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Cell Growth & Differentiation Vol. 12, 419-426, August 2001
© 2001 American Association for Cancer Research

Role of Increased Basal Expression of Heat Shock Protein 72 in Colonic Epithelial c2BBE Adenocarcinoma Cells1

Mark W. Musch2, Brian Kaplan and Eugene B. Chang

The Martin Boyer Laboratories, Department of Medicine, University of Chicago, Chicago, Illinois 60637


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Although the expression of heat shock proteins (hsps) can be induced by a variety of stressful stimuli, certain neoplasms, including human intestinal T84, HT-29, and Caco2 adenocarcinoma cell lines, express constitutively high levels even under nonstress conditions. In this study, we examine the functional significance of increased hsp72 in spontaneously differentiating Caco2bbe (C2) cells. The expression of hsp72 in these cells was specifically inhibited by hsp72 antisense transfection. The loss of hsp72 expression did not affect growth rate, contact inhibition, morphological development, or functional differentiation. In contrast, these cells were significantly more sensitive to the injurious effects of oxidants and tumor necrosis factor (TNF) but not doxorubicin. To investigate potential mechanisms of action, a number of steps in the TNF-mediated cell death was measured. Antisense reduction of hsp72 did not alter activation of I{kappa}B. In contrast, mitochondrial cytochrome c release and activation of caspase 9 were significantly delayed in hsp72 antisense cells stimulated either with TNF or monochloramine. In conclusion, high endogenous expression of hsp72 by intestinal adenocarcinoma cells appears to confer selective survival advantage but does not affect their growth and differentiation.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Adenocarcinoma cells are remarkably resistant to injury by radiation and systemic, immunological, and chemotherapeutic agents. As a consequence, these tumors are difficult to treat and often proliferate rapidly, even under conditions that may adversely affect normal cells. The mechanisms underlying the survival advantage may be in part related to the high endogenous expression of stress proteins, which include members of the hsp3 family. Some of these proteins are expressed constitutively and act as chaperones, but others are selectively expressed under conditions of stress (1 , 2) . These proteins are believed to bind and protect critical cellular proteins, preventing their denaturation by adverse factors or conditions. In many intestinal epithelial cells (3, 4, 5) , hsp72 appears to be a major inducible hsp, its expression associated with enhanced survival to conditions such as oxidant- and thermal-induced stress. In this study, we report that several transformed intestinal epithelial cell lines express high endogenous levels of hsp72 under basal, nonstress conditions in marked contrast to normal, diploid intestinal epithelial cells. This increase in hsp72 expression in intestinal neoplastic cell lines correlated with that reported in colonic tumors in vivo (6 , 7) . However, the significance of increased basal expression of hsp72 in the biology and function of these cancer cells is not known. Because the functional and developmental characteristics of human intestinal epithelial C2 cells are highly reproducible and readily assessed, we used them to assess the role of constitutively expressed hsp72 on cellular proliferation, differentiation, and survival.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Hsp72 Expression in Nontransformed and Transformed Intestinal Cell Lines.
An initial survey of hsp72 expression was performed in the human colonic adenocarcinomas cell lines HT-29, T84, and C2 and in the nontransformed rat small intestinal cell lines RIE-1 and IEC18. As shown in Fig. 1Citation , the nontransformed cell lines expressed little, if any, hsp72 protein under basal conditions. In contrast, all three carcinoma cell lines exhibited high endogenous levels of this major stress protein under basal conditions. This increase was specific, as the levels of the constitutive homologue, hsc73, were similar in all five cell lines. Thermal stress (42°C x 23 min) caused an additional increase in endogenous hsp72 expression in all three human adenocarcinoma cell lines, as well as inducing a significant response in RIE-1 and IEC18 cells (data not shown).



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Fig. 1. Western blots demonstrating levels of hsp72 and hsc73 in five intestinal cell lines. Equivalent amounts of protein were analyzed in all cases, and the images presented are representative of three different experiments.

