A proteomic study of resistance to deoxycholate-induced apoptosis

Harris Bernstein1,2, Claire M. Payne1,2, Kathleen Kunke1, Cara L. Crowley-Weber1, Caroline N. Waltmire1, Katerina Dvorakova1, Hana Holubec1, Carol Bernstein1,8, Richard R. Vaillancourt3, Deborah A. Raynes6, Vincent Guerriero6,7 and Harinder Garewal2,4,5

1 Department of Microbiology and Immunology, 2 Arizona Cancer Center, 3 Department of Pharmacology and Toxicology and the Center for Toxicology, 4 Department of Internal Medicine, College of Medicine, University of Arizona, Tucson, AZ 85724, USA, 5 Tucson Veterans Affairs Medical Center, Section of Hematology/Oncology, Tucson, AZ 85723, USA, 6 Department of Animal Science and 7 Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA

8 To whom correspondence should be addressed Email: bernstein3{at}earthlink.net


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
The development of apoptosis resistance appears to be an important factor in colon carcinogenesis. To gain an understanding of the molecular pathways altered during the development of apoptosis resistance, we selected three cell lines for resistance to induction of apoptosis by deoxycholate, an important etiologic agent in colon cancer. We then evaluated gene expression levels for 825 proteins in these resistant lines, compared with a parallel control line not subject to selection. Eighty-two proteins were identified as either over-expressed or under-expressed in at least two of the resistant lines, compared with the control. Thirty-five of the 82 proteins (43%) proved to have a known role in apoptosis. Of these 35 proteins, 21 were over-expressed and 14 were under-expressed. Of those that were over-expressed 18 of 21 (86%) are anti-apoptotic in some circumstances, of those that were under-expressed 11 of 14 (79%) are pro-apoptotic in some circumstances. This finding suggests that apoptosis resistance during selection among cultured cells, and possibly in the colon during progression to cancer, may arise by constitutive over-expression of multiple anti-apoptotic proteins and under-expression of multiple pro-apoptotic proteins. The major functional groups in which altered expression levels were found are post-translational modification (19 proteins), cell structure (cytoskeleton, microtubule, actin, etc.) (17 proteins), regulatory processes (11 proteins) and DNA repair and cell cycle checkpoint mechanisms (10 proteins). Our findings, overall, bear on mechanisms by which apoptosis resistance arises during progression to colon cancer and suggest potential targets for cancer treatment. In addition, assays of normal-appearing mucosa of colon cancer patients, for over- or under-expression of genes found to be altered in our resistant cell lines, may allow identification of early biomarkers of colon cancer risk.

Abbreviations: DOC, deoxycholic acid; 5-LOX, 5-lipoxygenase; MEK-2, mitogen-activated protein kinase kinase 2; NaDOC, sodium deoxycholate; PI3K, phosphatidylinositol 3-kinase; PKR, protein kinase R


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Malignant transformation of colorectal epithelial cells to colorectal cancer involves genetic and environmental factors. Diet appears to be a major environmental factor in colon carcinogenesis. A high fat diet, as is typical of Western societies, is associated with an increased risk of colorectal cancer (1). An underlying cause of this association appears to be the elevated level of bile acids in the colon of Westerners (1). Deoxycholic acid (DOC) is the bile acid present at highest concentration in the human colon (2), and sodium deoxycholate (NaDOC) is especially high in individuals with adenomatous polyps and colon cancer (3). In a rat model of colon cancer, bile acids have a tumor promoting effect (4). After humans ingest high levels of dietary fat, DOC in fecal water ranges up to 0.73 mM (5). We, and others, found that DOC, at 0.10–1.0 mM, induces apoptosis in colonic epithelial cells in vitro or ex vivo using colonic biopsies (68). Furthermore, we showed that epithelial cells of the flat mucosa in the colons of individuals with colon cancer have reduced capacity to undergo bile salt-induced apoptosis (8,9). This is in accord with the recent finding that patients with colorectal cancer, but generally not other individuals, shed apoptosis-resistant cells into their stools (10).

