Idiopathic granulomatous mastitis: experience at a New York hospital.

INTRODUCTION: Idiopathic granulomatous mastitis (IGM) often mimics breast cancer. Presentation includes pain, palpable mass, suppuration or suspicious imaging. Widely reported in Asia and the Middle East, IGM is diagnosed after excluding specific granulomatous mastitis (SGM). Aetiology remains unknown. Lactation, prolactinaemia, ethnicity, autoimmune disease and Corynebacteria are associated. Treatment is controversial and the prevalence rising. Surgery and non-operative treatments including antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs), steroids, methotrexate and observation have advocates. METHODS: A retrospective chart review of 63 patients with IGM from 2008 to 2018 was undertaken focusing on birthplace, age, clinical presentation, wound cultures, imaging, treatments and outcomes. RESULTS: Sixty-one of 63 patients were Hispanic; 53 were Mexican-born women aged 23-46. Clinical presentation included pain, painful mass, painless mass, suppuration and abnormal imaging. Some 31/61 ultrasound examinations and 17/33 mammograms were deemed Breast Imaging Reporting and Data System (BI-RADS) score 4 or 5. Management included antibiotics (43), incision and drainage (24), NSAIDs (29), steroids (8), lumpectomy (18) and observation (12). Some 12/20 patients with painless masses resolved with observation, 3 received NSAIDs, 2 received steroids and 3 underwent lumpectomies. Antibiotics resolved 8/43 cases, 5 needed incision and drainage, 26 received NSAIDs, 6 received steroids and 5 underwent lumpectomies. Nineteen patients had indolent disease or recurrence. CONCLUSIONS: Excluding malignancy is critical, treatment challenging and recurrence common in IGM. A preponderance of patients were Mexican-born, similar to other reports from the USA. Over 50% of IGM cases had suspicious BI-RADS scores. Best management remains a challenge and ranges from observation to lumpectomy.

The prognostic impact of consensus molecular subtypes (CMS) and its predictive effects for bevacizumab benefit in metastatic colorectal cancer: molecular analysis of the AGITG MAX clinical trial.

Background: The consensus molecular subtypes (CMS) is a transcriptome-based classification of colorectal cancer (CRC) initially described in early-stage cohorts, but the associations of CMS with treatment outcomes in the metastatic setting are yet to be established. This study aimed to evaluate the prognostic impact of CMS classification and its predictive effects for bevacizumab benefit in metastatic CRC by correlative analysis of the AGITG MAX trial. Patients and methods: The MAX trial previously reported improved progression-free survival (PFS) for the addition of bevacizumab (B) to chemotherapy [capecitabine (C)±mitomycin (M)]. Archival primary tumours from 237 patients (50% of trial population) underwent gene expression profiling and classification into CMS groups. CMS groups were correlated to PFS and overall survival (OS). The interaction of CMS with treatment was assessed by proportional hazards model. Results: The distribution of CMS in MAX were CMS1 18%, CMS2 47%, CMS3 12%, CMS4 23%. CMS1 was the predominant subtype in right-sided primary tumours, while CMS2 was the predominant subtype in left-sided. CMS was prognostic of OS (P = 0.008), with CMS2 associated with the best outcome and CMS1 the worst. CMS remained an independent prognostic factor in a multivariate analysis. There was a significant interaction between CMS and treatment (P-interaction = 0.03), for PFS, with hazard ratios (95% CI) for CB+CBM versus C arms in CMS1, 2, 3 and 4: 0.83 (0.43-1.62), 0.50 (0.33-0.76), 0.31 (0.13-0.75) and 1.24 (0.68-2.25), respectively. Conclusions: This exploratory study found that CMS stratified OS outcomes in metastatic CRC regardless of first-line treatment, with prognostic effects of CMS groups distinct from those previously reported in early-stage cohorts. In CMS associations with treatment, CMS2 and possibly CMS3 tumours may preferentially benefit from the addition of bevacizumab to first-line capecitabine-based chemotherapy, compared with other CMS groups. Validation of these findings in additional cohorts is warranted. Clinical trial number: This is a molecular sub-study of MAX clinical trial (NCT00294359).

Vascular endothelial growth factor D expression is a potential biomarker of bevacizumab benefit in colorectal cancer.

