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• W2010050000 abstract "The level of endogenous photosensitiser, protoporphyrin IX (PPIX), can be enhanced in the cells by 5-aminolevulinic acid (ALA). We investigated the effect of critical parameters such as growth state of the cells and availability of intracellular iron in modulating the level of PPIX, in human primary cultured skin fibroblasts (FEK4) maintained either in exponentially growing or growth-arrested phase, following treatment with ALA. The addition of ALA to exponentially growing cells increased the level of PPIX 6-fold relative to control cells; however, in growth-arrested cells the same treatment increased the level of PPIX up to 34-fold. The simultaneous addition of the hydrophilic iron-chelator Desferal with ALA, boosted the level of PPIX up to 47-fold in growing cells and up to 42-fold in growth-arrested cells, suggesting that iron is limiting under the latter conditions. The strict dependence of PPIX enhancement on free available iron levels was examined by the level of activation of iron regulatory protein in band shift assays. This analysis revealed that the basal level of iron regulatory protein in growth-arrested cells was 6-fold higher than in growing cells, reflecting the influence of the free available iron pool in exponentially growing cells. Interestingly, the same ratio was found between the basal level concentration of PPIX in growing and growth-arrested cells. We propose that iron regulatory protein activation could serve as a marker for developing photodynamic therapy protocols because it identifies cells and tissues with a propensity to accumulate PPIX and it is therefore likely to predict the effectiveness of such therapies. The level of endogenous photosensitiser, protoporphyrin IX (PPIX), can be enhanced in the cells by 5-aminolevulinic acid (ALA). We investigated the effect of critical parameters such as growth state of the cells and availability of intracellular iron in modulating the level of PPIX, in human primary cultured skin fibroblasts (FEK4) maintained either in exponentially growing or growth-arrested phase, following treatment with ALA. The addition of ALA to exponentially growing cells increased the level of PPIX 6-fold relative to control cells; however, in growth-arrested cells the same treatment increased the level of PPIX up to 34-fold. The simultaneous addition of the hydrophilic iron-chelator Desferal with ALA, boosted the level of PPIX up to 47-fold in growing cells and up to 42-fold in growth-arrested cells, suggesting that iron is limiting under the latter conditions. The strict dependence of PPIX enhancement on free available iron levels was examined by the level of activation of iron regulatory protein in band shift assays. This analysis revealed that the basal level of iron regulatory protein in growth-arrested cells was 6-fold higher than in growing cells, reflecting the influence of the free available iron pool in exponentially growing cells. Interestingly, the same ratio was found between the basal level concentration of PPIX in growing and growth-arrested cells. We propose that iron regulatory protein activation could serve as a marker for developing photodynamic therapy protocols because it identifies cells and tissues with a propensity to accumulate PPIX and it is therefore likely to predict the effectiveness of such therapies. 5-aminolevulinic acid iron responsive element iron regulatory protein 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide photodynamic therapy protoporphyrin IX transferrin receptor Protoporphyrin IX (PPIX), an endogenous photosensitiser, is the direct precursor of heme in mammalian cells. This natural photodynamic agent is the crucial chromophore involved in the photosensitization driven by 5-aminolevulinic acid (ALA) supplementation. The addition of exogenous ALA to cells bypasses the negative feedback control of heme biosynthesis, leading to intracellular accumulation of photosensitizing concentrations of PPIX. The resulting photosensitization provides a basis for using ALA-induced PPIX for photodynamic therapy (PDT) of cancer (Kennedy et al., 1990Kennedy J.C. Pottier R.H. Pross D.C. Photodynamic therapy with endogenous protoporphyrin IX. Basic principles and present clinical experience.J Photobiol Photochem B Biol. 1990; 6: 143-148Crossref PubMed Scopus (1396) Google Scholar;Wolf and Kerl, 1991Wolf P. Kerl H. Photodynamic therapy in patients with xeroderma pigmentosum.Lancet. 