Peter L. Gutierrez

BCL-2 is involved in preventing oxidant-induced cell death and in decreasing oxygen radical production

Abstract It has been hypothesized that programmed cell death is mediated, in part, through the formation of free radicals via oxidative pathways. Furthermore, it has been proposed that BCL-2 acts to inhibit cell death by interfering with the production of oxygen-derived free radicals induced by a wide variety of stimuli. In order to examine the antioxidant function of BCL-2, we transfected mouse epidermal cells JB6 clone 41 with the expression vector pD5-Neo-BCL-2 and studied the effect of BCL-2 overexpression on oxidant-induced cell death and on the production of reactive oxygen species. Compared to Neo control cells, BCL-2-expressing cells are more resistant to the killing and growth retardation induced by hydrogen peroxide, superoxide, or by the oxygen radical-generating quinone-containing compounds menadione, diaziquone and adriamycin. The latter compounds generate reactive oxygen species during bioreductive metabolism. In addition, the exposed cells die by necrosis rather than apoptosis. Hydroxyl radical levels generated by the quinone-containing agents were low in BCL-2-expressing JB6 cells compared to control Neo cells. BCL-2, however, does not change the activities of the major cellular antioxidant enzymes superoxide dismutase, catalase or glutathione peroxidase. On the other hand, the glutathione concentrations increased in BCL-2 overexpressing cells after oxidative challenge, while the opposite was true for control cells. Thus, our results suggest that BCL-2 inhibition of oxidant-induced cell death is mediated, at least in part, through an antioxidant pathway, and that this pathway involves glutathione.

Interleukin-10 gene transfer inhibits murine mammary tumors and elevates nitric oxide.

Transfection of cDNA for IL‐10 into line 66.1 murine mammary tumor cells results in marked suppression of tumor growth and metastasis. Others have reported that nitric oxide has potent antitumor activity and IL‐10 is known to regulate the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We identified nitric oxide production in mammary tumors as indicated by electron paramagnetic resonance detection of nitric oxide‐hemoglobin (NO‐Hb). IL‐10 expression resulted in elevated levels of NO‐Hb in mammary tumors. Immunohistochemical examination of mammary tumors for iNOS protein revealed few positively staining cells in parental or control neo‐transfected tumors but strong iNOS staining in all IL‐10 transfected tumors, consistent with the NO‐Hb data. To determine if mammary epithelial tumor cells themselves, express nitric oxide synthase activity, cultured tumor cells were treated with pro‐inflammatory cytokines and nitrite accumulation was assessed in the conditioned medium. All IL‐10 producing cell lines accumulated uM concentrations of nitrite in response to short term (24 hr) cytokine stimulation. Cells not expressing IL‐10 (parental and neo‐transfectants) accumulated no nitrite under similar culture conditions. After longer stimulation (48 hr), parental and 66‐neo cells accumulated lower amounts of nitrite. IL‐10 gene transfer is associated with increased iNOS protein expression and enzymatic activity detected both in vitro<0R> and in vivo<0R>. Our findings suggest that the antimetastatic and antitumor activity of IL‐10 is related to enhanced production of nitric oxide. Int. J. Cancer 76:713–719, 1998.© 1998 Wiley‐Liss, Inc.

Correlation of expression of BP1, a homeobox gene, with estrogen receptor status in breast cancer.

BackgroundBP1 is a novel homeobox gene cloned in our laboratory. Our previous studies in leukemia demonstrated that BP1 has oncogenic properties, including as a modulator of cell survival. Here BP1 expression was examined in breast cancer, and the relationship between BP1 expression and clinicopathological data was determined.MethodsTotal RNA was isolated from cell lines, tumors, and matched normal adjacent tissue or tissue from autopsy. Reverse transcription polymerase chain reaction was performed to evaluate BP1 expression. Statistical analysis was accomplished with SAS.ResultsAnalysis of 46 invasive ductal breast tumors demonstrated BP1 expression in 80% of them, compared with a lack of expression in six normal breast tissues and low-level expression in one normal breast tissue. Remarkably, 100% of tumors that were negative for the estrogen receptor (ER) were BP1-positive, whereas 73% of ER-positive tumors expressed BP1 (P = 0.03). BP1 expression was also associated with race: 89% of the tumors of African American women were BP1-positive, whereas 57% of those from Caucasian women expressed BP1 (P = 0.04). However, there was no significant difference in BP1 expression between grades I, II, and III tumors. Interestingly, BP1 mRNA expression was correlated with the ability of malignant cell lines to cause breast cancer in mice.ConclusionBecause BP1 is expressed abnormally in breast tumors, it could provide a useful target for therapy, particularly in patients with ER-negative tumors. The frequent expression of BP1 in all tumor grades suggests that activation of BP1 is an early event.

The reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells: free radical formation and NAD(P)H: quinone-acceptor oxidoreductase (DT-diaphorase) activity.

