L5178Y-R Cells Research Articles

L5178Y sublines: a look back from 40 years. Part 2: Response to ionizing radiation

The aim was to review and summarize the results of 40 years of study concerning the response to ionizing radiation of the pair of L5178Y (LY) sublines, LY-R and LY-S, that differ in sensitivity to various DNA-damaging agents, among them X- and γ-rays. The reviewed data indicate the key importance of DNA damage repair and fixation for the ultimate fate of the irradiated LY cell. The cause of slow double-strand break (DSB) repair in LY-S cells is not identified, but a defect in non-homologous end-joining (NHEJ) would explain most features of the cellular response of LY-S cells to irradiation, as compared with repair-competent LY-R cells. The most prominent features are the very high radiosensitivity of G1 cells, extensive poly(ADP-ribose)-dependent damage fixation, long G2 arrest, considerable chromosomal damage seen as premature chromatin condensation (PCC) fragments and aberrations in metaphase cells. The main cause of radiosensitivity difference between LY sublines is in DNA repair/damage fixation ability. At the level of damage corresponding to a comparable lethal effect, the type of death differs between LY sublines; LY-S cells die in considerably greater proportion by apoptosis than LY-R cells, whereas the latter die in greater proportion by necrosis. This observation is consistent with differential expression of proteins that are pro- or anti-apoptotic. The prominent role of poly(ADP-ribosylation) in the response of LY-S cells apparently is connected with damage fixation, but is in contrast to other cell lines hypersensitive to X- or γ-radiation with DSB repair defects.

L5178Y sublines: a look back from 40 years. Part 1: General characteristics

The aim was to review and summarize the results of studies done over the last 40 years concerning the general characteristics and response to ultraviolet C (UV-C) radiation and hydrogen peroxide of the pair of L5178Y (LY) sublines, LY-R and LY-S, that differ in their sensitivity to various DNA damaging agents. (The response of the sublines to ionizing radiation is described in the second part of the paper.) Comparison of subline karyotypes shows a number of differences in their banding patterns. The sublines differ in their ion transport, the ganglioside pattern of plasma membranes, and in the content and turnover rate of poly(adenosine diphosphoribose) polymers. Nuclear matrix proteins show a differential affinity to these polymers. A unique property of the pair of LY sublines is an inverse cross-sensitivity to X-rays and hydrogen peroxide, with cross-sensitivities to hydrogen peroxide and UV-C, as well as to UV-C and a platinum (Pt) complex (cisplatin analogue). Initial DNA damage and repair and various aspects of the cellular response of the sublines were determined in cells damaged with these agents. The higher sensitivity of LY-R cells to hydrogen peroxide, as compared with LY-S cells, is causally related to the higher content of iron ions in these cells and a less efficient anti-oxidant defence system (including a lower catalase activity). Sensitivity of LY-R cells to UV-C radiation and Pt complexes is explained by impaired excision repair (the incision step is missing).

In vitro immunopotentiating properties and tumour cell toxicity induced by Lophophora williamsii (peyote) cactus methanolic extract.

Lophophora williamsii, also known as peyote, is found primarily in dry regions from Central Mexico, including the Mexican States of Nayarit, San Luis Potosí, Zacatecas, Nuevo León, Chihuahua, Coahuila and Tamaulipas, to Texas particularly in regions along Rio Grande. Peyote extracts have been associated with stimulating the central nervous system and regulating blood pressure, sleep, hunger and thirst. However, there is no evidence of any effect of peyote on the immune system or against tumour cell growth. The present study was designed to evaluate the in vitro effects of peyote methanolic extracts on some parameters of mouse and human leukocyte immunocompetence and tumour cell growth. Peyote extract (0.18-18 micro g/mL) activated nitric oxide production by murine macrophages, and stimulated up to 2.4-fold proliferation of murine thymic lymphocytes. In addition, peyote extract induced up to 1.85-, 2.29- and 1.89-fold increases in mRNA signal of IL-1, IL-6 and IL-8 by human leukocytes. Also examined were the effects of peyote extracts on murine lymphoma L5178Y-R and fi broblastoma L929, and human myeloid U937 and mammary gland MCF7 tumour cell growth using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Peyote extracts were toxic for MCF7, L5178Y-R, U937 and L929 (18 mg/mL peyote extract caused 1.3%, 8%, 45% and 60% viability respectively) cell lines.

