LIMITED CELL-CYCLE DEPENDENCE OF THE MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF L1210 LEUKEMIA CELLS

L1210 leukemia cells were synchronized by a double thymidine block technique and then characterized with regard to their susceptibility to merocyanine 540 (MC540)-sensitized photoinactivation. Cells harvested 5 (G2/M phase) h after release from the second thymidine block were most susceptible to MC540-sensitized photoinactivation followed, in order of decreasing sensitivity, by cells harvested 2 (S phase) h and by cells harvested 7 (G1 phase) h after release from the second block. The expression of dye-binding sites changed very little during the cell cycle.     

MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF LEUKEMIA CELLS: EFFECTS OF DOSE FRACTIONATION

The differential sensitivity to merocyanine 540 (MC540)-sensitized photoirradiation of leukemia cells, selected solid tumor cells, and normal pluripotent hematopoietic stem cells has been successfully exploited for the extracorporeal purging of simulated autologous remission bone marrow grafts. In this communication, we compare the effects of fractionated vs continuous irradiation upon the MC540-sensitized photoinactivation of L1210 and K562 leukemia cells. Exposure to MC540 (15 micrograms/mL) and fractionated doses of white light inactivated fewer in vitro clonogenic cells than exposure to an equivalent dose of continuous irradiation, provided the irradiation doses were small (8.1-16.2 kJ/m2) and spaced 1-2 h apart. The dye-sensitized photoinactivation of leukemia cells was enhanced when cells were stored at 4 degrees C instead of 37 degrees C between irradiation periods, most likely in part because the cells were unable to repair sublethal photodynamic damages at the lower temperature. These data suggest that cells can recover from sublethal damage inflicted by the plasma membrane-active photosensitizer, MC540.     

MODULATION BY THIOLS OF THE MEROCYANINE 540-SENSITIZED PHOTOLYSIS OF LEUKEMIA CELLS, RED CELLS, AND Herpes simplex VIRUS TYPE 1

This paper reports on the role of endogenous and exogenous thiols in the merocyanine 540 (MC 540)-sensitized photoirradiation of L1210 leukemia cells, human erythrocytes, and human Herpes simplex virus type 1. Several measures taken to decrease the intracellular content of glutathione enhanced the cells' sensitivity to MC 540-sensitized photoirradiation while stimulation of glutathione biosynthesis or supplementation of the extracellular or extraviral thiol content decreased the photosensitivity of cells and viruses. Taken together, these data suggest that endogenous and exogenous thiols can modulate the sensitivity of cells and enveloped viruses to MC 540-sensitized photoirradiation. They also pose new questions as to the mechanism of MC 540-sensitized photolysis.     

REACTION OF SINGLET OXYGEN WITH CHOLESTEROL IN LIPOSOMAL MEMBRANES. EFFECT OF MEMBRANE FLUIDITY ON THE PHOTOOXIDATION OF CHOLESTEROL

Abstract— The photosensitized oxygenation of cholesterol in liposomal membranes results in the formation of the characteristic singlet oxygen product, 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide. The yield of this product was found to be strongly temperature dependent with 6- to 7-fold increase above the transition temperature of the membrane using 1,2-dipalmitoyl- sn -glycero-3-phosphorylcholine or 1.2-dimyristoyl- sn -glycero-3-phosphorylcholine. In contrast, the formation of the radical autoxidation products of cholesterol was not significantly enhanced in these systems by higher temperatures. We conclude from the results that the oxidation of cholesterol by singlet oxygen in the artificial membranes is affected by both membrane fluidity and amount of hematoporphyrin incorporated into the membrane. The ratio of cholesterol to phospholipid which affects the morphology of membranes is a crucial factor in determining the yield of the singlet oxygen product.     

CYTOPROTECTION AGAINST MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF THE Na+,K+-ADENOSINE TRIPHOSPHATASE IN LEUKEMIA CELLS: GLUTATHIONE AND SELENOPEROXIDASE INVOLVEMENT

When irradiated with broad-band visible light in the presence of merocyanine 540 (MC540), murine leukemia L1210 cells grown under selenium-deficient conditions (Se(-) cells) accumulated lipid hydroperoxides and lost viability more rapidly than selenium-satisfied controls (Se(+) cells). These findings suggest that cytoprotection against photoperoxidation and photokilling is mediated at least in part by selenoperoxidase (SePX) action. Similar protection against photoinactivation of an intrinsic membrane enzyme, the Na⁺,K⁺-ATPase, has been observed. Thus, irradiation of MC540-sensitized Se(-) cells resulted in an immediate and progressive inactivation of ouabain-sensitive Na⁺, K⁺-ATPase; by contrast, activity loss in Se(+) cells was preceded by a prominent lag. Enzyme photoinactivation in Se(-) cells was inhibited by ebselen, an SePX mimetic, confirming that SePX(s) is (are) involved in natural protection. Desferrioxamine treatment (iron sequestration/inactivation) resulted in higher hydroperoxide levels and slower Na⁺,K⁺-ATPase inactivation during MC540/light exposure, whereas ferric-8-hydroxyquinoline treatment (iron supplementation) had the opposite effect. Thus, iron appears to play an important role in both of these processes. In contrast, photoinactivation of another intrinsic enzyme in L1210 cells, acetylcholinesterase (AChE), was unaffected by selenium or iron manipulation. On the basis of these findings, we propose that lipid peroxidation plays an important role in the photoinactivation of Na⁺,K⁺-ATPase, but not AChE. This is consistent with the fact that Na⁺, K⁺-ATPase's active site lies within the membrane bilayer, whereas AChE's active site lies outside the bilayer.     