Decreased ability to undergo apoptosis is a risk factor in colon carcinogenesis (8,9,11,12). Loss of apoptosis capability is associated with increased genomic instability (e.g. aneuploidy, point mutations, loss of heterozygosity) (13). Increased genomic instability may include mutations affecting expression of genes related to apoptosis and allow for further selection for apoptosis resistance leading to colon carcinogenesis (14,15). We have proposed that frequent exposure of the colonic epithelium of an individual to high concentrations of cytotoxic bile acids can select for an apoptosis-resistant cell population, making that individual at risk for colon cancer (8,9).

The experiments described here were performed to clarify the mechanisms by which apoptosis resistance can arise. Although apoptosis resistance could well arise to a variety of cytotoxic agents, we chose to investigate resistance to DOC-induced apoptosis. This choice was made since DOC occurs at apoptosis-inducing concentrations in response to high fat diets, and resistance to DOC-induced apoptosis was found to characterize the normal-appearing flat colonic mucosa of patients at increased risk for colon cancer (8,9). We selected for apoptosis resistance by exposing apoptosis-competent cells (reflective of the normal in vivo situation) to increasing concentrations of the cytotoxic bile salt NaDOC (16). Numerous gene products have been reported to have effects on apoptosis. It was initially not known if, during the development of resistance to apoptosis, few or many changes in gene expression would occur, and whether these changes would be similar or divergent among independently selected lines. Thus, selection was performed on three serially passaged, independent cultures of HCT-116.

A previous analysis of these apoptosis-resistant cell lines by our laboratory utilized 2-D gel electrophoresis/MALDI-mass spectroscopy to detect differences in abundantly expressed proteins (16). We identified five proteins with altered expression in that study. We have now greatly extended our proteomic study to include a large number (825) of monoclonal antibodies in conjunction with western blot analyses, so that we can detect differences in expression of a wide range of proteins. Significant increases or decreases in gene expression, in at least two of the three independent resistant cell lines (and therefore likely to be generally relevant to apoptosis resistance) were found for 82 proteins. We first grouped these 82 over- or under-expressed proteins into functional categories. We then identified 35 as having a previously reported role in apoptosis, and six as specifically responding to bile acids. We also illustrated probable pathways by which the apoptosis-related and DNA repair-related proteins may give rise to apoptosis resistance through their changed levels of expression. These findings may be applicable to development of early biomarkers of colorectal cancer risk. In addition, the over- and under-expressed proteins may suggest potential targets for cancer treatment.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cell lines, media and chemicals
HCT-116, a colon adenocarcinoma cell line [American Type Culture Collection (ATCC), Manassas, VA; ATCC # CCL 247] and apoptosis-resistant HCT-116 cell lines developed in our laboratory (16) were maintained in DMEM supplemented with 10% fetal calf serum (Omega Scientific, Tarzana, CA), 1% MEM non-essential amino acids, 100 µg/ml streptomycin, 100 U/ml penicillin and 3.44 mg/ml L-glutamine. Media components were from Gibco BRL Life Technologies (Grand Island, NY).

Apoptosis-resistant cell lines
The development of these cell lines was described previously (16), and is briefly summarized here. Early passage HCT-116 cells were split and seeded into four culture flasks. The cells in flask A were not exposed to NaDOC and serially passaged as an untreated control along with the other three cultures. After 48 h, the other three culture flasks (B, C and D) were treated with NaDOC. Initially, B cells were treated with 0.02 mM NaDOC, C cells with 0.1 mM NaDOC and D cells with 0.2 mM NaDOC. Treatment lasted for 48 h, after which the medium plus NaDOC was removed and fresh medium added. The remaining attached cells were allowed to grow until they reached ~80% confluency, after which they were split and passaged. Cells of the A line were passaged weekly, while the NaDOC treated cells took longer to recover from the NaDOC treatment and so were passaged less often. When the cells in a particular flask could survive a 48-h treatment with NaDOC, the concentration used to treat them in the following passage was increased. This was continued until all three lines were determined to be resistant to 0.5 mM NaDOC. This procedure was followed for a duration of 40–46 weeks at which time it was determined that the cell lines were stably resistant to NaDOC-induced apoptosis (16). That is, after growth for at least 4 weeks in non-selective conditions, in media without NaDOC, lines B, C and D were re-tested and still resistant to 0.5 mM NaDOC.