1991; 337: 1613-1614Abstract PubMed Scopus (41) Google Scholar;Peng et al., 1992Peng Q.J. Moan T. Warloe J. Nesland M. Rimington C. Distribution and photosensitizing efficiency of porphyrins induced by application of exogenous 5-aminolevulinic acid in mice bearing mammary carcinoma.Int J Cancer. 1992; 52: 433-443Crossref PubMed Scopus (144) Google Scholar). RecentlyHua et al., 1995Hua Z.S. Gibson S.L. Foster T.H. Hilf R. Effectiveness of δ-aminolevulinic acid-induced protoporphyrin as a photosensitiser for photodynamic therapy.In Vivo. Cancer Res. 1995; 55: 1723-1731PubMed Google Scholar have investigated the mechanism(s) of ALA-based PDT in order to evaluate its relative effectiveness in controlling tumor growth in vivo. They have demonstrated that PPIX is the predominant porphyrin species formed in tumors after ALA administration. Although an understanding of heme biosynthesis has helped to advance the field of ALA/PDT, the mechanism of the preferential accumulation of PPIX in tumor cells after ALA treatment is still unknown. In some studies, a correlation between cell proliferation rates and PPIX synthesis has been suggested (Rebeiz et al., 1992Rebeiz N. Rebeiz C.C. Arkins S. Kelley K.W. Rebeiz C.A. Photodestruction of tumor cells by induction of endogenous accumulation of protoporphyrin IX. Ehancement by 1,10-phenantroline.Photochem Photobiol. 1992; 55: 431-435Crossref PubMed Scopus (68) Google Scholar;Malik et al., 1989Malik Z. Ehrenberg B. Faraggi A. Inactivation of erythrocytic, lymphocytic and myelocytic leukemic cells by photoexcitation of endogenous porphyrins.J Photochem Photobiol B. 1989; 4: 195-205Crossref PubMed Scopus (72) Google Scholar), but these studies lack an explicitly stated proliferation rate. The accumulation of PPIX in the cells is not only influenced by the ratio of ALA conversion to PPIX, but is also related to the ferrochelatase-mediated insertion of ferrous iron into the porphyrin macrocycle to produce the heme molecule. It has been shown that differences in available iron in cells may influence the patterns of PPIX accumulation in vivo (Licznerski et al., 1993Licznerski B. Shanler S.D. Paszkiewicz G. Whitaker J.E. Wan W. Oseroff A.R. Effect of available iron on the accumulation of protoporphyrin IX, an endogenously synthesized photosensitiser produced from exogenous δ-aminolevulinic acid.Proc Annu Meet Am Assoc Cancer Res. 1993; 34: 363Google Scholar); however, the selective accumulation of PPIX in some malignant cells is attributed to their low ferrochelatase activity (Schoenfeld et al., 1988Schoenfeld N.S. Epstein O. Lahav M. Mamet R. Shaklai M. Atsmon A. The heme biosynthetic pathway in lymphocytes of patients with malignant lymphoproliferative disorders.Can Lets. 1988; 43: 43-48Abstract Full Text PDF PubMed Scopus (84) Google Scholar;El-Sharabasy et al., 1992El-Sharabasy M.M.H. El-Waseef A.M. Hafez M.M. Salim S.A. Porphyrin metabolism in some malignant diseases.Br J Cancer. 1992; 65: 409-412Crossref PubMed Scopus (146) Google Scholar). In some ALA/PDT studies, iron-chelators have been used to enhance the PPIX level by decreasing the amount of available iron in the cells (Licznerski et al., 1993Licznerski B. Shanler S.D. Paszkiewicz G. Whitaker J.E. Wan W. Oseroff A.R. Effect of available iron on the accumulation of protoporphyrin IX, an endogenously synthesized photosensitiser produced from exogenous δ-aminolevulinic acid.Proc Annu Meet Am Assoc Cancer Res. 1993; 34: 363Google Scholar;Hanania and Malik, 1992Hanania J. Malik Z. The effect of EDTA and serum on endogenous porphyrin accumulation and photodynamic sensitization of human K562 leukemic cells.Can Lets. 1992; 65: 127-131Abstract Full Text PDF PubMed Scopus (108) Google Scholar;He et al., 1993He D. Sassa S. Lim H.W. Effect of UVA and blue light on porphyrin biosynthesis in epidermal cells.Photochem Photobiol. 1993; 57: 825-829Crossref PubMed Scopus (36) Google Scholar;Iinuma et al., 1994Iinuma S. Farshi S.S. Ortel B. Hasan T. A mechanistic study of cellular photodestruction with 5-aminolevulinic acid-induced porphyrin.Br J Cancer. 