Labile iron pool correlates with iron content in the nucleus and the formation of oxidative DNA damage in mouse lymphoma L5178Y cell lines.

Labile iron pool (LIP) constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need for iron, its uptake and storage. In this study we investigated oxidative DNA damage in relation to the labile iron pool in a pair of mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S) differing in sensitivity to hydrogen peroxide. The LY-R cells, which are hydrogen peroxide-sensitive, contain 3 times more labile iron than the hydrogen peroxide-resistant LY-S cells. Using the comet assay, we compared total DNA breakage in the studied cell lines treated with hydrogen peroxide (25 microM for 30 min at 4 degrees C). More DNA damage was found in LY-R cells than in LY-S cells. We also compared the levels of DNA lesions sensitive to specific DNA repair enzymes in both cell lines treated with H(2)O(2). The levels of endonuclease III-sensitive sites and Fapy-DNA glycosylase-sensitive sites were found to be higher in LY-R cells than in LY-S cells. Our data suggest that the sensitivity of LY-R cells to H(2)O(2) is partially caused by the higher yield of oxidative DNA damage, as compared to that in LY-S cells. The critical factor appears to be the availability of transition metal ions that take part in the OH radical-generating Fenton reaction (very likely in the form of LIP).

Inhibition of glycolipid shedding rescues recognition of a CD1+ T cell lymphoma by natural killer T (NKT) cells.

Neoplastic transformation of cells is accompanied by an aberration of cell surface glycolipid composition. These tumor-associated, altered glycosphingolipids are often shed into the tumor cell microenvironment and mediate immunosuppressive activity. The nature and form of glycolipids shed by a variety of tumor cell lines and the mechanism(s) of shedding have been well characterized. The murine T cell lymphoma line, L5178Y-R, is known to shed a tumor-associated glycolipid, gangliotriaosylceramide, into the culture medium. We analyzed the effect of glycolipids from L5178Y-R on antigen presentation by murine CD1d1 molecules. CD1d1 molecules present glycolipid antigens to a specialized class of T cells called natural killer T (NKT) cells that mainly express a T cell receptor alpha chain (Valpha14Jalpha281) associated with Vbeta chains of limited diversity. In the current report, we found that L5178Y-R cells express CD1 on their cell surface yet are unable to stimulate CD1d1-specific NKT cells. We hypothesized that the glycolipid(s) shed by L5178Y-R inhibited antigen presentation by CD1d1. Pretreatment of CD1d1(+) cells with conditioned medium from L5178Y-R inhibited CD1-specific stimulation of canonical (Valpha14(+)) but not noncanonical (Valpha5(+)) NKT cells. Exogenous addition of lipids extracted from L5178Y-R cells as well as purified gangliotriaosylceramide mimicked this effect. Inhibition of glycolipid shedding in L5178Y-R cells with d-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol resulted in the rescue of CD1d1 recognition by canonical (but not noncanonical) NKT cells. These results suggest that one means by which certain tumor cells can evade the host's innate antitumor immune response is by shedding glycolipids that inhibit CD1-mediated antigen presentation to NKT cells.

Poly ADP-ribosylation in two L5178Y murine lymphoma sublines differentially sensitive to DNA-damaging agents

Purpose : To characterize the response to X-irradiation of the poly ADP-ribosylation system in two closely related murine lymphoma sublines, L5178Y-R (LY-R) and L5178Y-S (LY-S), with differential sensitivity to various DNA damaging agents (UV-C and ionizing radiation, hydrogen peroxide). Materials and methods : LY cells were X-irradiated (2 Gy). NAD + was determined in cell extracts by high-pressure liquid chromatography. ADP-ribose polymers were purified and analysed by densitometry after polyacrylamide gel electrophoresis. Nuclear matrix proteins were separated by SDS-polyacrylamide gel electrophoresis and processed for ADP-ribose polymer blots to estimate their ability to bind poly(ADP-ribose). Results : In the radiosensitive LY-S cells, the constitutive levels of ADP-ribose polymers were twofold higher than in radioresistant LY-R cells, but unresponsive to a challenge with 2 Gy X-rays. The concentrations of NAD + - the substrate for poly(ADP-ribose) synthesis - were identical in the two cell lines. X-rays (2 Gy) depleted NAD + only in LY-S cells. These cells also produced shorter poly(ADP-ribose) molecules as compared with LY-R cells. Nuclear matrix preparations of LY-S cells exhibited lower poly(ADP-ribose)-binding capacity than those of LY-R cells. Conclusion : The results demonstrate disturbances in the poly ADP-ribosylation response of the radiosensitive LY-S cells and reduced poly(ADP-ribose)-binding affinity of the nuclear matrix of these cells.