Preparation of lysates
Briefly, HCT-116 long-passage-sensitive and NaDOC-resistant cells were lysed using lysis buffer [50 mM Tris (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 3 mM phenylmethylsulfonyl fluoride, 3 µg/ml leupeptin and 3 µg/ml aprotinin] as described previously (17). Protein concentrations were determined using the bicinchonic acid system (Pierce Biotechnology, Rockford, IL). Samples were diluted to 2 mg/ml using lysis buffer and sent overnight on dry ice to BD Transduction Laboratories (Lexington, KY).

PAGE and western blotting
In order to accommodate the 825 different monoclonal antibodies used in the PowerBlotTM western array, five gels and western blots were run for each lysate (procedures performed at BD Transduction Laboratories). In addition, each cell lysate was run in triplicate. Each gel is 16 x 16 cm, 5–15% gradient SDS–polyacrylamide, and 1 mm thick. A gradient system allows a wide range of proteins to be detected on one gel. 400 µg of protein were loaded in one well across the entire width of the gel. (This is the equivalent of ~15 µg/lane on a standard 25 well gel.) The gel is run overnight at constant milliamps. The proteins separated in the gel were transferred to an Immobilon-P membrane (Millipore, Bedford, MA) overnight at 200 mA with the TE Series wet electrophoretic transfer apparatus (Hoefer/Amersham Biosciences, Piscataway, NJ). After transfer, the membrane was blocked for 1 h with 5% milk. Next, the membrane was clamped with a western blotting manifold that isolates 45 channels across the membrane. In each channel, a cocktail of one to seven monoclonal antibodies was added and allowed to hybridize for 1 h. The blot was removed from the manifold, washed and hybridized for 30 min with horseradish peroxidase-conjugated goat anti-mouse secondary antibody. The membrane was washed and developed with the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). A cocktail of the following protein standards was added to lane 45: Adaptin-ß, 106 kDa; STAT-3, 92 kDa; PTP1D, 72 kDa; mitogen-activated protein kinase kinase 2 (MEK-2), 46 kDa; RACK-1, 36 kDa; GRB-2, 24 kDa; Rap2, 21 kDa. Each step was carried out at room temperature.

A separate western blot was performed to confirm relative levels of one protein, HspBP1, in resistant cell line HCT116C and sensitive cell line HCT116A. In this assay, the antibody used was from Novus Biologicals, Littleton, CO.

Data analysis
Electronic images of blots were captured using the OdysseyTM Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Images were subjected to automatic spot finding and spot matching using PDQuest 2-D Analysis Software (Bio-Rad Laboratories, Hercules, CA). The data analyses of densitometry and confidence level of changes in protein expression levels were performed at BD Transduction Laboratories. Each comparison of a protein in a sample from a resistant cell line to the level of the same protein in the sensitive cell line was assigned a semi-quantitative ‘fold change value’. These fold changes were also assigned a confidence level. The highest confidence level of 5 corresponded to changes >2-fold in triplicate from good quality signals; 4 was for changes 1.5–1.9-fold in triplicate from good quality signals; 3 was for changes >2-fold in triplicate from low signals; 2 was for changes 1.25–1.5-fold in triplicate; and 1 was for changes >2-fold in duplicate from good quality signals. We then added our own requirement that, to be considered as important for further study of apoptosis resistance, a protein must have changed in the same direction (up or down) in at least two of the three independent apoptosis-resistant cell lines.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Three HCT-116 cell lines, designated B, C and D, resistant to NaDOC-induced apoptosis were selected by a process started with three different concentrations of NaDOC, as described in the Materials and methods. Thus, our resistant populations probably evolved towards apoptosis resistance by somewhat different pathways. We then tested the apoptosis-resistant cell lines for over- and under-expressed proteins, in comparison with cells, line A, that had been grown in parallel with the resistant lines, and which maintained the sensitivity to NaDOC-induced apoptosis of the parental line of HCT 116 cells. The proteomic analysis was performed three times, in each analysis comparing a sample of protein isolated from one of the three resistant cell lines to a sample of protein isolated from the sensitive cell line. Protein isolation from each of the cell lines A, B, C and D was done once.