 
Effect of Hsp72 Expression on Growth and Differentiation.
To assess the importance of hsp72 expression on growth, differentiation, and cell resistance to injury, C2/AS were established. C2 cells are most effectively transfected through electroporation (250 0µ0 F and 250 V), allowing us to establish a number of hygromycin-resistant C2/AS. Several clones demonstrated complete elimination of basal hsp72 expression (Fig. 2)Citation ,of which two were selected for additional analysis. The antisense effect was specific, as no changes in the constitutively expressed homologue, hsc73, were observed.



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Fig. 2. Western blots demonstrating specific reduction of hsp72 in C2/AS cells. C2/CEP4 and C2/AS (clones E3 and E5) were harvested near confluence, and equivalent amounts of protein were analyzed for hsp72 and hsc73. Images shown are representative from three separate experiments over 15 passages of the cells.

 
To determine whether basal hsp72 expression affected cell growth rates, cell proliferation of C2/CEP4 and C2/AS cells was assessed by the XTT assay. As shown in Fig. 3Citation , no differences were noted between these groups at 2, 4, or 7 days after plating. To determine whether the level of endogenous hsp72 expression affected the morphological development or the degree of contact inhibition of cell monolayers, electron micrographs of cell monolayers were prepared at preconfluence (2 days after plating), early confluence (7 days), and late confluence (14 and 21 days) (Fig. 4)Citation . Even at late confluence, contact inhibition of cell growth was evident in both groups, i.e., cultured cells remained a monolayer without evidence of palisading. In addition, the time course and degree of microvillar, terminal web, and development of the junctional complex appeared to be similar between the groups. No significant differences in the number, density, and height of microvilli could be detected. The degree of cellular polarity and monolayer height was also not affected by antisense inhibition of hsp72 (data not shown). Hsp72 inhibition by antisense transfection did not alter the development of barrier permeability in these cells. Both C2VC and C2/AS cells began to develop TER by day 3 after plating. By day 5, TERs in C2VC and C2/AS were 117 ± 14 and 124 ±12 12 {Omega}-cm2, respectively, and at day 7, the TERs were 184 ± 25 and 192 ± 22. Maximal TERs were attained around day 10 (231 ± 24 and 244 ± 39 {Omega}-cm2) and were maintained for at least another 10 days.



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Fig. 3. Effect of antisense inhibition of hsp72 expression on proliferation rates. Cells were plated at 20–30% confluence, and, at the times designated, proliferation was measured by the XTT assay as described in "Materials and Methods." Cells were >95% confluent by day 7. Values shown are means ± SE for three experiments.

 


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Fig. 4. Electron micrographs of C2/CEP4 and C2/AS cells at 2, 7, and 14 days after plating. Cells were plated at 20–30% confluence on permeable Transwell supports and fixed at the times indicated. Cells were fed every 2–3 days. Images shown are representative of at least five sections taken from each monolayer fixed.

 
As another measure of differentiation, brush border enzyme activities were measured. The progressive increase in the specific activities of these enzymes has been used as an indicator of cellular differentiation, correlating with the development of brush border microvilli. As shown in Fig. 5Citation , the specific activities of the brush border hydrolases, alkaline phosphatase, and sucrase-isomaltase progressively increased in cellular monolayers over a 21-day period, as reported previously (8 , 9) . However, no differences in the kinetics or degree of increased brush border hydrolases were observed between C2/AS and C2/CEP4 cells.



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Fig. 5. Differentiation-dependent brush border hydrolase activities in C2/CEP4 and C2/AS cells. Cells were plated at 20–30% confluence on plastic and harvested at the designated days. Enzyme activities were measured in homogenates of the cells and expressed as activity per cell protein. Values shown are means ± SE for three experiments.

 
Resistance to Injury.
To determine whether the level of endogenous hsp72 conferred survival advantage to these adenocarcinoma cells, C2 transfectants were exposed to varying concentrations of the oxidant H2O2. The degree of cell injury was assessed by the XTT assay, reflected by the reduced capacity to convert XTT to the formazan dye measured by absorbance at 490 nm. After a 30-min exposure to this oxidant, significant injury of C2/CEP4 cells was apparent only at H2O2 concentrations >1 mM (Fig. 6)Citation . In contrast, C2/AS cells were far more sensitive to the injurious effects of the oxidant. Cell injury was evident at concentrations as low as 0.1 mM with a half-maximal effect observed at a concentration of 0.3 mM. Therefore, the selective reduction of hsp72 in C2/AS cells makes them more sensitive to oxidant injury.



