Paul J. Boor

Regression of Lung and Colon Cancer Xenografts by Depleting or Inhibiting RLIP76 (Ral-Binding Protein 1)

Ral-binding protein 1 (RALBP1) is a stress-responsive and stress-protective multispecific transporter of glutathione conjugates (GS-E) and xenobiotic toxins. It is frequently overexpressed in malignant cells and plays a prominent antiapoptotic role selectively in cancer cells through its ability to control cellular concentration of proapoptotic oxidized lipid byproducts. In the absence of chemotherapy, depletion or inhibition of RALBP1 causes regression of syngeneic mouse B16 melanoma. Because RALBP1 transports anthracycline and Vinca alkaloid drugs, as well as GS-E, and because it confers resistance to these drugs, we proposed that depletion or inhibition of RALBP1 should cause regression of human solid tumors that overexpress RALBP1 and augment chemotherapy efficacy. Non-small-cell lung cancer (NSCLC) H358 and H520 and colon SW480 cell lines were used. Cytotoxic synergy between anti-RALBP1 immunoglobulin G (IgG), cis-diammine-dichloroplatinum (II) [CDDP], and vinorelbine was examined in cell culture and xenografts of NSCLC cells. Effects of RALBP1 depletion by antisense were examined in xenografts of NSCLC H358, NSCLC H520, and colon SW480 cells. RALBP1 depletion by phosphorothioate antisense was confirmed and was associated with rapid, complete, and sustained remissions in established s.c. human lung and colon xenografts. RALBP1 inhibition by anti-RALBP1 IgG was equally as effective as antisense and enhanced CDDP-vinorelbine in lung cancer xenografts. These studies show that RALBP1 is a transporter that serves as a key effector function in cancer cell survival and is a valid target for cancer therapy, and confirm that inhibitory modulation of RALBP1 transport activity at the cell surface is sufficient for antitumor effects.

The role of amine oxidases in xenobiotic metabolism

The amine oxidases of mammalian tissues are a heterogeneous family of enzymes that metabolise various monoamines, diamines and polyamines produced endogenously, or being absorbed as dietary or xenobiotic substances. The heterogeneous class of amine oxidases can be divided on an arbitrary basis of the chemical nature of their cofactors into two types. Monoamine oxidase (MAO) and an intracellular form of polyamine oxidase (PAO) contain flavin adenine dinucleotide (FAD) as their cofactor, whereas a second group of amine oxidases without FAD contain a cofactor possessing one or more carbonyl groups, making them sensitive to inhibition by carbonyl reagents such as semicarbazide; this group includes semicarbazide-sensitive amine oxidase (SSAO) and the connective tissue enzyme, lysyl oxidase. This article focuses on the general aspects of MAOs contribution to the metabolism of foreign toxic substances including toxins and illegal drugs. Another main objective of this review is to discuss the properties of PAO and SSAO and their involvement in the metabolism of xenobiotics.

Glutathione-S-transferase protects against oxidative injury of endothelial cell tight junctions.

Oxidative damage of endothelial tight junction permeability is involved in the pathophysiology of a variety of vascular diseases. The authors studied the role of the antioxidant enzyme, human glutathione-S-transferase A4-4 (hGSTA4-4), in regulating expression of major molecules of tight junction in vascular endothelial cells under oxidative stress induced by H(2)O(2). A vascular endothelial cell line, mouse pancreatic endothelial cells (MS1), was transduced with recombinant adenoviral vector containing hGSTA4-4 gene. hGSTA4-4 induced expression of tight junction proteins occludin and zonula occludens (ZO)-1 under oxidative stress. Increased hGSTA4-4 expression correlated with increased transepithelial electrical resistance and decreased tyrosine phosphorylation of occludin and ZO-1 following exposure to H(2)O(2). In addition, morphologic dissociation of occludin, ZO-1, and F-actin during oxidative stress was reduced in hGSTA4-4-expressing cells. To explore a genetic approach for vascular diseases associated with disruption of tight junction proteins, we introduced the same viral vector to blood vessels of mice, rats, and rabbits ex vivo and found strong expression of hGSTA4-4 in endothelial cells. These results demonstrate that oxidative stress mediated disruption of tight junctions in endothelial cells may be attenuated by hGSTA4-4 expression.