Although 825 antibodies were used to test for over- or under-expression, only 454 of the corresponding proteins were detected in cell lines A, B, C or D. Of the 454 proteins detected, 213 were not significantly changed in any of the three resistant lines. Of the 241 proteins with significant changes, 91 proteins were significantly over- or under-expressed in at least two of the three resistant cell lines. Of these, 43 were significantly over-expressed, 39 were significantly under-expressed and nine were significantly over-expressed in at least one resistant line and significantly under-expressed in at least one resistant line. We focused on the 82 (43 + 39) proteins that were consistently over- or under-expressed in at least two of the three resistant lines (and were therefore most likely to be of general importance to apoptosis resistance). Figure 1 shows examples of some of these over- and under-expressed protein spots.



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Fig. 1. Comparison of western blots from apoptosis-sensitive cell line HCT-116 A and apoptosis-resistant cell lines HCT-116 B, C and D. Cell lysates from apoptosis-sensitive cell line A, and NaDOC resistant cell lines B, C and D were screened for changes in the levels of protein expression. Results from four gels, all from gel ‘Template C’, from a Power BlotTM Western Array (BD Transduction Laboratories, Lexington, KY) are shown. Some of the proteins [gelsolin, PI3K, protein kinase R (PKR), MEK2, 5-LOX], which are listed in Tables IGoGoIV and are significantly increased in two or more resistant cell lines are circled and named on these blots.

 

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Table I. Proteins over- or under-expressed in at least two of the three resistant cell linesa

 

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Table II. Proteins that are increased in resistant cells and reported in the literature to have a role in apoptotsis

 

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Table III. Proteins that are decreased in resistant cells and reported in the literature to have a role in apoptosis

 

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Table IV. Proteins over- or under-expressed in at least two of the three resistant cell lines and reported to be related to deoxycholate or bile acids

 
Two of us (D.A.R. and V.G.) were the discoverers of one of the proteins, HspBP1 (18), which was under-expressed, by our proteomics analysis, in apoptosis-resistant cell lines HCT116B and HCT116C. Thus, we further analyzed HspBP1 by an independent western blot assay. Using a Novus Biologicals antibody, we confirmed that HspBP1 was under-expressed in apoptosis-resistant cell line HCT116C compared with apoptosis-sensitive cell line HCT116A. Further, using an ELISA assay, we quantified the levels of HspBP1 as 11.3 ng/mg total protein in HCT116A and the under-expressed amount of 4.0 ng/mg total protein in HCT116C.

The proteins can be grouped into functional categories as indicated in Table I. The nine proteins that were significantly changed in at least two of the three resistant lines, but whose changes were in opposite directions, i.e. over-expressed in one line but under-expressed in another, are not listed in the table. Table I also indicates in which resistant line (B, C or D) an increase (+) or decrease (–) in gene expression was observed, as well as a brief description of each protein's function. Nine of the proteins are listed twice in the table, because they fall into two different functional categories.

