Image cytometry was applied to study the intracellular localization of autofluorescence and the influence of an oxidative stress on this emission. K562 erythroleukemia cancer cells were analyzed with a microspectrofluorometer, coupled with a Argon laser (Ar+) (363 nm). From each cell, 15 x 15 emission spectra were recorded in the 400-600 nm spectral range to generate a spectral image of autofluorescence. The intracellular locations of the autofluorescence emission and of the specific mitochondrial probe rhodamine 123 (R123) were matched. Under a 363 nm excitation, all spectra from K562 cells show equivalent profiles with a 455 nm maximum emission, near of reduced nicotinamide adenine dinucleotide-(Phosphate) solution (NAD(P)H) (465 nm maximum emission). The spatial distribution of autofluorescence is homogeneous and different from the one of R123. Hydrogen peroxide (H2O2) (200 microM) and menadione (Men) (5 microM) induce a weak spectral change and a decrease in autofluorescence intensity, down to 40% of the initial emission. Doxorubicin (Dox) induces a dose-dependent decrease in autofluorescence emission and a release of intracellular free radicals. When cells were pre-treated 1 h with 1 mM glutathione (GSH), Dox induces a lower free radicals release, no significant variation of autofluorescence intensity and a lower growth inhibitory effect. Images cytometry of autofluorescence suggest that the intracellular NAD(P)H would not be restricted to mitochondrial compartments. The release of free radicals was associated with a decrease in autofluorescence intensity, mainly attributed to NAD(P)H oxidation both inside and outside mitochondria.

1α,25-dihydroxyvitamin D 3 (VD 3) and the EB1089 analog are well known for their roles in the modulation of proliferation and the differentiation of several malignant cells. In addition, VD 3 or EB1089 displayed a high disposal of oxidant features and the ability to cause release of reactive oxygen species (ROS). We attempted to enhance HL60 cell differentiation and to limit ROS generation, by the association of deltanoids with doxorubicin and the antioxidants catalase (CAT), superoxide dismutase (SOD) and N-acetyl cystein (NAC). Differentiation of HL60 cells into monocytic lineage was studied by expression of mRNA, protein CD14 and functional differentiation by the nitroblue tetrazolium assay. The 2',7'-dichlorodihydrofluorescein diacetate (H 2-DCFDA) dye allowed to evaluate in situ ROS generation. When associated with 0.1 nM EB1089, 15 nM doxorubicin induced an increase of differentiated cell percentage from 29% to 87% and did not affect VD 3-treated cells. The association with doxorubicin also induced a significant increase of ROS release (p<0.05) versus VD 3 and EB1089-treated cells. These results correspond to additivity of individual effects of doxorubicin and deltanoids. Antioxidant agents (10 nM NAC, 50 U/ml SOD or 2000 U/ml CAT) were associated with 10 nM VD 3 or 1 nM EB1089 for 72 h. Compared to VD 3 and EB1089 treatments, associations with antioxidants induced a slight increase of differentiated cells and a significant increase of CD14 mRNA. The highest differentiation effect occurred in the case of the EB1089-NAC association. Antioxidants induced a decrease (p<0.05) in ROS release generated by VD 3 or EB1089 near the level of untreated cells. Thus, antioxidant agents demonstrated a protective effect against VD 3 and EB1089 oxidative cytotoxicity and an enhancement of the monocyte differentiation. Combinations of antioxidants with deltanoids could dissociate the oxidative stress and differentiation.

We attempted to characterize the cellular autofluorescence phenomenon of living HL-60 cells and to appraise its modifications under oxidative stress conditions induced by 1α,25(OH) 2 D 3 (VD 3 ) and its analog EB1089. Autofluorescence emission spectra of human promyelocytic HL-60 leukemic cells were monitored using laser scanning confocal microspectrofluorometry under UV excitation. Evaluation of reactive oxygen species (ROS) release was performed using the 2',7'-dichlorodihydrofluorescein diacetate (H 2 -DCFDA) staining and fluorescence emission measurement. VD 3 (1, 10, 100 nM) or EB1089 (0.1, 1 and 10 nM) induces a decrease in autofluorescence emission intensity that can be attributed to the oxidation of the coenzyme nicotinamide adenine dinucleotide (phosphate) NAD(P)H into NAD(P) + . A dose-dependent increase (p<0.05) in ROS release is observed in VD 3 -and EB1089-treated cells. As compared with VD 3 -or EB1089-treated cells, doxorubicin-VD 3 or doxorubicin-EB1089 treatments strongly decrease the autofluorescence intensity and induce a higher release of ROS (p<0.05). The association of antioxidants (N-acetyl cysteine, superoxide dismutase, catalase) with VD 3 or EB1089 induce a more limited autofluorescence decrease and a weaker ROS generation, as compared with VD 3 and EB1089 treated cells. In conclusion, the free radicals release, generated by VD 3 and EB1089, was associated with the decrease in autofluorescence emission and can be modulated by doxorubicin and antioxidants.