BACKGROUND: Bevacizumab prolongs progression-free survival (PFS) in patients with metastatic colorectal cancer. We analysed the protein expression levels of vascular endothelial growth factor (VEGF) ligands and receptors to determine their prognostic and predictive effects. METHODS: We graded expression of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-R1, and VEGF-R2 to assess whether overexpression predicted bevacizumab resistance in samples from 268 of 471 patients randomised to capecitabine (C), capecitabine and bevacizumab (CB), or CB and mitomycin (CBM) in the MAX trial and extended the analysis to the CAIRO-2 population. RESULTS: Patients with low expression of VEGF-D (0, 1þ) benefited from bevacizumab treatment (PFS hazard ratio (HR) (C vs CBþCBM), 0.21; 95% CI, 0.08­0.55; overall survival (OS) HR, 0.35; 95% CI, 0.13­0.90). Patients with higher VEGF-D expression received less benefit (VEGF-D 2þ PFS HR, 0.67; 95% CI, 0.45­1.00; OS HR, 0.82; 95% CI, 0.52­1.30; VEGF-D 3þ PFS HR, 0.77; 95% CI, 0.50­1.17; OS HR, 1.28; 95% CI, 0.79­2.09) (P interaction o0.05). In CAIRO-2, there was no difference in PFS or OS according to VEGF-D expression. CONCLUSIONS: The predictive value of VEGF-D expression for bevacizumab may depend on the chemotherapy backbone used. Further evaluation is required before clinical utilisation.

Telomere length is a novel predictive biomarker of sensitivity to anti-EGFR therapy in metastatic colorectal cancer.

BACKGROUND: Telomeres are TTAGGG tandem repeats capping chromosomal ends and partially controlled by the telomerase enzyme. The EGFR pathway putatively regulates telomerase function, prompting an investigation of telomere length (TL) and its association with anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (mCRC). METHODS: Colorectal cancer cell lines were treated with multiple drugs and sensitivity determined. Clinical information was gathered from 75 patients who had received anti-EGFR drugs. Telomere length was measured using a validated qRT-PCR technique. RESULTS: In CRC cell lines, TL independently predicted cetuximab sensitivity. Cells with shorter TL had growth inhibition of 18.6±3.41% as compared with 41.39±8.58% in longer TL (P=0.02). These in vitro findings were validated clinically, in a robust multivariate model. Among patients with KRas WT tumours, those with longer TL had a superior median progression-free survival (PFS) of 24.9 weeks than those with shorter TL; median 11.1 weeks, HR 0.31; P=0.048. CONCLUSION: Telomere length could be a potential unique biomarker predictive of clinical benefit (PFS) of mCRC patients treated with anti-EGFR therapy. This is the novel demonstration of a complex hitherto undescribed interaction, placing anti-EGFR therapy, EGFR pathway, and the telomerase complex within a clinical context.

PR55α-containing protein phosphatase 2A complexes promote cancer cell migration and invasion through regulation of AP-1 transcriptional activity.

The proto-oncogene c-Jun is a component of activator protein-1 (AP-1) transcription factor complexes that regulates processes essential for embryonic development, tissue homeostasis and malignant transformation. Induction of gene expression by c-Jun involves stimulation of its transactivation ability and upregulation of DNA binding capacity. While it is well established that the former requires JNK-mediated phosphorylation of S63/S73, the mechanism(s) through which binding of c-Jun to its endogenous target genes is regulated remains poorly characterized. Here we show that interaction of c-Jun with chromatin is positively regulated by protein phosphatase 2A (PP2A) complexes targeted to c-Jun by the PR55α regulatory subunit. PR55α-PP2A specifically dephosphorylates T239 of c-Jun, promoting its binding to genes regulating tumour cell migration and invasion. PR55α-PP2A also enhanced transcription of these genes, without affecting phosphorylation of c-Jun on S63. These findings suggest a critical role for interplay between JNK and PP2A pathways determining the functional activity of c-Jun/AP-1 in tumour cells.

Role of the faecolith in modern-day appendicitis.