As shown in Table I, the largest general functional category (19 proteins) includes enzymes that catalyze post-translational modification of proteins, such as kinases [e.g. in Figure 1, see increased expression of PI3K (phosphatidylinositol 3-kinase) and MEK2 (mitogen-activated protein kinase kinase 2)], phosphatases, farnesylating and ubiquitin conjugating enzymes. We have also indicated pathways in which some of these proteins are active in Figure 2 (and some of these proteins and pathways are discussed at greater length in the Supplementary Material Online). The next largest class (17 proteins) includes structural proteins such as those involved in cytoskeleton and microtubule assembly. The next functional groups in Table I are regulatory proteins such as transcription factors (11 proteins), DNA repair and cell cycle checkpoint control (10 proteins), and proteins employed in lysosomes, Golgi, endocytosis and exocytosis (10 proteins). The remaining 24 proteins were distributed into 10 functional classes. The number of proteins (n = 82) that behaved consistently in the selected lines is much greater than those that behaved inconsistently (n = 9). This observation agrees with the expectation that the over- or under-expression of proteins in the resistant lines should ordinarily be causally related to the apoptosis-resistance phenotype rather than being a random change.



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Fig. 2. Diagram of some established signal-transduction pathways responsible for the induction of, or protection from, apoptosis induced by hydrophobic bile acids. Proteins named in red are over-expressed and those named in blue are under-expressed in at least two of the apoptosis-resistant cell lines B, C or D. The signaling pathways that involve mitochondrial damage, ER stress and the activation of caspases that probably lead to cell death are shown. Some of the potential anti-apoptotic pathways shown include the EGFR/MAPK pathway, NF-{kappa}B activation and transcription of anti-apoptotic genes, increased nitric oxide synthase (NOS) activity with the generation of the anti-apoptotic molecule NO, and ionic changes that prevent apoptosome formation. See Supplementary Material Online for more details on these signaling pathways.

 
Over- or under-expressed proteins involved in apoptosis
The proteins with altered expression, listed in Table I, have the major functions indicated there. However, using PubMed, we identified an apoptosis-related function, in addition, for many of these proteins. Our expectation was that previous work on the role of these proteins in apoptosis might provide insight into how apoptosis resistance arose in the resistant cell lines and, more importantly, how it might arise during progression to colon cancer. For a subset of 35 proteins of the total 82 (43%), evidence in the literature indicates a role in apoptosis. Of these 35 proteins, 21 were over-expressed (Table II) and 14 were under-expressed (Table III).

Among the 21 over-expressed proteins with a role in apoptosis, evidence indicates that 12 are anti-apoptotic, six are either anti- or pro-apoptotic depending on specific conditions such as cell type and different apoptosis-inducing agents, and three are pro-apoptotic. Table II gives a brief description and references to the apoptotic behavior of these proteins. The fact that 86% of these over-expressing proteins have anti-apoptotic effects is consistent with the expectation that the apoptosis resistance phenotype results, in part, from over-expression of anti-apoptotic proteins. It is also of interest that four of the 12 over-expressed, anti-apoptotic proteins (5-LOX, PI3K, P-cadherin and cyclin D3) are reported to be present at an increased level in cancer cells (see Table II for references), which may reflect the development of apoptosis resistance in these cancers. The possible role of two of these proteins (5-LOX and PI3K) in apoptosis resistance is indicated in Figure 2.

Among the 14 under-expressed proteins, four proteins were pro-apoptotic, seven were pro- or anti-apoptotic depending on circumstance and three were anti-apoptotic. The proteins, which are pro-apoptotic and under-expressed may contribute to the apoptosis-resistance of these cell lines.

It is not surprising that some proteins are observed to be anti-apoptotic and pro-apoptotic under different circumstances. We have reviewed evidence recently for a class of bi-functional proteins that promote DNA repair and cell cycle checkpoint control (anti-apoptotic functions) but also respond to high levels of DNA damage by inducing apoptosis (19). hRad9 (Table II) appears to be an example of such a protein.

