Autosomal dominant acute necrotizing encephalopathy.

OBJECTIVE: To define the clinical and biochemical abnormalities of an autosomal dominant form of acute encephalopathy. METHODS: The clinical details of 11 affected family members in comparison with 63 unaffected relatives were analyzed. RESULTS: Affected children become comatose after onset of a febrile illness. Outcomes include full recovery, permanent neurologic impairment, and death. Recurrences produce more severe impairments. Lesions of necrotizing encephalopathy of the thalamus and brainstem are present on autopsy and MRI. Oxidative phosphorylation of intact mitochondria from a muscle biopsy shows loose coupling. Unaffected family members, including obligate carriers, share no clinical characteristics, demonstrating incomplete penetrance. CONCLUSIONS: Characteristic pathology and MRI findings define this disorder of autosomal dominant acute encephalopathy. Leigh syndrome and sporadic acute necrotizing encephalopathy share similarities but are distinct.

beta-Lapachone-induced apoptosis in human prostate cancer cells: involvement of NQO1/xip3.

beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.

NAD(P)H:Quinone oxidoreductase activity is the principal determinant of beta-lapachone cytotoxicity.

beta-Lapachone activates a novel apoptotic response in a number of cell lines. We demonstrate that the enzyme NAD(P)H:quinone oxidoreductase (NQO1) substantially enhances the toxicity of beta-lapachone. NQO1 expression directly correlated with sensitivity to a 4-h pulse of beta-lapachone in a panel of breast cancer cell lines, and the NQO1 inhibitor, dicoumarol, significantly protected NQO1-expressing cells from all aspects of beta-lapachone toxicity. Stable transfection of the NQO1-deficient cell line, MDA-MB-468, with an NQO1 expression plasmid increased apoptotic responses and lethality after beta-lapachone exposure. Dicoumarol blocked both the apoptotic responses and lethality. Biochemical studies suggest that reduction of beta-lapachone by NQO1 leads to a futile cycling between the quinone and hydroquinone forms, with a concomitant loss of reduced NAD(P)H. In addition, the activation of a cysteine protease, which has characteristics consistent with the neutral calcium-dependent protease, calpain, is observed after beta-lapachone treatment. This is the first definitive elucidation of an intracellular target for beta-lapachone in tumor cells. NQO1 could be exploited for gene therapy, radiotherapy, and/or chemopreventive interventions, since the enzyme is elevated in a number of tumor types (i.e. breast and lung) and during neoplastic transformation.

MRI-guided radiofrequency thermal ablation of implanted VX2 liver tumors in a rabbit model: demonstration of feasibility at 0.2 T.

Successful radiofrequency (RF) thermal ablation was performed on VX2 tumors implanted in 23 rabbit livers under magnetic resonance (MR) guidance using a C-arm-shaped low-field 0.2 T system. RF application and immediate postprocedure MRI of all animals was performed [T2-weighted, turbo short tau inversion recovery (STIR), T1-weighted before and after gadopentetate dimeglumine administration). Follow-up MRI with a superparamagnetic iron oxide (SPIO) contrast medium was performed in nine rabbits at 2 weeks and in four rabbits at 1 month post RF ablation. All livers were harvested for pathologic examination. T2-weighted and turbo-STIR images demonstrated the highest tumor-to-RF-thermal lesion contrast-to-noise ratios (CNRs; means 4.5 and 3.8, respectively) on postprocedure images; this was redemonstrated at 2- and 4-week follow-up imaging. T2-weighted imaging never overestimated pathologic lesion size by more than 2 mm, and the radiologic-pathologic correlation coefficient was not less than 0.90. In conclusion, MRI-guided RF thermal ablation in implanted liver tumor is feasible using a C-arm-shaped low-field 0.2 T system. The thermal lesion size can be most accurately monitored with T2-weighted and turbo-STIR images.

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 LD99.9 dose 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%, and via Complex 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 consumption via electron 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.

Liver biopsy: effects of biopsy needle caliber on bleeding and tissue recovery.