Cellular differentiation in three-dimensional lung cell cultures.

Introduction: Aspects of human biology that are not sufficiently addressed by current cell culture models are cellular differentiation and three-dimensional (3-D) structural organization. A model that more closely associates the presence and biology of organelles to molecular expressions relevant to these organelles may provide evidence of cellular differentiation and the beginning steps in the construction of a 3-D architecture. The development of a new model — 3-D cell cultures — may ultimately provide a better understanding of lung biology and pathobiology. Purpose: The purpose of this study was to develop both traditional monolayer and 3-D cell cultures of a known and well documented normal lung cell line and then to determine similarities and differences between these cultures in terms of differentiation and molecular marker expression. Methods: An immortalized cell line was grown as a traditional monolayer (ML) in culture flasks and as 3-D cultures in rotating walled vessels and incubated under identical conditions. Comparison for presence of differentiation and marker expression between these cultures and control tissue collected from surgical patient specimens was studied. Electron microscopy for identification of ultra structures, and immunohistochemistry (ZO-1, EMA, ICAM-1, villin, tubulin, CK 18, VWF, Collagen IV and human mucin) for phenotypic comparisons between cells in ML and 3-D cell cultures was conducted. Results: Electron microscopy identified presence of lipid inclusion, microvilli, extra cellular matrix, and tight junctions in the 3-D cultures; differentiation not seen in ML cultures. The degree of differentiation determined by immunohistochemistry when the cell line was grown as ML or 3-D cultures shows that ultra-structure and marker expressions were more representative of control tissue than when cells were grown in 3-D than as MLs. Summary: The development of 3-D cell cultures will provide for a new and more powerful tool in the study of lung biology and pathobiology.

Immuno- and Hepato-Toxicity of Dichloroacetic Acid in MRL+/+ and B6C3F1 Mice

Dichloroacetic acid (DCA) is a by-product of chlorination that occurs in drinking water disinfected with chlorine. Metabolism of trichloroethene (TCE) also generates DCA. TCE exposure is associated with the development of autoimmune diseases, which may be induced by TCE metabolites, such as DCA. Thus, it is important to understand immunotoxic responses to DCA. We chose 2 murine models, autoimmune-prone MRL+/+ and normal B6C3F1 mice. Both strains of mice were exposed to DCA for 12 weeks. Following DCA treatment, liver weights and liver-to-body weight ratios were significantly increased in both strains of mice when compared to their respective controls. The serum activity of alanine and aspartate aminotransferases was not significantly altered in either strain. In MRL+/+ mice, the serum concentrations of IgG and IgM were significantly increased, whereas in B6C3F1 mice, only serum IgG3 was increased. DCA treatment did not change the levels of inflammatory cytokines in the serum. However, independent of treatment, the concentrations of G-CSF in the serum were lower in MRL+/+ mice than in B6C3F1 mice, whereas IL-12 serum levels were higher in MRL+/+ mice. DCA treatment decreased IL-10 and KC chemokine concentrations in the livers of MRL+/+ mice, whereas T-helper cell cytokines (IL-4, IL-5, IL-10, IFNγ, and GM-CSF), pro-inflammatory cytokines (IL-6, IL-12, and G-CSF), and KC chemokine were increased in the livers of DCA-treated B6C3F1 mice. Stimulation of splenic T-lymphocytes with antibodies against CD3 and CD28 resulted in a marked difference in the secreted cytokines between the two strains of mice. T-lymphocytes from MRL+/+ mice secreted more IL-2, IL-4 and IL-10, but less IFNγ and GM-CSF, than did T-lymphocytes from B6C3F1 mice. Thus, the cytokine levels in serum and liver, and the cytokine secretion patterns from stimulated splenic T-lymphocytes suggested a higher propensity of inflammatory responses in B6C3F1 than in MRL+/+ mice. Treatment with DCA also affected lipid accumulation in the liver more severely in B6C3F1 than in MRL+/+ mice. Thus, these results indicate that DCA induced stronger inflammatory responses leading to more severe hepatotoxicity in B6C3F1 mice than in MRL+/+ mice, and more pronounced immune responses in the latter.