1994; 70: 21-28Crossref PubMed Scopus (219) Google Scholar;Berg et al., 1996Berg K. Anholt H. Bech Ø Moan J. Influence of iron chelators on the accumulation of protoporphyrin IX in 5-amino-levulinic acid-treated cells.Br J Cancer. 1996; 74: 688-697Crossref PubMed Scopus (125) Google Scholar). A clear model does not emerge from these studies, probably because of the difficulty in comparing the results obtained from tumors of diverse origin, where the rate of PPIX accumulation is dictated by a varying combination of specific cellular and tissue characteristics. There is a strong requirement to find cell and tissue markers that will indicate which tumors are suitable for ALA/PDT, because the effectiveness of ALA in inducing PPIX in the cells is cell-, tissue- and organ-specific (Kennedy et al., 1990Kennedy J.C. Pottier R.H. Pross D.C. Photodynamic therapy with endogenous protoporphyrin IX. Basic principles and present clinical experience.J Photobiol Photochem B Biol. 1990; 6: 143-148Crossref PubMed Scopus (1396) Google Scholar). Recently,Rittenhouse-Diakun et al., 1995Rittenhouse-Diakun K. van Leengoed H. Morgan J. Hryhorenko E. Paszkiewicz G. Whitaker J.E. Oseroff A.R. The role of transferrin receptor (CD71) in photodynamic therapy of activated and malignant lymphocytes using the heme precursor δ-aminolevulinic acid (ALA).Photochem Photobiol. 1995; 61: 523-528Crossref PubMed Scopus (123) Google Scholar have used the transferrin receptors (TfR, also designated CD71) of the cells as a marker for the intracellular level of iron in ALA/PDT. They have shown that some activated normal and malignant lymphocytes that have increased CD71 expression, can be identified as cells highly susceptible to ALA/PDT and therefore could serve as a PDT target. This marker may have limited usefulness, however, because in some malignant cell lines the presence of elevated TfR expression is unrelated to internal iron stores (Neckers, 1991Neckers L.M. Regulation of transferrin receptor expression and control of cell growth.Pathobiol. 1991; 59: 11-18Crossref PubMed Scopus (46) Google Scholar). Direct measurement of intracellular levels of free iron is extremely difficult. The protein central to iron homeostasis in the cell is the iron regulatory protein (IRP), which when activated in response to low iron levels binds to iron responsive elements (IRE) of ferritin and TfR mRNA. This results in either an inhibition of ferritin synthesis or an increase in the stability of the TfR mRNA, both of which will lead to increased levels of free intracellular iron. An extremely sensitive estimate of free iron levels can be made using IRP/IRE bandshift assays. In this study, we examined the accumulation of PPIX in the primary fibroblast FEK4 cell line maintained in both a growing (full serum) and a growth-arrested (several days in low serum) state. The correlation between the enhancement of PPIX levels and the level of the free iron pool in the cells after various treatments was monitored by the level of activation of IRP in bandshift assays. All biochemicals were from Sigma (Poole, U.K.) except where indicated. The normal human skin fibroblast cell line (FEK4) was cultured in Earle’s modified minimal essential medium (EMEM; Life Technologies, Paisley, Scotland) supplemented with 15% fetal calf serum (Seromed, Germany), L-glutamine (Life Technologies), sodium bicarbonate (Life Technologies), penicillin, and streptomycin (Life Technologies). For growing-phase experiments, cells were grown to 80% confluency in 15 cm dishes for 3 d. At day 3, each dish contained ≈1 × 106 fibroblasts. For experiments with growth-arrested cells, cells were first cultured as described above and then the 15% fetal calf serum conditioned media was replaced by fresh EMEM containing 0.5% fetal calf serum and incubated for 10 more days. The cells were counted each day of incubation either in full serum for 3 d (15% fetal calf serum) or in low serum for 10 d (0.5% fetal calf serum). The results (not shown) revealed that in full serum conditions, the number of cells doubled each 22 h until day 3 (80% confluency) and, when incubated until day 4 (100% confluency), cells stopped dividing. After changing the full serum media at day 3 for media containing 0.5% fetal calf serum, cells stopped dividing and the number of cells stayed constant for the next 10 d. For this study we refer to cells incubated