NON-NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m² for the UVASUN lamp, 75 to 390 J/m² for the FS20 lamp and 3.8 to 17.2 J/m² for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.     

TIME-RESOLVED STUDIES OF SINGLET-OXYGEN EMISSION FROM L1210 LEUKEMIA CELLS LABELED WITH 5-(N-HEXADECANOYL)AMINO EOSIN. A COMPARISON WITH A ONE-DIMENSIONAL MODEL OF SINGLET-OXYGEN DIFFUSION AND QUENCHING

Abstract— Time-resolved measurements were made of near-infrared emission from 5-( N -hexadecanoyl)amino-eosinlabeled L1210 leukemia cells following pulsed-laser excitation. The cells were suspended in phosphate-buffered saline made with deuterium oxide solvent. A significant fraction of the emission occuring10–80 μs after the laser pulse was due to singlet oxygen. This singlet-oxygen emission is believed to result from singlet oxygen generated near the cell-membrane surface, where 5-( N -hexadecanoyl)amino eosin is known to concentrate, and then diffusing out into the buffer. The intensity and the kinetics of the experimentally observed singlet-oxygen emission were in excellent agreement with the predictions of a theoretical one-dimensional model of singlet-oxygen diffusion and quenching.
During the10–80 μs time period studied, most of the singlet oxygen was located in the buffer. Thus, the use of water-soluble singlet-oxygen quenchers, such as histidine, provide one means of separating the singlet-oxygen emission quenchers, such as histidine, provide one means of separating the singlet-oxygen emission from other sources of light during this time interval.     

A FLAVOPROTEIN MAY MEDIATE THE BLUE LIGHT-ACTIVATED BINDING OF GUANOSINE 5'-TRIPHOSPHATE TO ISOLATED PLASMA MEMBRANES OF Pisum sativum L.*

Abstract— A blue light photoreceptor has not been identified in higher plants. Most proposals for a blue light-absorbing chromophore lack evidence for a direct connection between the putative chromophdre and a biological effect. Fluorescence data for the plasma membrane from etiolated buds of Pisum sativum L. suggest that we are measuring fluorescence emission of flavin species, and probably not pterin species. Fluorescence data indicate that a putative flavin exists associated with a protein or protein complex in the plasma membrane. Excitation of plasma membranes that were boiled in the presence of 0.1% sodium dodecyl sulfate and treated with blue light yields a fluorescence band with a maximum of approximately 552 nm. This fluorescence emission can be rapidly quenched by the flavin antagonists phenylacetic acid (PAA) and KI. Blue light-enhanced binding of guanosine 5'-[Γ-thio]triphosphate (GTPγS) to a protein in the plasma membrane is strongly inhibited by PAA, KI, and NaN₃, all quenchers of flavin excited states, indicating that a chromophore for this photoreaction may be a flavin associated with a plasma membrane protein. The above evidence is consistent with the participation of a flavin as the chromophore for the light-induced GTP-binding reaction in pea plasma membrane.     

CORRELATION BETWEEN ENDOGENOUS GLUTATHIONE CONTENT AND SENSITIVITY OF CULTURED HUMAN SKIN CELLS TO RADIATION AT DEFINED WAVELENGTHS IN THE SOLAR ULTRAVIOLET RANGE

Abstract— Glutathione depletion of cultured human skin fibroblasts by treatment with buthionine-S,R-sulfoximine (BSO) sensitises them to radiation at a series of defined wavelengths throughout the solar UV range. We now show that there is a close quantitative correlation between cellular glutathione content (as depleted by BSO) and sensitivity to radiation at 365 nm. A weaker correlation is observed when cells are depleted of glutathione using diethylmaleimide. Both fibroblasts and epidermal keratinocytes derived from the same foreskin biopsy are sensitised to radiation at 313 nm by glutathione depletion. However, the keratinocytes are sensitised to a much lesser extent, an observation which agrees quantitatively with the higher residual levels of cellular glutathione remaining after maximum depletion by BSO (approximately 25% for the keratinocytes vs less than 5% for the fibroblasts). At low to intermediate fluence levels, 10 mM cysteamine present during irradiation at 302 nm is able to almost completely reverse the sensitising effects of glutathione depletion suggesting that the endogenous thiol protects against radiation at this wavelength by a free radical scavenging mechanism. At 313 nm, the sensitisation is not reversed by cysteamine suggesting that glutathione plays a more specific role in protection against radiation at longer wavelengths. Xeroderma pigmentosum group A fibroblasts (excision deficient) are also sensitised to radiation at 313 and 365 nm by depletion of glutathione but since the sensitization is less than that observed for the normal strain, we cannot conclude that glutathione protects against a sector of DNA damage susceptible to excision repair. The results provide further evidence that endogenous glutathione is involved in protecting human skin cells against a wide range of solar radiation damage and suggest that while free radical scavenging is involved at the shortest wavelength (302 nm) tested, a more specific role of glutathione is involved in protection against radiation at longer wavelengths.