Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa.

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (gammaH2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, approximately 50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and gammaH2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.

Molecular and cellular characterization of imexon-resistant RPMI8226/I myeloma cells.

Imexon is an aziridine-containing iminopyrrolidone with selective growth-inhibitory potency for multiple myeloma. Our previous research indicates that imexon induces mitochondrial alterations, oxidative stress, and apoptosis. This drug represents an interesting model drug with a nonmyelosuppressive profile to study the basic mechanisms leading to antitumor activity and resistance. The major purpose of this study was to characterize an imexon-resistant RPMI8226/I cell line that was developed from RPMI8226 cells by continuous exposure to imexon. No significant differences were observed in the sensitivity to several cytotoxic drugs, including mitoxantrone, mitomycin C, melphalan, methotrexate, cytarabine, cisplatin, vincristine, and paclitaxel, in the imexon-resistant cells. However, RPMI8226/I cells were cross-resistant to arsenic trioxide, doxorubicin, fluorouracil, etoposide, irinotecan, and especially IFN-alpha. The data from DNA microarray and Western blot analyses indicated that the levels of antiapoptotic proteins Bcl-2 and thioredoxin-2, which reside mainly in the mitochondria, are increased in RPMI8226/I cells. In addition, increased levels of lung resistance protein were detected in imexon-resistant cells. Expression of P-glycoprotein was not detected in RPMI8226/I cells. No loss of mitochondrial membrane potential or increase in the levels of reactive oxygen species was observed in RPMI8226/I cells after exposure to imexon; however, the levels of glutathione are increased in the RPMI8226/I cells. Transmission electron microscopy revealed significant changes in the mitochondrial morphology of RPMI8226/I cells, whereas no ultrastructural changes were observed in other cellular compartments. Imexon-resistant RPMI8226/I myeloma cells appear to have a unique mechanism of resistance that is associated with morphological alterations of mitochondria, increased protection against oxidative stress, elevated levels of glutathione, and enhanced expression of antiapoptotic mitochondrial proteins.

Role of mitochondrial complexes I and II, reactive oxygen species and arachidonic acid metabolism in deoxycholate-induced apoptosis.

Bile acids are promoters of colon cancer; however, the mechanism(s) of action of this tumor promoter are largely unknown. Bile acids induce apoptosis in colon epithelial cells and it is probable that the modulation of apoptosis contributes, in part, to colon carcinogenesis. We tested the hypothesis that damage to mitochondria is an upstream event in sodium deoxycholate (NaDOC)-induced apoptosis and that a pro-oxidant state of the cell favors survival. NaDOC-induced damage to mitochondria was assessed by a decrease in mitochondrial membrane potential using flow cytometry and an increase in megamitochondria formation using transmission electron microscopy. We found that inhibition of mitochondrial complexes I and II with rotenone and thenoyltrifluoroacetone, respectively, dramatically protected HT-29 cells against NaDOC-induced apoptosis. Antioxidants (e.g. lazaroids U-74389G and U-8389G), however, sensitized cells to NaDOC-induced apoptosis, in spite of a reduction in reactive oxygen/nitrogen species. Lazaroid pre-treatment caused a marked decrease in NaDOC-induced activation of the anti-apoptotic transcription factor, NF-kappaB, which may provide the basis for the sensitization to apoptosis caused by these antioxidants. Inhibitors of arachidonic acid metabolism (e.g. esculetin, sulindac sulfide, NS-398) also sensitized HT-29 cells to NaDOC-induced apoptosis. These results indicate that the life/death decision is the result of a shift in the balance between specific anti-apoptotic and pro-apoptotic factors, respectively, that may have significance to colon carcinogenesis.

A proteomic study of resistance to deoxycholate-induced apoptosis.

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.

Development and molecular characterization of HCT-116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate.

Evidence from live cell bioassays shows that the flat mucosa from patients with colon cancer exhibits resistance to bile salt-induced apoptosis. Three independent cell lines derived from the colonic epithelial cell line HCT-116 were selected for resistance to bile salt-induced apoptosis. These cell lines were developed as tissue culture models of apoptosis resistance. Selection was carried out for resistance to apoptosis induced by sodium deoxycholate (NaDOC), the bile salt found in highest concentrations in human fecal water. Cultures of HCT-116 cells were serially passaged in the presence of increasing concentrations of NaDOC. The resulting apoptosis resistant cells were able to grow at concentrations of NaDOC (0.5 mM) that cause apoptosis in a few hours in unselected HCT-116 cells. These cells were then analyzed for changes in gene expression. Observations from cDNA microarray, 2-D gel electrophoresis/MALDI-mass spectroscopy, and confocal microscopy of immunofluorescently stained preparations indicated underexpression or overexpression of numerous genes at either the protein or mRNA level. Genes that may play a role in apoptosis and early stage carcinogenesis have been identified as upregulated in these cell lines, including Grp78, Bcl-2, NF-kappaB(p50), NF-kappaB(p65), thioredoxin peroxidase (peroxiredoxin) 2, peroxiredoxin 4, maspin, guanylate cyclase activating protein-1, PKCzeta, EGFR, Ras family members, PKA, PI(4,5)K, TRAF2 and BIRC1 (IAP protein). Under-expressed mRNAs included BNIP3, caspase-6, caspase-3 and serine protease 11. NF-kappaB was constitutively activated in all three resistant cell lines, and was responsible, in part, for the observed apoptosis resistance, determined using antisense oligonucleotide strategies. Molecular and cellular analyses of these resistant cell lines has suggested potential mechanisms by which apoptosis resistance may develop in the colonic epithelium in response to high concentrations of hydrophobic bile acids that are associated with a Western-style diet. These analyses provide the rationale for the development of hypothesis-driven intermediate biomarkers to assess colon cancer risk on an individual basis.