DNA lesion complexity and induction of apoptosis by ionizing radiation

Purpose : To determine whether murine lymphoid cell lines can discriminate between high- and low-LET (linear energy transfer) radiation-induced DNA lesions. Materials and methods : Sensitivity to killing by DNA-incorporated 3 H and 125 I decays, accumulated during storage in the gas phase of a liquid nitrogen tank, was determined by clonogenic survival assay. Results : Induction of a lethal event in the STRij-4-2.2, WEHI-22.1, and L5178Y-R cell lines required approximately 30 times more 3 H than 125 I decays. Hence, the same ratio of 3 H to 125 I decays was found irrespective of whether the cell lines contained mutant or wild-type p53 and irrespective of whether they underwent rapid interphase or mitosis-related apoptosis after irradiation. The 18-81 cell line differed in showing a ratio of around 21 and it is argued that this may be a consequence of v-ABL over-expression. The assumption that DNA-incorporated 3 H and 125 I decays are low- and high-LET-like events respectively was confirmed by the similar sensitivity of L5178Y-R and -S cells to killing by 125 I decays in contrast with their difference in sensitivity to 3 H decays. Conclusions : The difference in lethal effectiveness between DNA-incorporated 3 H and 125 I decays can be explained by the hypothesis that simple DSB (double-strand breaks) are non-lethal and that cell killing is attributable to complex DSB. The low-LET radiation-specific sensitization of L5178Y-S cells may reflect defective repair of a DNA lesion class (presumably simple DSB) that is differentially induced by high- and low-LET radiation and is non-lethal to cells with normal repair capacity.

Caffeine-inhibitable control of the radiation-induced G2 arrest in L5178Y-S cells deficient in non-homologous end-joining.

The two L5178Y (LY) sublines bear a heterozygous Tp53 mutation that affects its transactivation function. LY-S (radiation-sensitive) cells are deficient in double strand break (DSB) repair by non-homologous end-joining (NHEJ) and do not express p21WAF1 (Cdkna1) either constitutively or after x-irradiation, in contrast to their radiation-resistant counterpart LY-R cells, which express p21WAF1 constitutively. Radiation-induced G2 arrest in LY-S cells is very long (11 h/Gy) but 2 mM caffeine treatment shortens it, decreases the fraction of G2 cells and increases the fraction of apoptotic cells. The treatment also increases the DNA damage that is estimated with the comet assay 18 h after irradiation with 5 Gy (ca. 23% of the initial value for x-rays and ca. 47% for x-rays plus caffeine). This indicates that either the repair has not been completed or the apoptotic DNA fragmentation has been initiated (or both). The same treatment applied to x-irradiated (5 Gy) LY-R cells (G2 arrest, 4 h/Gy) has no radiosensitising effect, induces no apoptosis and does not alter the amount of DNA damage left unrepaired (ca. 28%). The results are compatible with the assumption that inhibition of the Atm-dependent homologous recombination repair by caffeine, brings differential effects in LY sublines because of the defect of the alternative DNA repair system (NHEJ) in LY-S cells.

Dissociation of mitochondrial depolarization from cytochrome c release during apoptosis induced by photodynamic therapy.