Over- or under-expressed proteins with a functional relationship to bile acids
Since we used NaDOC to obtain the apoptosis-resistant lines, we explored the literature on the 82 over- or under-expressed proteins to learn if any had a functional relationship to bile acids. The six proteins found with such a relationship (and references) are given in Table IV.

Bid appears to be necessary for bile acid-induced apoptosis (Figure 2) and is under-expressed in the apoptosis-resistant lines (Table I). Bile acid activation of JNK-1 may induce apoptosis (Figure 2) and it is under-expressed in the resistant lines (Table I). PI3K is activated by bile acids, inhibits apoptosis (Figure 2) and is over-expressed in the resistant lines (Table I and Figure 1). 5-LOX helps to protect against deoxycholate-induced apoptosis (Figure 2) in colon epithelial cells and is over-expressed in all three resistant lines (Table I and Figure 1). The two other proteins with a relationship to bile acids (Table IV), LXR an oxysterol receptor, and sodium potassium ATPase (Na+,K+-ATPase), which regulates K+ ions in the cytosol, are described further near the end of the Supplementary Material Online.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
The apoptosis resistance phenotype selected for in our three cell lines B, C and D is a stable feature, persisting for at least a month after removal of NaDOC. Thus, the resistance phenotype is probably due to localized mutations, epigenetic changes (e.g. alterations in methylation patterns) and/or aneuploidy. Mutations or epigenetic alterations may be within the promoter or the coding region of genes whose expression levels have changed or within gene(s) regulating their expression. A mutation or epimutation in a key regulatory protein or transcription factor, or aneuploidy, may affect expression of many proteins.

Because mutations/epimutations/aneuploidy occur randomly, the sequence of their occurrence is likely to have differed between the three resistant cell lines. In addition, since the initial exposures to NaDOC differed for the three lines, the initial selective pressures also differed. Thus, the patterns of gene expression in the three resistant lines might be expected to differ from each other. In fact, 57 of the 82 proteins listed in Table I are only over- or under-expressed in two of the three apoptosis-resistant cell lines. This indicates that there is more than one assortment of altered protein patterns that can give rise to high levels of apoptosis resistance.

The 82 proteins altered in either two or three of the apoptosis-resistant cell lines are likely to be of general interest in apoptosis resistance, since they occurred in multiple cell populations during development of apoptosis resistance. The 57 proteins that were under- or over-expressed in only two of the three cell populations, however, may only sometimes be altered during selection for apoptosis resistance. It is important to note, nevertheless, that a substantial overlap in gene expression was found among the three independent apoptosis- resistant populations, which implies that the changes that can provide effective apoptosis-resistance in these particular cell types are limited.

Thus, the observed changes in protein levels in our resistant cell lines may shed light on the alterations in gene expression that are responsible for the observed apoptosis resistance in the flat mucosa of colon cancer patients (8,9). In particular, one of the proteins with increased expression in the apoptosis- resistant lines, FPTase {alpha} (farnesyl transferase) (Figure 2, Tables I and II) is the target of farnesyl transferase inhibitors currently in Phase II clinical trials as anticancer agents (20,21). Other proteins with altered expression may also be potential targets of anticancer agents or may have potential as biomarker(s) for early detection of colon cancer risk.

Of the 82 significantly and concordantly over- or under-expressed proteins, 35 proteins (43%) are reported to have a role in apoptosis. Of these 35 proteins, 21 are over-expressed and most (18/21) have an anti-apoptotic effect under some conditions. Among the 14 proteins under-expressed in the resistant lines, most (11/14) had a pro-apoptotic effect under some conditions. Thus, the increase in resistance to bile acid-induced apoptosis that occurs during progression to colon cancer may similarly be accompanied by an increase in the levels of numerous anti-apoptotic proteins and decreases in numerous pro-apoptotic proteins.