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Fig. 6. Effect of the oxidant H2O2 on viability of C2/CEP4 and C2/AS cells. Cells were used at 7 days after plating and injured for 30 min with various amounts of H2O2 in HBSS with Ca and MgCl2 for 30 min. The HBSS was replaced with phenol red-free DMEM with XTT reagents, and the conversion to the formazan dye was followed for 120 min. Values shown are means ± SE for three experiments. Control, 0.03 mM, 0.1mM; 0.3 mM 1mM, 3mM, .

 
C2 cells can also be injured by other agents, including the cytokine TNF-{alpha}a and the chemotherapeutic agent doxorubicin. After 2 days of exposure to these agents, the C2/AS cells demonstrated significantly greater sensitivity to injury by TNF-{alpha} but not to doxorubicin (Fig. 7)Citation . Failure to observe an effect in the latter group may be attributable to the high levels of the multidrug-resistant protein P-glycoprotein commonly expressed by these cells (10 , 11) . This membrane protein is known to be able to rapidly transport xenobiotics, such as doxorubicin, out of neoplastic cells, thereby substantially reducing their toxic effects.



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Fig. 7. Effect of the cytokine TNF or the chemotherapeutic agent doxorubicin on viability of C2/CEP4 and C2/AS cells. Cells were treated with varying concentrations of TNF or doxorubicin for 2 days, 7 days after plating. Media was removed and replaced with phenol red-free DMEM with XTT reagents, and the conversion to the formazan dye was followed for 60 min. Values shown are means ± SE for three experiments. *,P < 0.05 compared with CEP4 transfected cells.

 
Mechanism of Hsp72 Protection.
To determine where hsp72 confers cytoprotection, cells were treated with either TNF{alpha} or monochloramine, and specific biochemical changes were determined. TNF{alpha} regulates activation of NF{kappa}B by stimulating phosphorylation and release of I{kappa}B from NF{kappa}B; therefore, I{kappa}B levels were measured at early times points after TNF{alpha} (30 ng/ml) stimulation in C2/AS and C2/CEP4 cells (Fig. 8)Citation . l{kappa}B rapidly decreased in both C2/CEP4 and C2/AS cells, reaching the lowest level at 30 min. I{kappa}B was rapidly resynthesized in both the CEP4 and AS cells, demonstrating a functional NF{kappa}B response (NF{kappa}B positively regulates I{kappa}B resynthesis). Because the kinetics of I{kappa}B activation are not altered in C2/AS, a distal step appears to be involved.



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Fig. 8. Time course of I{kappa}B degradation in TNF-stimulated C2VC and C2/AS cells. Cells were stimulated with TNF (30 ng/ml), and total cell proteins were isolated and solubilized at varying times. Samples were run on SDS-PAGE and blotted, and Western blots were probed with a monoclonal anti-I{kappa}B antibody. Images shown are representative of those of three separate experiments.

 
Because TNF-{alpha} is known to stimulate oxidant formation in a number of cells (12 , 13) , cellular levels of TBARS were measured after TNF stimulation. Products of lipid peroxidation in total cell homogenates of C2/AS and C2/CEP4 cells treated with TNF-{alpha} (30 ng/ml) were measured by the TBARS procedure, which assesses levels of cellular oxidant formation (14) . No differences between the two groups were noted in the TBARS reactive substances, demonstrating that the presence of hsp72 does not inhibit oxidant generation from endogenous sources after TNF-{alpha} (Fig. 9)Citation These studies thus implicated a step distal to oxidant generation.



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Fig. 9. Time course of generation of lipid peroxides in TNF-stimulated C2VC and C2/AS cells. Cells were stimulated with TNF (30 ng/ml) and homogenized at varying times to measure lipid peroxides. Lipid peroxides were measured using the thiobarbituric acid procedure, and standard curves were generated with malondialdehyde. Values shown are means ± SE for three separate experiments.