The S9 fraction of MCF-7 human breast carcinoma cells has NAD(P)H (quinone-acceptor) oxidoreductase activity as measured by the reduction of dichlorophenol-indophenol (DCPIP). This reduction is dependent on the activators Tween-20 and bovine serum albumin and it is inhibitable by dicumarol. The S9 fraction also has cytochrome c reductase activity which is approximately 29 times less than the two-electron reduction activity of NAD(P)H (quinone-acceptor) oxidoreductase. Diaziquone (AZQ) is a substrate for this NAD(P)H oxidoreductase active S9 fraction as judged by its enzymatic reduction detected spectrophotometrically and by electron spin resonance spectroscopy. Two-electron mediated enzymatic reduction of AZQ was evidenced by the formation of the colorless dihydroquinone (AZQH2) which could be followed at 340 nm. The production of the dihydroquinone was inhibitable by dicumarol implicating NAD(P)H oxidoreductase in its formation. Under aerobic conditions, electron spin resonance spectroscopy showed evidence for the production of AZQ semiquinone (AZQH) and oxygen radicals. Under anaerobic conditions no oxygen radicals were observed, but the semiquinone was stable for hours. These results are also inhibitable by dicumarol and suggest a two-step one-electron oxidation process of the dihydroquinone. The production of semiquinone and oxygen radicals as detected by electron spin resonance spectroscopy was more sensitive to dicumarol when NADPH was used as cofactor (68% inhibition of OH and 65% inhibition of AZQH) than when NADH was used (28% inhibition of OH and 5% inhibition of AZQH). This suggests that NADH flavin reductases play a more important role in the one-electron reduction pathway of AZQ in MCF-7 S9 fraction than NADPH reductases. The reduction of AZQ by NAD(P)H (quinone-acceptor) oxidoreductase may play an important role in the bioreductive alkylating properties of AZQ.

Essential role of nitric oxide and interferon-γ for tumor immunotherapy with interleukin-10

Several laboratories have reported anti-tumor activity for high levels of interleukin-10 (IL-10) expressed as a transgene or administered as recombinant protein. The authors have reported a positive correlation for nitric oxide production and anti-tumor activity of IL-10 in a murine model of breast cancer. In the current study, they sought evidence of a mechanistic role for nitric oxide in IL-10–mediated tumor growth inhibition. They wanted to determine whether pharmacologic inhibition of nitric oxide synthase (NOS) activity reverses the therapeutic effect of IL-10. Administration of either of two NOS inhibitors, aminoguanidine (AG) or L-lysine, N6-1-iminoethyl-dihydrochloride, appears to abrogate in part the tumor growth inhibition observed when IL-10 is overexpressed as a transgene in two murine mammary tumor cell lines. Nitric oxide levels were assessed at the tumor site by measuring nitrosylated heme levels by electron spin resonance spectroscopy. Nitric oxide hemoglobin levels were lower in tumors from aminoguanidine-treated mice, indicating that effective inhibition of nitric oxide production occurred at the tumor site. Previous investigations showed that the inducible form of NOS protein (iNOS), but not constitutive NOS, was expressed at higher levels in IL-10–expressing tumors. Because iNOS is regulated at the transcriptional level, the authors compared iNOS mRNA levels in IL-10 and control tumors. Northern analysis revealed strong iNOS message expression in all six IL-10–expressing tumors examined, whereas message was faintly detected in parental or 66-neo tumors. The inducible form of NOS is responsive to induction by interferon-&ggr; (IFN-&ggr;). The role of IFN-&ggr; in IL-10–mediated tumor inhibition and iNOS mRNA induction was determined. When tumors were transplanted to IFN-&ggr; mutant mice, the tumor-inhibitory activity of IL-10 was lost. Furthermore, iNOS mRNA was no longer induced in the absence of host expression of IFN-&ggr;. These data indicate that nitric oxide contributes to the anti-tumor activity of IL-10 and that expression of iNOS in this context depends on IFN-&ggr;.

In vitro action of continuous-wave ultrasound combined with adriamycin, X rays or hyperthermia

We compared the ability of continuous-wave ultrasound to enhance cytotoxicity from X irradiation, hyperthermia or exposure to adriamycin. The survival of CHO cells exposed in culture medium to these agents was determined with and without continuous-wave ultrasound (1.62 or 1.765 MHz). In water-filled transmission exposure vessels with 2-cm-diameter Mylar end windows, 10-min insonation not producing cytotoxicity could produce .OH radicals (measured by electron paramagnetic resonance) even at 0.4 W/cm2. Ultrasound at intensities ranging between 1 and 2.5 W/cm2 increased the clonogenic cytotoxicity of adriamycin (P = 0.0023 by paired t test) but not of X rays (2-10 Gy) or hyperthermia (44 degrees C for 10-50 min). The only significant action of continuous-wave ultrasound under similar test conditions was the potentiation of adriamycin-induced clonogenic cytotoxicity, possibly mediated by cavitational activity.