Cardiac Preconditioning With Local Laser-Induced Hyperthermia

BACKGROUNDnWhole body hyperthermia has been shown to be highly effective in preconditioning cardiac tissue against reperfusion injury. The current study was aimed to assess the ability of regional sublethal laser-induced hyperthermia to precondition cardiac tissue against ischemic-induced myocardial infarction.nnnMETHODS AND RESULTSnDiode laser radiation was used to locally heat (42 degrees C) the left ventricle by irradiating the epicardial surface of rat heart for 20 min. As control, another group of animals were treated with whole-body hyperthermia (WBH) for 20 min. After a 4-h recovery period, the left coronary artery was occluded for 30 min followed by 4 h of reperfusion. A significant degree of reduction in infarct size as assessed by percent of at-risk area was observed in animals that were subjected to laser-induced local hyperthermia (15.4 +/- 3.1; n = 5) versus the sham group (49.8 +/- 6.6; n = 5). The degree of cardiac protection induced by local hyperthermia was similar to the protection observed when the animals were preconditioned with WBH. Furthermore, regional laser-induced hyperthermia and WBH induced similar degree of up-regulation of heat shock protein 70 in cardiac tissue. Regional hyperthermia up-regulated heat shock protein in regions of the heart beyond that directly subjected to laser-induced heat stress.nnnCONCLUSIONnSublethal local heating of the heart with diode laser irradiation can improve myocardial salvage and may provide a practical approach for tissue preconditioning against reperfusion injury.

Role of the Media in Vascular Injury: Atherosclerosis and Dissection

Although a metabolic role for endothelium in toxic injury has been well established, a similar role has not been as thoroughly explored for the vascular media. In this study two forms of vascular medial involvement in toxic injury are examined. Early atherosclerotic plaques are studied by immunohistochemistry for an α class glutathione-S-transferase (GST) isozyme known as hGSTA4-4, which has preferential metabolic activity for α, β-unsaturated aldehydes derived from lipid peroxidation, especially 4-hydroxy-2-nonenal. Findings in human plaque indicate that hGST A4-4 is highly upregulated in vascular smooth muscle cells (VSMCs) within the plaque and in the medial VSMCs underlying plaque. Endothelial cells, while not expressing hGST A4-4 distant from plaques, were found to express the isozyme in cytoplasm overlying plaque. In a series of second experiments, we illustrate a developmental model of dissecting aortic aneurysm (DAA) obtained by administering semicarbazide, an inhibitor of the little-studied VSMC enzyme semicarbazide-sensitive amine oxidase (SSAO), to pregnant rats during the last trimester of development. Newborn rats consistently developed DAA which is characterized by splitting of media of ascending thoracic aorta and extensive blood collections surrounding the vessel. These experimental examples emphasize the potential role of the media in toxic insults to blood vessels. Also, the potential importance of toxic injury to developing blood vessels by in utero exposure to xenobiotic substances is illustrated.

Morphological Evaluation of the Heart and Blood Vessels

Methods essential for the morphological evaluation of the heart and blood vessels in toxicological studies are reviewed in this chapter. Focus is on tissue analysis rather than isolated cell studies. The elements of an accurate and thorough morphologic examination are defined. Concepts of gross examination and proper tissue fixation for microscopic and other studies are presented. The general methods of microscopic study including routine histology and special histologic and histochemical preparations are reviewed. Immunohistochemical, electron microscopic, and specialized ultrastructural studies are addressed. Throughout the chapter, basic concepts, the importance of careful sample preparation, and inclusion of proper controls are emphasized.