 
Release of cytochrome c from mitochondria has been recognized as an important factor in cell death (15) . The oxidant monochloramine causes a rapid release of cytochrome c from mitochondria (Fig. 10)Citation . The oxidant-stimulated release of cytochrome c is faster in C2/AS cells, suggesting that hsp72 might protect mitochondrial integrity. Cytochrome c release caused by TNF-{alpha} stimulation took much longer. Nevertheless, cytochrome c release again was more rapid in C2/AS cells (Fig. 10)Citation . Release of cytochrome c results in a number of processes, including the activation of the initiator caspase 9 (16) . To determine whether the presence of hsp72 also delayed the kinetics of caspase 9 activation, caspase 9 activity was measured in C2/AS and C2/CEP4 cells stimulated with TNF-{alpha} (30 ng/ml). As shown in Fig. 11Citation , activation of caspase 9 was delayed in the presence of hsp72, corresponding in time to cytochrome c release.



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Fig. 10. Time course of release of mitochondrial cytochrome c from C2VC and C2/AS cells. Cells were stimulated with TNF (30 ng/ml), and total cell proteins were isolated and solubilized at varying times. Samples were run on SDS-PAGE and blotted, and Western blots were probed with a monoclonal anti-cytochrome c antibody. Images shown are representative of those of three separate experiments.

 


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Fig. 11. Time course of caspase 9 activation in TNF-stimulated C2VC and C2/AS cells. Cells were stimulated with TNF (30 ng/ml), and cytosolic fractions were isolated at varying times. Caspase 9 activity was measured using a fluorometric substrate. Values shown are means ± SE for three separate experiments. *, P < 0.05 and ++, P 0.01 compared with vector transfected cells by paired Student’s ttest.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The increased expression of hsp72 has been reported in several neoplastic cell types, although the functional and biological significance of these observations has not been determined (17, 18, 19, 20) . In this study, we demonstrate that the high endogenous expression of hsp72 appears to confer a survival advantage to intestinal epithelial cancer cells but does not alter other biological functions, such as cellular proliferation, morphological development, and functional differentiation. We chose to study the C2bbe subclone of the Caco2 human colonic adenocarcinoma because it is a more homogeneous population than the parental cell line (8) . Furthermore, these cells are an excellent model for studying the development of the brush border (hence, bbe = brush border expresser) and other mature phenotypes and functional characteristics of intestinal epithelial cells.

Our studies also show that hsp72 reduction diminishes cell survival to oxidant- and TNF-induced stress. Susceptibility to TNF-induced cellular injury or apoptosis is affected by the cellular level of hsp72 (21, 22, 23, 24, 25) . In Walter and Euza Hail Institute fibroblasts, e.g., cells transfected with the human-inducible hsp72 cDNA exhibit a 1000-fold reduction in sensitivity to killing by TNF{alpha} (21) . This effect was highly specific, as the transfection of another hsp, hsp27, did not confer protection. TNF-{alpha} may cause cellular apoptosis, which is associated with the activation of c-Jun-NH2-terminal kinases, proteolytic caspases, and oxidant formation (24 , 25) and interaction of death domains. The present results suggest that early steps in signal transduction by TNF are not altered. Degradation of the transcription factor regulator I{kappa}B, which occurs after it dissociates from NF{kappa}B, is not altered in cells with reduced hsp72. In addition, generation of cellular oxidants is not altered in C2/AS, indicating a site of hsp72 action at a more distal step.

Pharmacological evidence for the importance of hsp72 {alpha}s well as potentially other hsps) can be inferred by the use of the flavonoid quercetin. We (3) and others (23 , 26) have shown that quercetin inhibits the production of hsp72. Quercetin treatment sensitizes Walter and Euza Hail Institute-S cells to the chemotherapeutic agents topotecan and gemcitabine (26) . In K562, MOLT, Raji, and MCAS tumor cells, quercetin inhibits the growth and induces apoptosis in the G1 and S phases (23) . Thus, it is possible that quercetin’s antitumor effects are mediated by decreased hsp72 production. Indeed, antisense inhibition of hsp72 enhances the induction of apoptosis by quercetin (23) , suggesting that decreased hsp72 may be an important step in its actions.