INTRODUCTION: The prevailing view on appendicitis is that the main aetiology is obstruction owing to faecoliths in adults and lymphoid hyperplasia in children. Faecoliths on imaging studies are believed to correlate well with appendicitis. METHODS: A retrospective chart review was conducted of 1,014 emergency appendicectomy patients between 2001 and 2011. Faecolith prevalence in adult and paediatric appendicectomy specimens with and without perforation was studied. The sensitivity and positive predictive value (PPV) of computed tomography (CT) for identifying faecoliths in the pathology specimen were examined. RESULTS: Overall, faecoliths were found in 18.1% (178/986) of appendicitis specimens and 28.6% (8/28) of negative appendicectomies. Faecolith prevalence for positive cases was 29.9% (79/264) in paediatric patients and 13.7% (99/722) in adults (p<0.05). Faecolith prevalence was 39.4% in perforated appendicitis but only 14.6% in non-perforated appendicitis (p<0.05). In adults, faecolith prevalence was 27.5% in perforated appendicitis and 12.0% in non-perforated appendicitis (p<0.05) while in paediatric patients, it was 56.1% in perforated appendicitis and 22.7% in non-perforated appendicitis (p=0.00). Sensitivity and PPV of preoperative CT in identifying faecoliths on pathology were 53.1% (86/162) and 44.8% (86/192) respectively. CONCLUSIONS: Faecolith prevalence is too low to consider the faecolith the most common cause of non-perforated appendicitis. Faecoliths are more prevalent in paediatric appendicitis than in adult appendicitis. Preoperative CT is an unreliable predictor of faecoliths in pathology specimens.

ß-catenin negatively regulates expression of the prostaglandin transporter PGT in the normal intestinal epithelium and colorectal tumour cells: a role in the chemopreventive efficacy of aspirin?

BACKGROUND: Levels of the pro-tumorigenic prostaglandin PGE(2) are increased in colorectal cancer, previously attributed to increased synthesis through COX-2 upregulation and, more recently, to decreased catabolism. The functionally linked genes 15-prostaglandin dehydrogenase (15-PGDH) and the prostaglandin transporter PGT co-operate in prostaglandin degradation and are downregulated in colorectal cancer. We previously reported repression of 15-PGDH expression by the Wnt/ß-catenin pathway, commonly deregulated during early colorectal neoplasia. Here we asked whether ß-catenin also regulates PGT expression. METHODS: The effect of ß-catenin deletion in vivo was addressed by PGT immunostaining of ß-catenin(-/lox)-villin-cre-ERT2 mouse tissue. The effect of siRNA-mediated ß-catenin knockdown and dnTCF4 induction in vitro was addressed by semi-quantitative and quantitative real-time RT-PCR and immunoblotting. RESULTS: This study shows for the first time that deletion of ß-catenin in murine intestinal epithelium in vivo upregulates PGT protein, especially in the crypt epithelium. Furthermore, ß-catenin knockdown in vitro increases PGT expression in both colorectal adenoma- and carcinoma-derived cell lines, as does dnTCF4 induction in LS174T cells. CONCLUSIONS: These data suggest that ß-catenin employs a two-pronged approach to inhibiting prostaglandin turnover during colorectal neoplasia by repressing PGT expression in addition to 15-PGDH. Furthermore, our data highlight a potential mechanism that may contribute to the non-selective NSAID aspirin's chemopreventive efficacy.

Negative appendicectomy rate as a quality metric in the management of appendicitis: impact of computed tomography, Alvarado score and the definition of negative appendicectomy.

INTRODUCTION: The negative appendicectomy rate (NAR) is a quality metric in the management of appendicitis. While computed tomography (CT) has been associated with a low NAR, Alvarado scoring produces an acceptable NAR. The definition of negative appendicectomy may affect the NAR. This study examined the impact of CT, Alvarado score and definition on the NAR. METHODS: The charts of 1,306 emergency appendicectomy patients from 1996 to 2010 were reviewed. Three five-year cohorts were created (Cohort A: 1996-2000, Cohort B: 2001-2005, Cohort C: 2006-2010) and the NAR was calculated for each cohort using two definitions of negative appendicectomy: absence of inflammation (NAR-STD) and absence of intramural neutrophils (NAR-STR). NAR-STD was correlated to the CT rate for Cohorts B and C and also to Alvarado score for Cohort C. RESULTS: When the definition of negative appendicectomy was changed, the NAR rose from 9.2% to 15.8% (p=0.0097) for Cohort A, from 2.8% to 8.6% (p=0.0180) for Cohort B (CT rate: 80.6%) and from 3.0% to 6.7% (p=0.0255) for Cohort C (CT rate: 92.4%). The introduction of CT lowered NAR-STD from 1996-2000 (9.2%) to 2001-2010 (2.9%) but increasing the CT rate from 2001-2010 had no impact on the NAR. The positive predictive value for Alvarado score (98.60%) and CT (99.03%) were similar. CONCLUSIONS: The definition of a negative appendicectomy determines the NAR. CT reduces the NAR regardless of definition but routine CT is unnecessary for male patients with positive Alvarado scores. Early/mild appendicitis may resolve without surgery and CT may contribute to unnecessary surgery. Alvarado scoring allows selective use of CT in suspected appendicitis.