PURPOSE: To evaluate different-caliber biopsy cutting needles in terms of the benefits and potential risk of bleeding in a swine model. MATERIALS AND METHODS: A total of 190 sequential liver biopsy specimens were obtained in 11 Yorkshire pigs (weight, 50-70 lb [22.5-31.5 kg]) by using 14-, 18-, and 20-gauge cutting needles. For each biopsy procedure, blood loss was determined by weighing sponges used to absorb bleeding, and sample-tissue DNA content was measured with spectrofluorometry. Analysis of variance was used to compare results. RESULTS: The larger the caliber of needle, the greater the absolute blood loss (for 14-gauge, 1.69 g; for 18-gauge, 0.74 g; for 20-gauge, 0.32 g) and DNA content per sample (for 14 gauge, 40.38 microg; for 18-gauge, 12.18 microg; for 20-gauge, 5.86 microg). The ratio of blood loss to amount of DNA recovered did not differ among the different-caliber needles. To obtain the same amount of diagnostic tissue, more passes were needed with the smaller-caliber needles. CONCLUSION: Use of larger-caliber needles is more efficient despite the greater amount of blood loss, because more tissue can be recovered and because fewer passes are necessary, which reduces the chances of complications.

Reduction of intracellular pH is not the mechanism for the synergistic interaction between photodynamic therapy and nigericin.

Previous studies showed that photodynamic therapy (PDT) sensitized by aluminum phthalocyanine can be dramatically potentiated by the K+/H+ ionophore nigericin. Nigericin equilibrates intracellular pH (pHi) and extracellular pH (pHe) and is most effective in potentiating PDT damage when cells are in an acidic environment (pH 6.5-6.7). We therefore hypothesized that the ability of nigericin to lower pHi is causally related to its ability to potentiate PDT. To test this, the pHi of A549 cells was reduced using pHe-adjusted growth medium, with or without addition of amiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibitors of the membrane-based exchangers responsible for regulating pHi. Using fluorescence ratio imaging, we found that pHi can be equilibrated to within +/- 0.05 pH unit, in the pH range of 6.0-6.8, for up to 1 h after pHe adjustment. Cells equilibrated to various pHi were subjected to PDT at various light fluences, then plated for clonogenic survival immediately after PDT treatment. There is no significant effect of lowering pHi, to values as low as 6.23, on the toxicity of PDT, regardless of whether pHi is lowered by adjustment of the medium alone or by addition of exchange inhibitors. However, cells equilibrated to pHi 6.0 are more sensitive to PDT, with survival reduced by 1 log at 20 kJ/m2 and 1.5 log at 30 kJ/m2, relative to cells treated at a pHi of 6.8 (controls). In contrast, 20 microM nigericin in medium at pHe 6.7 reduces pHi to 6.55, but reduces the surviving fraction at 20 kJ/m2 by nearly 3 logs relative to controls. These data conclusively demonstrate that the ability of nigericin to potentiate PDT is not directly related to its ability to lower pHi. Furthermore, they show that the expression of PDT damage is independent of pHi, except at the very low value of 6.0. Photodynamic therapy does not induce apoptosis in A549 cells, at surviving fractions of 0.1 to 0.01, under any of the treatment conditions used in this study.

Insulin and insulin-like growth factor-1 (IGF-1) inhibit repair of potentially lethal radiation damage and chromosome aberrations and alter DNA repair kinetics in plateau-phase A549 cells.

Plateau-phase A549 cells exhibit a high capacity for repair of potentially lethal radiation damage (PLD) when allowed to recover in their own spent medium. Addition of either insulin or insulin-like growth factor-1 (IGF-1) to the spent medium 60 to 120 min before irradiation significantly inhibits PLD repair. The 9-h recovery factor (survival with holding/survival without holding) is reduced from 10.8 +/- 0.7 to 3.4 +/- 0.3 by insulin and to 3.0 +/- 0.4 by IGF-1. Neither growth factor alters the cell age distribution of the plateau-phase cells, increases the rate of incorporation of 5-bromo-2'-deoxyuridine into DNA, or alters the extent of radiation-induced mitotic delay in cells subcultured immediately after irradiation. Both insulin and IGF-1 alter the kinetics for rejoining of DNA double-strand breaks (DSBs), slowing the fast component of rejoining significantly. However, these growth factors have no effect on the initial level of DSBs or on the percentage of residual unrejoined breaks at 120 min postirradiation. Both growth factors affect repair of lesions leading to dicentric, but not to acentric, chromosome aberrations significantly. In control cells (treated with phosphate-buffered saline, 90 min prior to irradiation), the half-time for disappearance of dicentrics was 4.1 h (3.4 to 5.1 h), and 47.1 +/- 3.7% of the residual damage remained at 24 h postirradiation. Insulin and IGF-1 increased the half-time for disappearance of dicentrics to 5.2 h (3.9 to 7.7 h) and 5.7 h (5.5 to 5.9 h), respectively, and increased residual damage to 56.1 +/- 5.9% and 60.8 +/- 6.0%, respectively. Overall, these data show that insulin and IGF-1 inhibit PLD repair in A549 cells by mechanisms which are independent of changes in cell cycle parameters. The data suggest that the growth factors act by inducing changes in chromatin conformation which promote misrepair of radiation-damaged DNA.