The mechanisms by which hsps protect cells are only recently becoming understood. Hsp72 directly associates with a number of cellular proteins and is capable of stabilizing their structure and protecting them from denaturing agents, such as oxidants. Many of the proteins may be critical for sustaining vital cell functions. Furthermore, hsp72 protects key cytoskeletal proteins that may be involved in cell structure, polarity, and formation of tight junctions. The induction of hsp72 has also been shown to protect mitochondrial function as evidenced by the XTT assay in this study, a readout dependent on mitochondrial function. The delay in release of cytochrome c from the mitochondria may be a pivotal step in the protection conferred by hsp72. A delay in cytochrome c release would furthermore slow the activation of caspase 9 as our data demonstrated. It is noteworthy that two recent studies also demonstrate that hsp70, through its chaperoning action, modulates the activity of caspase 9 (27 , 28) . Another hsp, hsp27, has also been shown to interact with and inhibit caspase 9, thus negatively regulating cell death (15) . Thus, hsp72 protection appears to be conferred at two points along this important apoptotic pathway.

Of interest, hsp72 expression did not ameliorate the injurious effects of the chemotherapeutic agent doxorubicin in our C2 cells. Hsp70 overexpression has conferred protection against doxorubicin-induced G2 cell cycle arrest in some cell lines (29) . We speculate these differences may be attributable to high levels of expression of the mdr protein in the C2 cell line that rids cells of xenobiotics by pumping them out. With high mdr expression preventing the toxic intracellular accumulation of chemotherapeutic agents, the actions of hsp72 may be superfluous. However, at very high chemotherapeutic concentrations, mdr and hsp72 protective functions can be overwhelmed.

The lack of effect on cell proliferation was unexpected, as interactions between hsp72 and the p53 have been reported in a number of cell types (17 , 30 , 31) . The p53 protein appears to be a pivotal regulator of cell growth through its role as a transcriptional activator. However, the Caco2 cell line, the parent line of the C2 cells, contains deleted and mutant p53 alleles. DNA damaging agents do not induce p53 in this cell line, suggesting the absence of a functionally active p53 protein. It is tempting to speculate that mutant p53 proteins may lose the ability to suppress transformation; however, they may gain new functions through interactions with other oncogenes, such as ras. We do not know if the mutated p53 in the C2 cells binds hsp72 or cooperates with ras, which is also mutated in Caco2 cells.

In this study, constitutive hsp72 expression in the C2 cell line leads to increased resistance to injury in vitro. Does this have any functional significance for tumor cells in vivo? In vivo, cells such as the monocyte and macrophage synthesize a number of mediators that are designed to injure their targets. Cells in the process of transformation, such as adenomas, may express proteins, allowing them to survive the defenses of the body. Could increased survival ability lead to cell transformation? Increased hsp72 expression has been noted in many potentially transformed cells in tissues as diverse as B lymphocytes (32) , colon (33 , 34) , breast (35 , 36) , ovarian (37) , and oral tissues (18 , 31) . Our studies suggest that increased sustained levels of hsp72 provide malignant cells with a survival advantage, particularly under adverse conditions. Whether this process can be exploited in the treatment of neoplasia remains to be determined.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture and Creation of Hsp72 Antisense Transfected Cells.
C2 (8) cells were grown in DMEM (high glucose) with 10% (volume for volume) fetal bovine serum and used between passages 50 and 80. The intestinal epithelial cell lines RIE-1 (rat small intestine), IEC18 (rat small intestine), and HT-29 (human adenocarcinoma) were also grown in the same medium. Human intestinal epithelial T84 (human adenocarcinoma) cells were grown in DMEM/F12 mix (50:50, volume for volume) with 10% newborn calf serum. Hsp72 was analyzed in all cells by Western blotting of 10 µg of cell protein as described previously (5) using the monoclonal antibodies C92, specific for hsp72, and 1B5, specific for hsc73 (StressGen, Victoria, British Columbia, Canada).