Photodynamic therapy (PDT) with the phthalocyanine photosensitizer Pc 4 induces rapid apoptosis in mouse lymphoma (LY-R) cells, initiating with the release of cytochrome c from mitochondria. It has been proposed that the opening of the mitochondrial membrane permeability transition pores, which results in the dissipation of the mitochondrial membrane potential (Δψm), is essential for the escape of cytochrome c from mitochondria into the cytosol as well as for apoptotic cell death. Therefore, we have assessed the correlation between the loss of Δψm and the release of cytochrome c following PDT. Treatment of LY-R cells with 300 nM Pc 4 and 60, 90 or 120 mJ/cm2of red light resulted in apoptosis of 80–90% of the cells, accompanied by >20-fold elevation in caspase-3-like activity within one h. At all 3 doses of PDT employed here, the majority of the cytochrome c was released from mitochondria at 15 min after irradiation, as determined by an immunohistochemical method. In contrast, the loss of Δψm following PDT, as monitored by the uptake of JC-1 or Rh-123, depended on the PDT dose and the post-treatment time. In spite of the release of cytochrome c at 15 min after each of the 3 doses, a corresponding loss of Δψm was observed only for those cells that received the highest dose of PDT. Virtually all cells that received one of the lower doses of PDT (300 nM Pc 4 plus 60 or 90 mJ/cm2) maintained normal Δψm. Hence, our results support the conclusion that the release of cytochrome c from mitochondria resulting from Pc 4-PDT-induced photodamage is independent of the loss of Δψm. Therefore, it is important to consider a range of doses of this or other apoptotic stimuli in deciphering the relationship of metabolic responses that contribute to apoptosis. © 2001 Cancer Research Campaign http://www.bjcancer.com

Phthalocyanine 4 photodynamic therapy-induced apoptosis of mouse L5178Y-R cells results from a delayed but extensive release of cytochrome c from mitochondria

Photodynamic therapy (PDT) activates the mitochondrial pathway of apoptosis, for which the release of cytochrome c into the cytosol is considered critical. To further elucidate the role of cytochrome c release in PDT-induced apoptosis, we monitored cytochrome c localization immunocytochemically and related it to nuclear apoptosis of the same cells. When mouse L5178Y-R cells were treated with 300 nM phthalocyanine (Pc) 4 and 0–75 mJ/cm 2 red light, cytochrome c release had a dose response similar to that of clonogenic cell killing, with nearly identical threshold doses. Within individual cells, the release of cytochrome c appeared to be an all-or-none phenomenon. Moreover, it was tightly associated with activation of a caspase-3-like protease and changes in nuclear morphology. Thus, in response to Pc 4-PDT, the release of cytochrome c from mitochondria is a key determinant of apoptotic cell death.

The response of L5178Y lymphoma sublines to oxidative stress: antioxidant defence, iron content and nuclear translocation of the p65 subunit of NF-kappaB.

We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radio-resistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-kappaB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.

Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: a study of L5178Y mouse lymphoma cell lines differentially sensitive to H2O2

The redox properties of iron make this metal a key participant in oxygen-mediated toxicity. Accordingly, L5178Y (LY) mouse lymphoma cell lines, which display a unique inverse cross-sensitivity to ionizing radiation (IR) and hydrogen peroxide (H(2)O(2)), are a suitable model for the study of possible differences in the constitutive control of intracellular iron availability. We report here that the level of iron in the cytosolic labile iron pool (LIP), ie, potentially active in the Fenton reaction, is more than 3-fold higher in IR-resistant, H(2)O(2)-sensitive (LY-R) cells than in IR-sensitive, H(2)O(2)-resistant (LY-S) cells. This difference is associated with markedly greater content of ferritin H-subunits (H-Ft) in LY-S than in LY-R cells. Our results show that different expression of H-Ft in LY cells is a consequence of an up-regulation of H-Ft mRNA in the LY-S mutant cell line. In contrast, posttranscriptional control of iron metabolism mediated by iron-responsive element-iron regulatory proteins (IRPs) interaction is similar in the 2 cell lines, although IRP1 protein levels in iron-rich LY-R cells are twice those in iron-deficient LY-S cells. In showing that LY cell lines exhibit 2 different patterns of intracellular iron regulation, our results highlight both the role of high LIP in the establishment of pro-oxidant status in mammalian cells and the antioxidant role of ferritin. (Blood. 2000;95:2960-2966)

Differential induction of apoptosis in x-irradiated L5178Y sublines bearing p53 mutation.

We examined apoptosis and expression of p53, E2F-1, bax, bclx(L) and bc12 proteins in two L5178Y (LY) murine lymphoma sublines, LY-R and LY-S, which differ in radiosensitivity and double-strand break (DSB) repair. Both sublines are heterozygous for a p53 mutation in codon 170 that precludes the transactivation function. Accordingly, there is no G1/S arrest after irradiation. We found that there is no change in expression of E2F-1, bax, bclx(L) or bc12 proteins in both LY sublines after x-irradiation. LY-R cells do not constitutively express bc12, whereas both sublines show high bax content. Radiation induces delayed apoptosis to a greater extent in LY-S than in LY-R cells. The apoptosis can be seen 24 h after irradiation (2 Gy) of LY-S cells, with a maximum at 48 h. LY-R cells need 5 Gy and 72 h post-irradiation incubation to show marked apoptosis (identified by the TUNEL method). The reported observations support the assumption that differential radiosensitivity of LY sublines is associated with the induction of apoptosis that is not related to transactivation by p53 and is primarily related to differential DNA repair ability.

Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy.

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH3)2-(CH2)3N(CH3)2 (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.

Promotion of photodynamic therapy-induced apoptosis by stress kinases.

Photodynamic therapy (PDT), a cancer treatment that employs a photosensitizer and visible light, induces apoptosis in murine LY-R leukemic lymphoblasts and in CHO cells, but the rate and extent of apoptosis are much greater in LY-R cells. Three MAPK family members, ERK1/ERK2, SAPK/JNK, and p38/HOG, are important intermediates in signal transduction pathways. To ascertain whether activation of one or more MAPKs could mediate PDT-induced apoptosis, Western blot analysis has been performed on the proteins of LY-R and CHO cells at various times following lethal (90 - 99% cell kill) doses of PDT photosensitized by the phthalocyanine Pc 4. The blots were probed with antibodies to each of the proteins as well as antibodies specific for the activated (phosphorylated) forms of each kinase. Of the three MAPK types, only the p46 and p54 SAPK/JNKs were found to be activated by PDT in LY-R cells, with a maximum approximately threefold increase in the content of the phosphorylated forms reached in 30 - 60 min. An even larger relative activation was observed in CHO cells. PDT did not affect ERK and p38/HOG activation in LY-R cells. In the case of CHO cells, however, ERK2 was slightly activated at 5 min post-PDT, then declined, and p38/HOG was strongly activated from 5 to 60 min post-PDT. A specific inhibitor (PD98059) of MEK1, the kinase that activates ERK, had little or no effect on PDT-induced apoptosis in either LY-R or CHO cells. In contrast, a specific inhibitor of p38/HOG (SB202190) blocked PDT-induced apoptosis in LY-R cells with a lesser effect in CHO cells. The results suggest that both the SAPK and p38/HOG cascades can be stimulated by PDT and that the latter participates in both rapid and slow PDT-induced apoptosis. Furthermore, the high level of constitutively active p38/HOG in LY-R cells may poise those cells for rapid activation of apoptosis following PDT.

Nuclear translocation of the p65 subunit of NF-kappaB in L5178Y sublines differing in antioxidant defense.

We examined the induction of nuclear translocation of the p65 subunit of NF-kappaB in L5178Y (LY) cells. We used two LY sublines which are inversely cross-sensitive to hydrogen peroxide and x-rays: LY-R cells are radioresistant and oxidant-sensitive, whereas LY-S cells are radiosensitive and oxidant-resistant. Hydrogen peroxide, phorbol ester and x-rays caused a marked translocation of p65-NF-kappaB in LY-R cells and a weak translocation in LY-S cells. By manipulating the antioxidant defense status, we obtained an alteration in the p65-NF-kappaB translocation induction in LY-R cells. A similar effect was achieved with lovastatin pretreatment (25 microM, 24 h, 37 degrees C). The response of LY-S cells under all these conditions was considerably weaker. We conclude that differential nuclear translocation of p65-NF-kappaB in LY sublines is not related to the lethal effect of the activating, damaging agent; rather it is due to the more efficient antioxidant defense in LY-S than in LY-R cells.

Chelating of iron and copper alters properties of DNA in L5178Y cells, as revealed by the comet assay

We have previously found different proportions of iron and copper in nuclei of two sublines of murine lymphoma L5178Y (LY) and proposed a model of chromatin organization with these metal ions at the DNA attachment sites. We now examine the effect of chelators, desferal (DFO, iron-specific) and neocupreine (NEO, copper-specific) on DNA of LY-R and LY-S cells, using the comet and micronuclei frequency tests. There is less copper and more iron in LY-R nuclei than in LY-S nuclei. Accordingly, the effect of NEO is more marked in LY-R than in LY-S cells and in both sublines it is expressed as enhanced tail moment (measure of DNA damage in the comet assay) and increased micronuclei frequency. On the contrary, the effect of DFO on the tail moment is less pronounced in LY-R than in LY-S cells. With increasing DFO concentrations, there is a gradual decrease in the tail moment values below the control level in LY-S cells. In LY-R cells the tail moment values initially increase, then gradually decrease, eventually falling below the control level. This points to a dramatic conformational change that masks the effect of DNA discontinuities. The presence of the latter is indicated by the increase in micronuclei frequency. These results support the postulated differential role of iron and copper ions in maintaining the higher order DNA structure in LY sublines.