for 3 d in full serum media as growing cells and as growth-arrested whenever they were incubated for 10 d in 0.5% fetal calf serum. Fibroblasts were passaged by trypsinization once a week and used for experiments between passages 10 and 14. Monolayers of cultured fibroblasts in growing (day 3, 80% confluence) or growth-arrested phase (day 10 in 0.5% fetal calf serum EMEM) were treated for 18 h in their respective conditioned media supplemented with ALA alone or combined with Desferal (Ciba-Geigy, Basel, Switzerland). The concentration of 100 μM was chosen for ALA and Desferal (see Results). After 18 h incubation in the dark with ALA or ALA plus Desferal in conditioned media, porphyrins were extracted out of cells according toShoenfeld et al., 1994Shoenfeld N. Mamet R. Nordenberg Y. Shafran M. Babushkin T. Malik Z. Protoporphyrin biosynthesis in melanoma B16 cells stimulated by 5-aminolevulininc acid and chemical inducers: characterization of photodynamic inactivation.Int J Cancer. 1994; 56: 106-112Crossref Scopus (64) Google Scholar. The porphyrin extracts were then diluted 1:30 in 1.5N HCl and the fluorescence was measured using a Perkin-Elmer LS5 luminescence spectrometer (404 nm excitation and detection at 604 nm). Concentration of porphyrins was determined using commercial PPIX as the standard and then normalized for cell number. The level of PPIX was also measured in the conditioned media of the growing and growth-arrested cells after addition of ALA alone or combined with chelators according toFukuda et al., 1993Fukuda H. Battle A.M.C. Riley P.A. Kinetics of porphyrin accumulation in cultured epithelial cells exposed to ALA.Int J Biochem. 1993; 25: 1407-1410Crossref PubMed Scopus (46) Google Scholar. The results (not shown) revealed minor traces of PPIX (less than 2% of accumulated PPIX in the cells) in media from growing cells and no traces in media from growth-arrested cells. This is probably because ALA/chelator treatments were always performed in conditioned media rather than fresh serum-containing media. Nevertheless this amount is included in the values given for PPIX accumulation in growing cells. In order to verify that iron-chelation prior to ALA administration increases the level of PPIX, cells in the growing state (day 3, 80% confluent) were pretreated with Desferal prior to ALA treatment: Cells were first incubated for 6 or 15 h with 100 μM Desferal and then washed twice with phosphate-buffered saline (PBS, Oxoid, Basingstoke, U.K.). After addition of conditioned media containing 100 μ M ALA, the cells were incubated for 18 h in the dark and porphyrins were extracted out of cells. The fluorescence was measured by a spectrofluorimeter. To test if the addition of free iron to the growth-arrested cells converts the accumulated PPIX to heme and therefore decreases the level of PPIX in these cells, we performed the following experiment: Cells growth-arrested for 10 d were pretreated for 6 or 15 h with 1, 10, and 50 μM Fe-citrate. For Fe-citrate treatments, a fresh 1 mM stock solution of Fe-citrate was prepared each time by equimolar mixing of 1 mM Na3 citrate and 1 mM FeCl3 solutions. After several rinses with PBS, the cells were incubated for 18 h under dark conditions in conditioned media supplemented with ALA (100 μM). The porphyrins were then extracted out of cells and their fluorescence was measured by a spectrofluorimeter. In order to follow changes in the basal level of porphyrins in growing and growth-arrested cells, the following time course experiment was designed: The Porphyrins of cells were either extracted in the growing state (in 15% fetal calf serum EMEM) at day 3 (G3, 80% confluency) or every 4 d in the growth-arrested state (in 0.5% fetal calf serum EMEM) till day 10 (A2, A6, A10). The level of porphyrins was determined by a spectrofluorimeter. In order to verify that differences in available iron under the above conditions are responsible for the variation of PPIX accumulation, we employed the RNA-bandshift assay in which the level of iron is monitored by the formation of complexes between IRP and the IRE of ferritin mRNA. For the time-course experiments, the cytoplasmic extracts were prepared at 4°C either from growing cells in day 3 (G3, 80% confluency), or every 4 d from growth-arrested cells (A2, A6, and A10) according toMüllner