To construct the hsp72 antisense DNA, the full-length human hsp72 cDNA was isolated as a 2.3-kb BamHI–HindIII fragment and cloned in the reverse direction into the eukaryotic expression vector pCEP4. This vector drives insert expression by the strong cytomegalovirus promoter and confers resistance to hygromycin. C2 cells were transfected with DNA constructs (either p2.3AS or pCEP4 control) by electroporation. Five million cells were pulsed at 250 µF at 250 V with 10 µg of DNA. Cells were allowed to equilibrate to 37°C for 10 min and then diluted with complete medium and plated. After 2 days, the cells were selected with fresh medium with the antibiotic hygromycin (250 µg/ml), and after another 2 days, the medium was changed. To allow clones to recover, hygromycin was omitted from the medium, and clones were allowed to grow to 3 mm in diameter. Single clones were trypsinized, propagated, and maintained on 50 µg/ml hygromycin. The basal level of hsp72 was measured in the clones by Western blotting with a specific monoclonal anti-hsp72 antibody as described previously (5) . The level of hsp72 was verified after three passages, and only clones that showed consistent expression were selected for additional study.

Functional and Developmental Assessment.
C2/CEP4 and C2/AS cells were grown on tissue culture-treated Transwells for studies of the morphology of the brush border. Cells were seeded at 100,000 cells/cm2 and then fixed for electron microscopy 2, 14, and 21 days after plating.

Brush border hydrolase activities were measured in cells grown for 2, 7, 14, and 21 days after plating. The expression of the enzymes is maturation dependent and has, therefore, been used as a measure of intestinal epithelial cell differentiation (8 , 9) . Scraped cell pellets were resuspended in homogenization buffer [10 mM Tris (pH 7.4), 3 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin and aprotinin], with protein measured by the Bradford assay (38) , sucrase measured by the microassay procedure of Messer and Dahlquist (39) , and alkaline phosphatase measured by the method of Cox (40) .

Development of tight junctions was functionally assessed by measuring TER using an EVOM (Millipore, Medford, MA).

Proliferation and Viability Studies.
For proliferation studies, C2/CEP4 and C2/AS cells were seeded into 96-well plates at a density of 2000 cells/well. After 2, 4, and 6 days, the number of cells in the wells was measured using the conversion of XTT to a formazan dye, a property of viable mitochondria. Because the number of mitochondria is proportional to the number of cells, the formation of the formazan dye (measured at 490 nm 45 and 90 min after adding reagents) should provide a measure of the number of cells present. In a limited number of experiments, cell counts were performed to confirm that the numbers of cells present correlated with the increases in the XTT signal.

For viability studies, cells were allowed to grow to near confluence (85–90%), usually occurring at 6 days after seeding. The baseline conversion of XTT to formazan was not different between C2/CEP4 and C2/AS cells. Cells were treated with varying concentrations of the oxidant H2O2 (0–3 mM) for 30 min before the addition of the XTT reagents. Conversion to the formazan dye was monitored at 30, 60, 90, and 120 min. To study the effects of TNF and doxorubicin on cell viability, cells were treated for 2 days with varying concentrations of TNF (0–300 ng/ml) or doxorubicin (0–10 µg/ml). XTT reagents were added as above, and conversion to the formazan dye was measured after 60 min. Previous studies from our laboratory demonstrated an excellent correlation between the XTT viability assay and the more cumbersome 51Cr release cytotoxicity assay after oxidant exposure (41) .

Activation of I{kappa}B.
TNF is known to activate the transcription factor NF{kappa}B by stimulating phosphorylation of the regulator I{kappa}B. I{kappa}B inactivation was measured by analyzing for levels of I{kappa}B at varying times after TNF stimulation. Cellular protein lysates were prepared essentially as above. Cell protein (10 µg) was resolved on 10% SDS-PAGE, and Western blots with an I{kappa}B monoclonal (H-4; Santa Cruz Biotechnology, Santa Cruz, CA) was used. Blots were performed and developed as described for hsp Western blots.