A 19S proteasomal subunit cooperates with an ERK MAPK-regulated degron to regulate accumulation of Fra-1 in tumour cells.

Fos-related antigen-1 (Fra-1) is a member of the Activator Protein-1 (AP-1) transcription factor superfamily that is overexpressed in a variety of cancers, including colon, breast, lung, bladder and brain. High Fra-1 levels are associated with enhanced cell proliferation, survival, migration and invasion. Despite its frequent overexpression, the molecular mechanisms that regulate the accumulation of Fra-1 proteins in tumour cells are not well understood. Here, we show that turnover of Fra-1, which does not require ubiquitylation, is cooperatively regulated by two distinct mechanisms-association with the 19S proteasomal subunit, TBP-1, and by a C-terminal degron, which acts independently of TBP-1, but is regulated by RAS-ERK (extracellular signal-regulated kinase) signalling. TBP-1 depletion stabilized Fra-1 and further increased its levels in tumour cells expressing RAS-ERK pathway oncogenes. These effects correlated with increased AP-1 transcriptional activity. We suggest that during Fra-1 degradation, association with TBP-1 provides a mechanism for ubiquitin-independent proteasomal recognition, while the C terminus of the protein regulates its subsequent proteolytic processing.

Heterogeneity of Jagged1 expression in human and mouse intestinal tumors: implications for targeting Notch signaling.

Inhibition of Notch signaling is effective in inhibiting colon tumorigenesis, but targeting specific components of the pathway may provide more effective strategies. Here we show that the expression of Jagged1, a ligand for canonical Notch signaling, was restricted to enteroendocrine cells or undetectable in the mucosa of the human small and large intestine, respectively. In contrast, increased expression characterized half of human colon tumors, although not all tumors with elevated Wnt signaling displayed elevated Jagged1. Increased Jagged1 was also present in intestinal tumors of Apc(1638N/+) and Apc(Min/+) mice, but to a higher level and more frequently in the former, and in 90% of mouse tumors Notch signaling was elevated when Jagged1 was elevated. In the human HT29Cl16E colonic carcinoma cell line, induction of goblet cell differentiation by contact inhibition of growth depended on the loss of Jagged1-mediated Notch activation, with signaling through Notch1 and Notch2 acting redundantly. Therefore, targeting of Jagged1 could be effective in downregulating Notch signaling in a subset of tumors, but may avoid the limiting gastrointestinal toxicity caused by pharmacological inhibition of Notch signaling.

Molecular mechanisms of action and prediction of response to oxaliplatin in colorectal cancer cells.

The platinum compound oxaliplatin has been shown to be an effective chemotherapeutic agent for the treatment of colorectal cancer. In this study, we investigate the molecular mechanisms of action of oxaliplatin to identify means of predicting response to this agent. Exposure of colon cancer cells to oxaliplatin resulted in G2/M arrest and apoptosis. Immunofluorescent staining demonstrated that the apoptotic cascade initiated by oxaliplatin is characterised by translocation of Bax to the mitochondria and cytochrome c release into the cytosol. Oxaliplatin treatment resulted in caspase 3 activation and oxaliplatin-induced apoptosis was abrogated by inhibition of caspase activity with z-VAD-fmk, but was independent of Fas/FasL association. Targeted inactivation of Bax or p53 in HCT116 cells resulted in significantly increased resistance to oxaliplatin. However, the mutational status of p53 was unable to predict response to oxaliplatin in a panel of 30 different colorectal cancer cell lines. In contrast, the expression profile of these 30 cell lines, assessed using a 9216-sequence cDNA microarray, successfully predicted the apoptotic response to oxaliplatin. A leave-one-out cross-validation approach was used to demonstrate a significant correlation between experimentally observed and expression profile predicted apoptosis in response to clinically achievable doses of oxaliplatin (R=0.53; P=0.002). In addition, these microarray experiments identified several genes involved in control of apoptosis and DNA damage repair that were significantly correlated with response to oxaliplatin.

c-Myc overexpression sensitises colon cancer cells to camptothecin-induced apoptosis.