Photodynamic Therapy-Induced Apoptosis in Lymphoma Cells: Translocation of Cytochrome c Causes Inhibition of Respiration as Well as Caspase Activation

L5178Y-R mouse lymphoma (LY-R) cells undergo rapid apoptosis when treated with photodynamic therapy (PDT) sensitized with the silicon phthalocyanine Pc 4. In this study we show that cytochrome c is released into the cytosol within 10 min of an LD99.9dose of PDT. Cellular respiration is inhibited by 42% at 15 min, and 60% at 30 min after PDT treatment, and caspase 3-like protease activity is elevated by 15 min post-PDT. In digitonin-permeabilized cells addition of cytochrome c to the respiration buffer reverses PDT-induced inhibition of state 3 respiration via Complex I by 40–60%, andviaComplex III by 50–90%. In contrast, extramitochondrial cytochrome c does not stimulate respiration in permeabilized control cells, and catalyzes only a low rate of oxygen consumptionviaelectron transfer to cytochrome b5 on the outer mitochondrial membrane. These results demonstrate that PDT-induced inhibition of respiration is primarily due to leakage of cytochrome c into the cytosol rather than to damage to the major enzyme complexes of the electron transport chain. Whether or not inhibition of respiration influences the time course or extent of Pc 4-PDT-induced apoptosis in LY-R cells is not clear at the present time.

Differential inhibitory effect of OK-1035 on DNA repair in L5178Y murine lymphoma sublines with functional or defective repair of double strand breaks

Radiosensitive L5178Y-S (LY-S) subline and its parental, more radioresistant L5178Y-R (LY-R) subline differ in DNA double strand break (DSB) rejoining. In this work we examined by comet assay the repair of X-ray-induced DNA damage in LY cells treated with OK-1035, a potent DNA-PK inhibitor. The unirradiated cells differ: the respective tail moment values for LY-R and LY-S cells were 9.62±2.84 and 3.52±0.1, reflecting the susceptibility to lysis conditions as well as the possible endogenous (oxidative) damage level. The level of initial DNA damage measured after irradiation (8 Gy) at DNA-denaturing pH was the same in both LY sublines: the mean tail moment values±SD were 92.93±10.39 for LY-R cells and 94.93±12.94 for LY-S cells. In LY-S cells the repair of 8 Gy X-ray-induced damage proceeded identically in the presence or absence of 2 mM OK-1035 to the same level of residual damage. In contrast, the level of residual damage in inhibitor treated LY-R cells was considerably higher than that in the untreated cells. Moreover, the inhibitor affected LY-R cells in G1 and S phases and not those in G2, in agreement with cell-cycle specificity of DNA-PK. These results may indicate that the DSB repair defect previously identified in LY-S cells is due to a lack of function of DNA-PK or its impaired activation in the irradiated cells.

Induction of DNA breakage in X-irradiated nucleoids selectively stripped of nuclear proteins in two mouse lymphoma cell lines differing in radiosensitivity.

The role of nuclear proteins in protection of DNA against ionizing radiation and their contribution to the radiation sensitivity was examined by an alkaline version of comet assay in two L5178Y (LY) mouse lymphoma cell lines differing in sensitivity to ionizing radiation. LY-S cells are twice more sensitive to ionizing radiation than LY-R cells (D0 values of survival curves are 0.5 Gy and 1 Gy, respectively). Sequential removal of nuclear proteins by extraction with NaCl of different concentrations increased the X-ray induced DNA damage in LY-R nucleoids. In contrast, in the radiation sensitive LY-S cell line, depletion of nuclear proteins practically did not affect DNA damage. Although there is no doubt that the main cause of LYS cells' sensitivity to ionizing radiation is a defect in the repair of double-strand breaks, our data support the concept that nuclear matrix organisation may contribute to the cellular susceptibility to DNA damaging agents.