level (**). (B) Quantification of PpIX signal by analysis of digital confocal images. Living cells on coverslips were analyzed with confocal microscopy and image processing, as described in Methods. Integrated fluorescence intensities (arbitrary fluorescence units) were calculated from 4 images/condition, corrected for cell number, and expressed relative to zero-MTX controls. Differences are significant at the p < 0.05 (*) or p < 0.005 (**) level.

(C) Methotrexate dose-ranging experiments to examine induction of PpIX in normal human keratinocytes, SCC13 cells, and HEK1 cells. Experiments in which the confocal image-processing method was used to determine relative PpIX concentration per cell were performed after 72 h incubation in MTX (concentrations shown on the x-axis as a logarithmic scale). Each data point is the mean ± SEM of 3 or more independent experiments (actual number in parentheses), with at least 3 confocal fields per experiment. All PpIX values were normalized to the zero-MTX NHEK control, which was arbitrarily set at 1.0.

(D) Methotrexate preconditioning selectively increases the expression of coproporphyrinogen oxidase (CPO) in carcinoma cells, while not affecting ferrochelatase (FC). Cells were incubated for 72 h in the presence of the following concentrations of MTX: Lane 1, 0 mg/L; Lane 2, 0.001 mg/L; Lane 3, 0.01 mg/L; Lane 4, 0.1 mg/L; Lane 5, 1.0 mg/L. Cells were then harvested and the lysates analyzed on Western blots using antisera specific to CPO, FC, or GAPDH (with bands detected at the expected sizes of ~38 kD, ~40 kD, and ~37 k, respectively). Graphs show the relative changes in band intensity at each MTX concentration, relative to the no-MTX control for each given cell type, as measured by gel densitometry.