et al., 1989Müllner E.W. Neupert B. Kühn L.C. A specific messenger-RNA binding-factor regulates the iron-dependent stability of cytoplasmic transferrin receptor messenger-RNA.Cell. 1989; 58: 373-382Abstract Full Text PDF PubMed Scopus (402) Google Scholar. The 32P-labeled RNA was transcribed in vitro from 1 μg of linearised (BamHI-cleaved) plasmid pGem-3Zf(+), containing the wild-type IRE (clone 42,Henderson et al., 1994Henderson B.R. Menotti E. Bonnard C. Kühn L.C. Optimal sequence and structures of iron-responsive elements, selection of RNA stem-loops with high affinity for iron regulatory factor.J Biol Chem. 1994; 268: 27327-27334Abstract Full Text PDF Google Scholar) from the 5′-untranslated region of human ferritin heavy chain mRNA, as described previously (Henderson et al., 1994Henderson B.R. Menotti E. Bonnard C. Kühn L.C. Optimal sequence and structures of iron-responsive elements, selection of RNA stem-loops with high affinity for iron regulatory factor.J Biol Chem. 1994; 268: 27327-27334Abstract Full Text PDF Google Scholar). RNA-protein complexes were analyzed according toMüllner et al., 1989Müllner E.W. Neupert B. Kühn L.C. A specific messenger-RNA binding-factor regulates the iron-dependent stability of cytoplasmic transferrin receptor messenger-RNA.Cell. 1989; 58: 373-382Abstract Full Text PDF PubMed Scopus (402) Google Scholar. The results were scanned with a PhosphorImager and quantitated with software Image Quant 3.3 (Molecular Dynamics, Sunyvale, CA). A Uvasun lamp (Mutzhas, Munich, Germany) was used as a source of broad spectrum UVA irradiation at fluences of 10, 20, and 50 kJ per m2, which were measured using an IL1700 radiometer (International Light, Newbury, MA). Irradiation was done at 25°C. Prior to irradiation, the media was removed and retained and the fibroblasts were covered with Ca2+/Mg2+ (0.01% each)-enriched PBS as described previously (Keyse and Tyrrell, 1989Keyse S.M. Tyrrell R.M. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide and sodium arsenite.Proc Natl Sci USA. 1989; 86: 99-103Crossref PubMed Scopus (1108) Google Scholar). Cultures of growing and growth-arrested FEK4 cells in 35 mm plates were first treated with ALA (± iron-chelator for 18 h, under dark conditions), and then irradiated with UVA at the indicated doses. The dark controls were prepared under the same conditions, except that they were not irradiated. After irradiation, the original media was added back to the fibroblasts and the cells were incubated from 4 to 28 h. At the end of incubation, the media was removed and the cells were covered with 1 ml serum-free media containing 100 μl of 5 mg MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) per ml. After 3 h, the MTT solution was removed and 1 ml of dimethylsulfoxide was added to the cells. Next, 100 μl of the solution was transferred into a 96 well plate and the plate was read on a ELISA-reader EAR 400 (SLT-Labinstruments, Vienna, Austria) using a 577 nm bandpass filter. The percentage of viable cells was calculated by comparing the absorbency of treated versus nontreated cell wells. In order to confirm the results obtained by MTT, cultures of growing and growth-arrested FEK4 cells in 10 cm plates were first treated for 18 h with ALA ± Desferal and then irradiated with UVA at indicated doses. After trypsinization, cells were diluted at 300–3000 cells per 10 cm plate and kept at 37°C under dark conditions for 14 d. Cells were then stained with crystal-violet in 1% methanol. The fraction of surviving cells was determined relative to the sham-exposed levels, which were normalized to 100%. The plating efficiency of the sham-exposed population was 72% ± 5% under these conditions. Stimulation of porphyrin biosynthesis in growing and growth-arrested cells was initially achieved by incubation of cells for various time (4–36 h) with different concentrations of ALA alone or combined with the iron-chelator Desferal. A comparison was made between two fluorimetric assays of spectrofluorimetry (using commercial PPIX as a calibration marker) and high performance liquid chromatography analysis in order to characterize the porphyrins extracted from the cells. These analyses (not shown) revealed a concentration of 100 μM to be optimal (low toxicity and high efficiency) for both ALA and Desferal. The incubation