Hyperresistance of leukemia cells to photodynamic inactivation after long-term exposure to hemin

Merocyanine 540 (MC540)-mediated photodynamic action is a novel approach for purging tumor cells from autologous remission bone marrow explants. The purpose of this study was to evaluate the effects of hemin (ferriprotoporphyrin IX), a potential source of pro-oxidant iron in bone marrow, on in vitro photodynamic inactivation of leukemia cells. Murine L1210 cells exhibited a progressive loss of clonogenicity when irradiated with broad-band visible light in the presence of MC540. Hemin had strikingly different effects on photokilling, depending on its contact time with cells, eliciting a sizable decrease in resistance after short-term (30-min) contact but a marked increase in resistance after long-term (24-h) contact. Similar trends were observed when cells were challenged with glucose/glucose oxidase, indicating that the responses apply to more than one type of oxidative stress. Immunoblot analyses revealed that the levels of inducible heme oxygenase (HO-1) and ferritin heavy (H) chain were substantially elevated 24 h after hemin addition. HO-1 increased relatively rapidly and maximized within 4 h after adding hemin, whereas H-ferritin increased more slowly in parallel with the development of hyperresistance, maximizing after 24-36 h. Desferrioxamine, an avid iron chelator, had no effect on HO-1 induction but inhibited both ferritin induction and the increase in cell resistance, suggesting that HO-mediated release of iron from hemin was necessary for triggering these responses. Spleen apoferritin was taken up by L1210 cells and strongly inhibited photokilling, further implicating ferritin involvement in hyperresistance. Photokilling was accompanied by free radical-mediated lipid peroxidation (thiobarbituric acid reactivity), which could be suppressed substantially by 24-h hemin preincubation. A plausible explanation for the long-term effects of hemin is that excess H-ferritin generated as a result of iron-regulatory protein deactivation sequesters toxic iron, which might otherwise catalyze damaging lipid peroxidation. Chronic oxidative release of hemin from bone marrow erythroid cells could compromise the efficacy of photopurging by making tumor cells more tolerant to photooxidative insult.