Elevation of GRP-78 and loss of HSP-70 following photodynamic treatment of V79 cells: sensitization by nigericin.

Chinese hamster V79 cells were treated with photodynamic therapy (PDT) sensitized by aluminum phthalocyanine (AlPc) or with the ionophore nigericin or with combinations of PDT and nigericin. We previously showed that PDT and nigericin interact synergistically in the killing of these cells; i.e. doses of PDT that kill no more than 10% of the cells in combination with nontoxic exposures to nigericin lead to a loss of clonogenicity of three to five orders of magnitude. Photodynamic therapy induces an enhanced rate of expression of the stress gene grp-78 both at the transcriptional and translational levels and causes a decrease in the synthesis of the constitutive heat shock protein HSP-70 as well as in expression of HSP-70 mRNA. When the cells are exposed to PDT in the presence of nigericin, these effects are elicited at three- to four-fold lower PDT doses. Thus, PDT in the presence of nigericin is much more effective in inducing the changes in gene expression than is PDT alone. In the absence of nigericin the PDT dose inducing a two-fold increase in GRP-78 accumulation causes little or no loss of clonogenicity. In the presence of nigericin, however, the PDT dose leading to a similar change in GRP-78 level produces up to a 50% loss of clonogenicity. The fact that nigericin is dose-modifying for both cell killing and stress responses suggests that nigericin either increases the yield of oxidative damage from a given dose of PDT or magnifies the cellular response to a constant level of oxidative stress.

Combined depletion of O6-alkylguanine-DNA alkyltransferase and glutathione to modulate nitrosourea resistance in breast cancer.

MCF-7 human breast cancer cells possess high levels of O6-alkylguanine-DNA alkyltransferase and moderate levels of glutathione, and are more resistant to chloroethylnitrosoureas (CNUs) than cells with low levels of either molecule. The role of each as a component of CNU resistance was assessed using O6-benzylguanine (O6-bG) or O6-methylguanine (O6-mG) to deplete the alkyltransferase and L-buthionine sulfoxamine (L-BSO) to deplete glutathione. O6-bG and O6-mG potentiated 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity, resulting in a dose modification factor of 5.4 and 2.3, respectively, which reflected the more potent inhibitory effect of O6-bG. L-BSO alone had little effect on the survival of MCF-7 cells following BCNU exposure, but when combined with O6-mG, BCNU cytotoxicity was additive, yielding a dose modification factor of 3.2. O6-bG or O6-mG and L-BSO acted independently, as neither class of inhibitor affected the other's mechanism of CNU resistance. Furthermore, MCF-7 cells overexpressing GST mu were not more resistant to BCNU than the parent cell line in either the presence or absence of O6-bG or L-BSO. These results indicate that on a relative basis in MCF-7 cells, the alkyltransferase is the cell's first line of defense against CNUs. This suggests that therapeutic trials based on O6-bG-induced biochemical modulation of CNU resistance may increase the efficacy of these chemotherapeutic agents against human malignant cells and that L-BSO may have little additive effect when used with these agents.

Effects of extracellular and intracellular pH on repair of potentially lethal damage, chromosome aberrations and DNA double-strand breaks in irradiated plateau-phase A549 cells.