KML001 Cytotoxic Activity Is Associated with Its Binding to Telomeric Sequences and Telomere Erosion in Prostate Cancer Cells

Purpose: KML001 (sodium metaarsenite) is an orally bioavailable arsenic compound that has entered phase I/II clinical trials in prostate cancer. In this study, we elucidated the mode of action of KML001 and investigated its effects on telomerase and telomeres. Experimental Design: We compared telomere length to KML001 cytotoxic activity in a panel of human solid tumor cell lines. Duration of exposure and concentrations of KML001 that affect telomerase and telomeres were evaluated in relation to established mechanisms of arsenite action such as reactive oxygen species–related DNA damage induction. Binding of KML001 to telomeres was assessed by matrix-assisted laser desorption/ionization mass spectrometry. Results: We established a significant inverse correlation (r2 = 0.9) between telomere length and cytotoxicity. KML001 exhibited activity in tumor cells with short telomeres at concentrations that can be achieved in serum of patients. We found that telomerase is not directly inhibited by KML001. Instead, KML001 specifically binds to telomeric sequences at a ratio of one molecule per three TTAGGG repeats leading to translocation of the telomerase catalytic subunit into the cytoplasm. In prostate cancer cells with short telomeres, KML001 caused telomere-associated DNA damage signaling as shown by γ-H2AX induction and chromatin immunoprecipitation assays as well as a rapid telomere erosion shown by metaphase fluorescence in situ hybridization. These effects were not seen in a lung cancer cell line with long telomeres. Importantly, arsenification of telomeres preceded DNA lesions caused by reactive oxygen species production. Conclusions: Sodium metaarsenite is a telomere targeting agent and should be explored for the treatment of tumors with short telomeres.

Trace determination of hydroxyl radical using fluorescence detection

Publisher Summary This chapter discusses a new, highly sensitive approach for detecting and quantifying radicals, hydroxyl radical (.OH), within biological systems. This approach is predicated on two well-known properties of the stable di-tertalkyl nitroxides: first, their rapid reaction with carbon-centered radicals to form stable O-alkylhydroxylamines (radical adducts), and second, their efficient quenching of excited singlet states. By covalently coupling a nitroxide at a short distance to a chromophore, fluorescence emission from the chromophore can be largely quenched. However, on reaction of radicals with the nitroxide moiety to form the diamagnetic (spin-paired) adducts, the intramolecular quenching pathway is eliminated and fluorescence emission increases, thus allowing radicals to be detected optically. The highly fluorescent radical adducts can then be separated by high-performance liquid chromatography (HPLC) and quantified fluorometrically. In most cases, mass spectrometry can be used to help identify or confirm the structure of the radical adduct. Because the reaction between .OH and the nitroxides does not lead to a stable product, an organic compound that reacts with .OH to generate a carbon-centered radical must be added.

Electron spin resonance of electrochemically generated free radicals from diaziquone and its derivatives

The one-electron electrochemical reduction of diaziquone (AZQ) and 12 analogs is analyzed using ESR spectroscopy and cyclic voltammetry. The hyperfine coupling constants arising from the interaction of the unpaired electron with the aziridine nitrogen nuclei fall within 1.20 and 2.26 G. Smaller couplings are observed arising from the protons and nitrogens in the carboethoxyamino groups. The in vitro activity of AZQ and its analogs is examined. Methyl groups in the aziridine rings increase the activity of some analogs. In the absence of aziridines, a chloroquinone compound with only carboethoxyamino groups was surprisingly active. This compound has a more positive cathodic peak than diaziquone.

Trace detection of hydroxyl radicals during the redox cycling of low concentrations of diaziquone: a new approach.

Quantifying oxygen radicals that arise during the redox cycling of quinone-containing anticancer agents such as diaziquone (AZQ) has been difficult, as has been their detection at low drug concentrations. This is due to the fact that EPR spin trapping, the method most often used for *OH detection, requires the use of high drug concentrations. Using a new highly sensitive technique that employs a fluorescamine-derivatized nitroxide, we show that low levels of NADPH-cytochrome P450 reductase (4.25 microg/ml) catalyze the production of hydroxyl radicals at very low, clinically relevant AZQ concentrations. Thus, at this enzyme concentration, we were able to detect a rate of 0.10 nM s(-1) hydroxyl radical production by 5 microM AZQ, a clinically relevant concentration. The Michaelis-Menten constants for AZQ-mediated hydroxyl radical production are: K(M) = 10.7 +/- 1.4 microM, and V(max) = 5.2 +/- 0.9 x 10(-8) M s(-1) (mg protein)(-1). Experiments employing catalase, superoxide dismutase, and NADPH-cytochrome P450 reductase, confirm the previously deduced conclusions from high drug concentrations, that is, that at low concentrations, AZQ acts to shuttle reducing equivalents from the enzyme to oxygen, thus generating the redox cycle. The data presented here suggest that the levels and locations of redox active metal ions may be the principal controlling factor in the pathway of AZQ activity that involves oxidative stress.