Although we first assigned the 82 over- and under-expressed proteins to 15 functional categories (Table I), another way of organizing their likely roles in apoptosis resistance is to place them in known pathways leading to increases or decreases in apoptosis. Figure 2 schematically indicates some well-known signal transduction pathways, which are likely to be responsible for the induction of apoptosis by hydrophobic bile acids. Evidence for some of these pro-apoptotic pathways has been presented previously by us and include mitochondrial (17) and endoplasmic reticulum (ER) stress response pathways (16,22). The proteins that were found to be decreased in the present study are indicated in blue in Figure 2.

Some of the possible pathways that are anti-apoptotic (blocking apoptosis and/or increasing survival) based on our published work and that of others are also indicated in Figure 2 (16,17,23). In particular, we reported that the activities of the cyclooxygenases, lipooxygenases and nitric oxide synthases and the activation of NF-{kappa}B protected cells against NaDOC-induced apoptosis. The proteins that were found increased in the present study are indicated in red in Figure 2. In the Supplementary Material Online we expand on the likely roles of some of the signal transduction proteins (with altered expression in our resistant cell lines) in the development of the apoptosis resistance phenotype.

In addition to signal transduction pathways, we also used DNA repair and check point pathways as a second area for organizing our understanding of the roles of the over- and under-expressed proteins. There were 10 proteins, among our 82, with DNA repair and cell cycle checkpoint functions (Table I). Defective expression of these types of protein is thought to often give rise to chromosome instability and to accelerate progression to cancer. The modes of action and relationships of these proteins are summarized in Figure 3. Four genes directly involved in DNA repair are under-expressed (shown in blue in the figure). These are hMSH3 (mismatch repair), XPA and p36 (nucleotide excision repair), and Ku80 (non-homologous end joining). Six genes have a role in cell cycle check point regulation. Three are over-expressed (shown in red in the figure), hRad9, Cyclin D3 and PI3K, and three are under-expressed (blue), checkpoint kinase 1 (Chk1), cyclin A and mitotic arrest-deficient 2 (MAD2). In the Supplementary Material Online we expand upon the probable roles of these 10 proteins in the development of the apoptosis resistance phenotype.



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Fig. 3. DNA repair and cell cycle checkpoint proteins that are over- or under-expressed in the apoptosis-resistant cell lines. Those named in red are over-expressed and those named in blue are under-expressed. Some of the modes of action and relationships of these proteins are indicated.

 
A third area in which our over- and under-expressed proteins can be organized is that of the secondary lysosomes. Previously, we presented electron microscopic evidence for the presence of large secondary lysosomes in all three apoptosis-resistant cell lines (16). Secondary lysosomes arise by the fusion of small primary lysosomes with damaged cellular organelles, cytoskeletal elements, ubiquitinated proteins or organelles, which provide a general housekeeping function, including fusion with endocytotic vesicles. Table I lists seven proteins associated with lysosomes or endocytototic vesicles. Five of these are over-expressed in at least two of the three resistant cell lines (cathepsin D, dynamin, lamp-1, PCLase and rab5), and two are under-expressed (adaptin {delta}, cathepsin L). These results suggest that some elements of apoptosis resistance are associated with altered lysosomal activity.

Overall, it appears that expression levels of multiple proteins are changed upon selection for apoptosis resistance in cultured colon epithelial cells. Similar changes may occur in the progression to colon cancer.


    Supplementary material
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Supplementary material can be found at http://www.carcin.oupjournals.org


    Acknowledgments
 
We acknowledge the assistance of Jennifer Stevenson of BD Transduction Laboratories with the statistical analysis of the protein changes associated with the Powerblots. This work was supported in part by NIH Institutional Core Grant #CA23074, NIH PPG #CA72008, Arizona Disease Control Research Commission Grants #10016 and #6002, VAH Merit Review Grant 2HG, and Biomedical Diagnostics and Research, Tucson, AZ.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received August 18, 2003; revised December 16, 2003; accepted December 19, 2003.





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