Plateau-phase A549 cells exhibit a high capacity for repair of potentially lethal radiation damage (PLD). Previously it was found that PLD repair could be partially inhibited by increasing the extracellular pH (pHe) of the spent medium from its normal value of 6.7-6.8 to 7.6 during postirradiation holding. The present study shows that PLD repair is also inhibited by reducing the pHe of the spent medium to 6.0. The effects of altering pHe on rejoining of DNA double-strand breaks (DSBs) as measured by neutral filter elution and on mitotic delay and chromosome aberrations seen after releasing cells from the plateau phase were investigated. Neither increasing nor decreasing the pHe of the spent medium had an effect on radiation-induced mitotic delay. Rejoining of DSBs was significantly inhibited by holding at pHe 6.0 but not affected by holding at pHe 7.6. At 2 h after irradiation about 51% of unrejoined breaks remained at pHe 6.0, compared to about 15% at pHe 6.7 or 7.6. However, holding at pHe 7.6 appeared to cause a marginal change in the kinetics of rejoining of DSBs. Repair of lesions leading to dicentric and acentric chromosome aberrations did not occur when cells were held at pHe 6.0, since less than 10% of these aberrations disappeared from cells held for 24 h before subculture. In contrast, holding plateau-phase cells at pHe 7.6 vs 6.7 caused a small but significant reduction in the disappearance of dicentrics but had no effect on the rate or extent of the disappearance of acentrics. These data have led us to hypothesize that inhibition of PLD repair by holding at pHe 6.0 is related both to inhibition of pH-dependent DNA repair enzymes and to induction of changes in DNA which lead to misrepair when the cells are released from plateau phase. Inhibition of PLD repair by holding at pHe 7.6, on the other hand, is related primarily to changes in DNA structure which promote misrepair.

Effect of the K+/H+ ionophore nigericin on response of A549 cells to photodynamic therapy and tert-butylhydroperoxide.

The K+/H+ ionophore nigericin dramatically increases killing of V79 cells and A549 cells by photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Previous studies suggested that the interaction between PDT and nigericin is related to the ability of this ionophore to reduce intracellular pH (pHi). The present study was undertaken to test the possibility that nigericin, by lowering pHi, inhibits reductive detoxification of PDT-produced peroxides by enzymes of the glutathione (GSH) redox cycle and the pentose cycle. To test this possibility we examined the effects of nigericin on the toxicity and metabolism of a model peroxide, tert-butylhydroperoxide (tert-BOOH), in A549 cells, a cell line in which the GSH redox cycle is known to be the principal pathway for reduction and detoxification of tert-BOOH. We found that nigericin equilibrates pHi of A549 cells with extracellular pH (pHe) in a time-dependent manner. It increases the toxicity of tert-BOOH toward A549 cells, inhibits loss of tert-BOOH from the buffer overlying the cells, and reduces the rate of 14CO2 release from radiolabelled glucose, which is a measure of pentose cycle activity. These effects are significantly greater at pHe 6.40 than at 7.40. Monensin, a Na+/H+ ionophore which does not reduce pHi, does not enhance the toxicity of tert-BOOH and has only a minimal effect on tert-BOOH reduction. These data suggest that nigericin-induced inhibition of peroxide detoxification is at least a plausible mechanism by which the ionophore might interact with PDT.

Sensitivity to chemical oxidants and radiation in CHO cell lines deficient in oxidative pentose cycle activity.

In this paper we examine the susceptibility of a series of G6PD- CHO cell lines to a variety of chemical oxidants. Addition of these drugs to K1D, the parental cell line, results in as much as a 20-fold increase in pentose cycle (PC) activity over control values. In two of our mutant lines, E16 and E48, little or no stimulation of PC activity is seen. These lines are shown to be much more susceptible to the toxic effects of the chemical oxidants t-butyl hydroperoxide and diamide. PC activity is also stimulated by ionizing radiation in K1D cells. One of the G6PD- cell lines has an increased aerobic radiation response compared to the parental line. However, since this is not the case with the other G6PD- cell lines, it is unclear whether this represents a difference in the absolute value of PC activity or some additional variable that may be influencing the results.

Mutagenicity of 2-amino-N6-hydroxyadenine at the tk locus in L5178Y strains differing in repair capabilities and karyotype.