time of 18 h was chosen to measure the PPIX level, because by 24 h this level was decreasing gradually. High performance liquid chromatography analysis of cells treated with ALA alone or combined with Desferal revealed that the overall porphyrin produced in the treated cells consisted of PPIX and therefore the porphyrin fluorescence in our extracts could be attributed to PPIX (data not shown). Table 1 shows the accumulation (pmoles) of PPIX in 107 growing and growth-arrested cells after treatment with ALA alone, Desferal alone, or their combination for 18 h. The treatment of growing cells with ALA alone increased the level of PPIX only 6-fold over untreated controls, whereas in growth-arrested cells the same treatment boosted the level of PPIX up to 34-fold over controls. The comparison of the basal level of PPIX in untreated growing and growth-arrested controls revealed that this level was 6-fold higher in growth-arrested cells. This basal level was boosted up to 935 pmoles in 107 growth-arrested cells with ALA, whereas in growing cells this level reached only a value of 31. PPIX accumulation is inversely dependent on available intracellular iron, which is required for the conversion of PPIX to heme. Upon ALA treatment, the transient accumulation of PPIX will increase if the rate of conversion of ALA to PPIX is greater than that of PPIX to heme. The latter conversion also involves the ferrochelatase enzyme that adds iron to PPIX to produce the heme molecule. In order to verify the role of intracellular iron on PPIX accumulation in growing and growth-arrested FEK4 cells, we combined the ALA treatment with the iron-chelator Desferal for 18 h. The results (Table 1) showed that simultaneous treatment of growing cells with ALA and Desferal was capable of boosting the level of PPIX up to 47-fold above control values, whereas in growth-arrested cells the same treatment resulted in a moderate increase from 34- (ALA alone) to 42-fold above the corresponding control values. Furthermore the treatment of growing and growth-arrested cells with Desferal, but no exogenous ALA, only slightly (up to 1.3-fold over controls) modulates the level of PPIX over corresponding control values, suggesting that iron starvation alone is not sufficient to boost the level of PPIX in the cells. Because the treatment of growth-arrested cells with ALA/chelator only moderately increased the PPIX level over that seen with ALA alone and the basal level of PPIX was higher in growth-arrested cells compared with growing cells, we conclude either that the ferrochelatase activity is very low in nondividing cells or that, in the growth-arrested state, the iron available in cells for conversion of PPIX to heme is limited.Table IModulation of PPIX levels following treatment of growing and growth-arrested (arrested) cells with ALA +/– DesferalDrugConcentration (μM)Incubation time (h)pmoles PPIX per 107 cells aPPIX values are means ± SEM, n = 8.Control/growing–184.8 ± 1Control/arrested–1827.3 ± 3Desferal/growing100185.7 ± 1Desferal/arrested1001828.4 ± 2ALA/growing1001831.5 ± 2ALA/arrested10018935.2 ± 60ALA + Desferal/growing10018225.8 ± 12ALA + Desferal/arrested100181158.9 ± 105a PPIX values are means ± SEM, n = 8. Open table in a new tab In order to distinguish between the possibilities mentioned above, we attempted to mimic the situation arising in growth-arrested cells, by generating conditions where iron is at a low level in growing cells. For this purpose we incubated the growing FEK4 cells with Desferal for either 6 or 15 h, prior to ALA treatment for 18 h. This was followed by spectrofluorimetric measurement of PPIX levels. The results (Figure 1a) showed that addition of Desferal alone to the cells for 6 or 15 h did not significantly increase the level of PPIX (0.98–1.3-fold over untreated control, respectively). When this treatment was followed by ALA administration for 18 h, however, the level of PPIX increased up to 87-fold over controls. The treatment with ALA alone for 18 h increased this level only six times and simultaneous treatment of growing cells with ALA and Desferal for the same period of time increased this level only up to 47-fold (results consistent with previous observations, Table 1). These results suggested that the level of accumulation of PPIX in cells, following ALA