Mitochondrial Cytochrome c Release.
Mitochondria were isolated from cells with or without TNF (30 ng/ml) or monochloramine (0.6 mM) for varying times. Cells were scraped into and washed with ice-cold saline and disrupted by 30 strokes with a tight-fitting Teflon pestle homogenizer. The resulting homogenate was centrifuged to obtain nuclei and unbroken cells (750 g for 5 min at 4°C). The resulting supernatant was removed, and mitochondria were separated from this fraction with a Percoll/metrizimide gradient as described previously (42) . For cytosolic fractions, cells incubated under identical conditions were homogenized as above with a Teflon pestle homogenizer and immediately spun in a microultracentrifuge at 100,000 x g for 15 min to obtain the cytosol as the supernatant. Both mitochondrial and cytosolic fractions were immediately prepared for SDS-PAGE after isolation and run on 12.5% SDS-PAGE. Protein concentrations in the mitochondrial and cytosolic fractions were determined by the bicinchoninic acid procedure. The antibody against cytochrome c oxidase was obtained from Transduction Laboratories (Lexington, KY). Cytosolic fractions (10 µmg) or 2 µmg of mitochondrial fractions were used for analysis.

Assay of Cellular Oxidation.
Oxidant generation in C2 cells proved to be problematic using the probe Rhodamine 123. Using attached cells on Leighton tubes designed for the flurometer, dye loading was not sufficient to get signal. To increase cell sample, detached (trypsinized) cells were harvested. However, these cells lost much of the dye when detached from the culture vessels. Therefore, lipid peroxidation was measured as an indicator of cellular oxidant generation using the lipid peroxidation assay kit (Calbiochem, San Diego, CA). Cells (AS and CEP4) were treated with TNF (30 ng/ml) for varying times and harvested for measurement of lipid peroxides in 50 mM Tris (pH 7.4) and 5 mM EDTA. Proteins were measured by the bicinchoninic acid procedure in one aliquot, and the cellular equivalent of 50 µg of protein was added to the reaction for the thiobarbituric acid reaction, which reacts with lipid peroxides producing malondialdehyde (14) . TBARS were measured photometrically using malondialdehyde as standard as directed by the assay manufacturer.

Caspase 9 Activation.
Cytosolic extracts from TNF or monochloramine-stimulated cells were isolated as described for analysis of cytochrome c release. Extracts were immediately assayed for caspase 9 activation using a fluorometric assay and the substrate Ac-Leu-Glu-His-Asp-AMC. A final concentration of a substrate of 50 µmM was used per manufacturer’s directions (Upstate Biotechnology, Lake Placid, NY). Protein (10 µg) was used per assay condition. The fluorescent product AMC was measured in a Hitachi F-2000 spectroflurometer at 460 nm. Caspase 9 activities were expressed as picomole AMC generated per h per mg protein.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the following grants: NIH DK-47722 (to E.B.C.), NIH Digestive Disease Research Core Center Grant (DK-42086), National Cancer Institute Grant (CA-14599; to the Cancer Research Center of the University of Chicago), and the Gastrointestinal Research Foundation of Chicago. Back

2 To whom requests for reprints should be addressed, at The Martin Boyer Laboratories, Department of Medicine, MC 6084, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. Phone: (773) 702-4708; Fax: (773) 702-2281; E-mail: mmusch{at}medicine.bsd.uchicago.edu Back

3 The abbreviations used are: hsp, heat shock protein; C2, Caco2/bbe; C2/AS, hsp72 antisense cells; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt; TER, transepithelial electrical resistance; H2O2, hydrogen peroxide; TNF, tumor necrosis factor; NF{kappa}B, nuclear factor {kappa}B; I{kappa}B, inhibitor of NF{kappa}B; TBARS, thiobarbituric acid reactive substances; mdr, multidrug resistance. Back

Received for publication 9/26/00. Revision received 4/27/01. Accepted for publication 6/ 4/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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