The cytotoxicity and mutagenicity of 2-amino-N6-hydroxyadenine (AHA) were measured in strains of L5178Y differing in repair capabilities and karyotype. Strain LY-R83 is monosomic for chromosome 11 and is therefore hemizygous for the tk gene, while strains LY-R16 and LY-S1 are TK+/- heterozygotes. Both strain LY-R83 and LY-R16 are sensitive to UV light and are presumed to be deficient in the excision of pyrimidine dimers as shown for the parental strain, LY-R (Hagen et al., 1988; Szumiel et al., 1988). Strain LY-S1 is sensitive to the cytotoxic effects of ionizing radiation and is presumed to be defective in the repair of radiation-induced DNA double-strand breaks, as shown for the parental strain, LY-S (Evans et al., 1987a; Wlodek and Hittelman, 1987). The sensitivities of the three strains to the cytotoxic effects of AHA were similar. After a 4-hour treatment with AHA at 37 degrees C, the D37 for all three strains was approximately 35 ng/ml. The AHA-induced mutant frequency was similar for the hemizygous TK+ strain LY-R83 and the heterozygous TK +/- strain LY-R16, but was slightly higher for strain LY-S1 than for either LY-R strain at an AHA concentration of 100 ng/ml. The proportion of AHA-induced LY-S1 TK -/- mutants forming colonies with diameters less than 0.3 mm was much lower than following treatment with X radiation (24% vs. 61% for AHA and X radiation, respectively). These results indicate that the vast majority of AHA-induced TK -/- mutants harbor single gene mutations. AHA did not result in cyanide-insensitive oxygen uptake, and treatment with this compound did not induce a significant number of DNA single-strand breaks, DNA alkali labile lesions, or DNA degradation in either strain. However, two hours after AHA removal, DNA single-strand breaks and/or alkali-labile lesions, possibly due to the occurrence of DNA repair, were apparent in the DNA of both strain LY-R16 and strain LY-S1.

Inhibition of recovery from potentially lethal radiation damage in A549 cells by the K+/H+ ionophore nigericin.

A549 cells held for 4 hr in Hank's balanced salt solution, after 10 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is dependent on extracellular pH (pHe). Recovery factors of 2.2 to 3.5 are observed when pHe is 6.40 to 7.30, but recovery factors of less than 1.0 are found when pHe is reduced to 6.20 or 6.00. The K+/H+ ionophore nigericin, when added to cells post-irradiation, inhibits PLDR in a pHe-dependent manner; it is increasingly more effective as pHe is reduced from 6.80 to 6.40. The presence of nigericin thus causes inhibition of PLDR at pHe's that normally promote recovery. The drug does not affect radiation response of A549 cells when present only during irradiation. Effects of low pHe buffer, with and without nigericin, on intracellular pH (pHi) and on ATP levels were examined in an effort to elucidate the mechanisms for inhibition of PLDR and enhancement of radiation response. Incubation of cells in pHe 6.00 buffer results in a slight decrease in pHi and does not induce a drop in ATP levels. In contrast, post-irradiation incubation of cells in pHe 6.40 buffer containing 2 microM nigericin causes an immediate and dramatic decrease in pHi, and a gradual loss of ATP to 30% of control levels by 4 hr. The data obtained so far suggest that a very slight lowering of pHi may influence post-irradiation holding recovery, and that the mechanisms by which pHe 6.00 buffer alone, or pHe 6.40 buffer containing nigericin, affect holding recovery are different.

Enhancement of photodynamic cell killing (with chloroaluminum phthalocyanine) by treatment of V79 cells with the ionophore nigericin.

The K+/H+ ionophore nigericin dramatically increases killing of V79 cells by photodynamic therapy (PDT), when cells pretreated with 1 microM chloroaluminum phthalocyanine are incubated with nigericin before exposure to red light. Nigericin affects primarily the shoulder of the PDT dose-response curve, reducing the surviving fraction from 0.90 to 0.02 after a fluence of 7 kJ/m2 and from 0.80 to 0.0003 after a fluence of 12 kJ/m2. Optimal enhancement of PDT occurs when cells are incubated with 2 microM nigericin, at pHe 6.7, for 30 to 60 min before irradiation. However, significant enhancement of PDT also occurs when nigericin is added immediately before irradiation. Treatments with chloroaluminum phthalocyanine and nigericin, nigericin alone, or nigericin and red light are not toxic to cells. Cells treated with the combined agents display a rounded morphology 2 h after light exposure and lyse within 12 h. However, rounding of cells is not accompanied by severe depletion of ATP or by permeabilization of the plasma membrane to trypan blue. These results, together with known metabolic effects of nigericin, suggest that nigericin potentiates PDT by perturbing ion transport across either mitochondrial or plasma membranes.

Role of glutathione in the aerobic radiation response.