induction, is directly related to the levels of available iron. We then tried the inverse procedure to mimic the situation arising in growing cells by treating the growth-arrested cells with various concentrations of Fe-citrate (1, 10, and 50 μM) for 15 h prior to ALA administration. The results (Figure 1b) showed that the addition of Fe-citrate alone for 15 h slightly modulated the basal level of PPIX in growth-arrested cells, where it was decreased to a 0.5-fold control value. Consistent with previous results (see Table 1), addition of ALA alone increased this level up to 34-fold above control values. When Fe-citrate treatment was followed by ALA treatment, however, the level of accumulated PPIX decreased gradually and in a dose-dependent manner up to 5-fold over nontreated controls. The fact that addition of Fe-citrate decreased the level of PPIX in growth-arrested cells following ALA treatment, suggested that a lack of iron rather than low ferrochelatase activity was responsible for the accumulation of PPIX upon ALA induction in these cells. In order to follow the rate of accumulation of PPIX under full or low serum conditions in FEK4 cells, we extracted the PPIX of the cells either in a growing (under full serum, G3) or every 4 d in a growth-arrested state (low serum condition). This provided a correlation between the modulation of the basal level of PPIX and the growth-state of the cells. The result (Figure 2a) showed that when cells enter the nondividing state (from day 2 of incubation in low serum, A2), they start gradually accumulating PPIX and at day 10 of incubation in low serum (A10), the level is 6-fold higher than in growing cells (G3). The level of free iron can be estimated in the cells by IRP/IRE bandshift assays. This level was followed in FEK4 cells under full serum conditions (growing day 3, G3) and under low serum conditions (arrested day 2, A2; day 6, A6; and day 10, A10) in order to demonstrate the relationship between the modulation of iron levels and the accumulation of the basal levels of PPIX according to different growth states of the cells (see Figure 2a). For this purpose the cytoplasmic extracts of cells in the growing and the growth-arrested states were isolated and complexed with the H-ferritin IRE probe. The results (Figure 2b) showed that the level of RNA-protein complexes increased when cells entered the nondividing state. The value obtained from scanning of the IRP/IRE signal at day 3 of growing (G3, 80% confluence, exponentially growing) was arbitrarily chosen as 1. This value increased up to 1.5-fold at day 2 (A2) and to 3.6 at day 6 (A6) of incubation in low serum. G3 cells incubated for longer periods in low serum conditions showed an increase of up to 6-fold at day 10 (A10). These experiments confirm that the level of available free iron in cells in the exponentially growing state (G3) is much higher than in cells in the nondividing state (A2→A10) and is consistent with the hypothesis that this change in available iron corresponding with growth state of the cells determines the rate of PPIX accumulation in the cells (Figure 2a). Similarly, IRP activity increased in the growing cells pretreated with Desferal as a result of scavenging the intracellular free iron pool (Figure 1c), and consequently led to an increased PPIX accumulation following ALA treatment (Figure 1a). Conversely, IRP activity that decreased in a dose-dependent manner in growth-arrested cells pretreated with increased concentrations of Fe-citrate (Figure 1d), led to a dose-dependent decrease in PPIX levels following ALA treatment (Figure 1b). These results further strengthen the conclusion that the modulation of IRP binding activity, which correlates directly with the level of intracellular iron, will determine the effectiveness of ALA in inducing PPIX in the cells. In order to establish a correlation between PPIX accumulation and photo" @default.
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• W2010050000 title "The Iron Regulatory Protein Can Determine the Effectiveness of 5-Aminolevulinic Acid in Inducing Protoporphyrin IX in Human Primary Skin Fibroblasts" @default.
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• W2010050000 doi "https://doi.org/10.1046/j.1523-1747.1999.00556.x" @default.
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