We will review the relationships between glutathione (GSH), protein thiols, and cellular responses to radiation, peroxides, and peroxide-producing drugs. Our primary interest involves the behavior of sulfhydryls as electron and hydrogen carriers, and their capacity to protect various target molecules against radiation and peroxidative damage. We used reagents such as L-buthionine sulfoximine (LBSO), alone and in combination with N-ethyl maleimide (NEM), diamide, and dimethylfumarate, to decrease GSH so that it could no longer participate in the electron transfer reactions. Our results indicate that aerobic sensitization produced by GSH depletion can be further enhanced if electron-accepting agents, such as tertiary butyl hydroperoxide (t-BOOH), are present during irradiation. Hydroperoxide is a substrate for glutathione peroxidase and diverts electrons and hydrogen away from target molecules during its reduction. Sensitivity to radiation seems to be due to the inhibition of the mitochondria's capacity to reduce hydroperoxide. We will also report the mitochondria's ability to reduce the oxygen radicals produced by radiation and drugs. Data also indicate that t-BOOH oxidizes protein thiols which are enzymatically involved in repair of DNA damage.

pH-dependent effects of the ionophore nigericin on response of mammalian cells to radiation and heat treatment.

The extracellular pH (pHe) in many solid tumors is often lower than the pH of normal tissues. The K+/H+ ionophore nigericin is toxic to CHO cells when pHe is below but not above 6.5, and thus it has potential for selective killing of tumor cells in an acidic environment. This study examines the pH-dependent effects of nigericin on the response of CHO cells to radiation and heat treatment. Cells held for 4 h in Hank's balanced salt solution, after 9 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is maximal at pHe 6.7-6.8. Addition of nigericin, postirradiation, not only inhibits PLDR when pHe is below 6.8, but interacts synergistically with radiation to reduce survival below that of cells plated immediately after irradiation when pHe is 6.4 or lower. Nigericin enhances heat killing of CHO cells perferentially under acidic conditions, and where neither heat nor drug treatment alone is significantly toxic. Survival of cells held for 30 min at 42.1 degrees C in the presence of 1.0 microgram/ml nigericin is 0.6, 0.08, 0.003, and 0.00003 at pHe 7.4, 6.8, 6.6, and 6.4, respectively, relative to survival of 1.0 in untreated cultures. The biochemical effects of nigericin at pHe 7.4 vs pHe 6.4 have been investigated. Nigericin inhibits respiration, stimulates glucose consumption, and causes dramatic changes in intracellular concentrations of Na+ and K+ at pHe 7.4 as well as 6.4. The drug reduces intracellular levels of ATP, GTP, and ADP but has more pronounced effects under acidic incubation conditions. Others have shown that nigericin equilibrates pHe and intracellular pH (pHi) only when pHe is 6.5 or lower. Our observations and those of others have led us to conclude that lowering of pHi by nigericin is either the direct or indirect cause of enhancement of radiation and heat killing of cells in an acidic environment.

Factors influencing the oxidation of the radioprotector WR-1065.

N-(2-Mercaptoethyl)-1,3-diaminopropane (WR-1065) is the free thiol form of the radio- and chemoprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Interest currently exists in the clinical use of WR-2721 and WR-1065 as radio- and chemoprotectors of normal tissues. However, measurement of plasma levels of WR-1065 has proven difficult, due to rapid drug oxidation. Therefore, we studied factors influencing the oxidation of WR-1065, in Hepes-buffered saline as well as in tissue culture media containing 10% fetal bovine serum. The rate of oxygen consumption by WR-1065, as determined using the Clark oxygen electrode system, was faster in medium plus serum than in Hepes-buffered saline. That this effect is largely due to the presence of trace metal ions in tissue culture media and serum was indicated by the observation that addition of Cu2+ or Fe3+ to buffer stimulated oxygen consumption. Addition of KCN inhibited the reaction of WR-1065 with oxygen, and this effect was dependent on KCN concentration. That KCN blocked WR-1065 oxidation to the disulfide was verified using Ellman's reagent to quantitate the free thiol form. The rate of oxygen consumption was shown to be affected by temperature as well as concentration of WR-1065. Catalase reduced the rate of oxygen consumption of WR-1065, indicating that peroxide is formed in this system. Superoxide dismutase had a stimulatory effect. WR-1065 was found to stimulate the hexose monophosphate shunt in A549 cells. Since this stimulation was prevented by the presence of catalase, it appeared to be due to the response of the cells to peroxide, formed as a result of WR-1065 autooxidation.