The proto-oncogene c-Myc is overexpressed in 70% of colorectal tumours and can modulate proliferation and apoptosis after cytotoxic insult. Using an isogenic cell system, we demonstrate that c-Myc overexpression in colon carcinoma LoVo cells resulted in sensitisation to camptothecin-induced apoptosis, thus identifying c-Myc as a potential marker predicting response of colorectal tumour cells to camptothecin. Both camptothecin exposure and c-Myc overexpression in LoVo cells resulted in elevation of p53 protein levels, suggesting a role of p53 in the c-Myc-imposed sensitisation to the apoptotic effects of camptothecin. This was confirmed by the ability of PFT-alpha, a specific inhibitor of p53, to attenuate camptothecin-induced apoptosis. p53 can induce the expression of p21(Waf1/Cip1), an antiproliferative protein that can facilitate DNA repair and drug resistance. Importantly, although camptothecin treatment markedly increased p21(Waf1/Cip1) levels in parental LoVo cells, this effect was abrogated in c-Myc-overexpressing derivatives. Targeted inactivation of p21(Waf1/Cip1) in HCT116 colon cancer cells resulted in significantly increased levels of apoptosis following treatment with camptothecin, demonstrating the importance of p21(Waf1/Cip1) in the response to this agent. Finally, cDNA microarray analysis was used to identify genes that are modulated in expression by c-Myc upregulation that could serve as additional markers predicting response to camptothecin. Thirty-four sequences were altered in expression over four-fold in two isogenic c-Myc-overexpressing clones compared to parental LoVo cells. Moreover, the expression of 10 of these genes was confirmed to be significantly correlated with response to camptothecin in a panel of 30 colorectal cancer cell lines.

p21(WAF1/cip1) is an important determinant of intestinal cell response to sulindac in vitro and in vivo.

Sulindac, a nonsteroidal anti-inflammatory drug, inhibits intestinal tumorigenesis in humans and rodents. Sulindac induced complex alterations in gene expression, but only 0.1% of 8063 sequences assayed were altered similarly by the drug in rectal biopsies of patients treated for 1 month and during response of colonic cells in culture. Among these changes was induction of the cyclin-dependent kinase inhibitor, p21(WAF1/cip1). In Apc1638(+/-) mice, targeted inactivation of p21 increased intestinal tumor formation in a gene-dose-dependent manner, but inactivation of p21 completely eliminated the ability of sulindac to both inhibit mitotic activity in the duodenal mucosa and to inhibit Apc-initiated tumor formation. Thus, p21 is essential for tumor inhibition by this drug. The array data can be accessed on the Internet at http://sequence.aecom.yu.edu/genome/.

Down-regulation of beta-catenin TCF signaling is linked to colonic epithelial cell differentiation.

The beta-catenin TCF pathway is implicated in the regulation of colonic epithelial cell proliferation, but its role in the regulation of cell differentiation is unknown. The colon carcinoma cell line, Caco-2, spontaneously undergoes G(0)/G(1) cell cycle arrest and differentiates along the absorptive cell lineage over 21 days in culture. In parallel, we show that beta-catenin-TCF activity and complex formation are significantly down-regulated. The down-regulation of beta-catenin-TCF signaling was independent of APC, which we characterized as having a nonsense mutation in codon 1367 in Caco-2 cells, but was associated with a decrease in TCF-4 protein levels. Total beta-catenin levels increased during Caco-2 cell differentiation, although this was attributable to an increase in the membrane, E-cadherin-associated, fraction of beta-catenin. Importantly, down-regulation of beta-catenin-TCF signaling in undifferentiated Caco-2 cells by three different mechanisms, ectopic expression of E-cadherin, wild-type APC, or dominant negative TCF-4, resulted in an increase in the promoter activities of two genes that are well-established markers of cell differentiation, alkaline phosphatase and intestinal fatty acid binding protein. These studies demonstrate, therefore, that in addition to its established role in the regulation of cell proliferation, down-regulation of the beta-catenin-TCF pathway is associated with the promotion of a more-differentiated phenotype in colonic epithelial cells.

Resistance to butyrate-induced cell differentiation and apoptosis during spontaneous Caco-2 cell differentiation.

BACKGROUND & AIMS: The short-chain fatty acid butyrate induces cell cycle arrest, differentiation, and apoptosis in colon cancer cells, but often induces opposite effects in normal colonic epithelial cells. We determined whether response to butyrate is dependent on the basal differentiation status of colonic epithelial cells. METHODS: Caco-2 cells at progressive stages of differentiation were treated with butyrate, and endpoints were measured. RESULTS: Response of Caco-2 cells to butyrate was dependent on their differentiation status. Butyrate maximally stimulated cell cycle arrest, apoptosis, alkaline phosphatase activity, transepithelial resistance, cell migration, urokinase receptor expression, and interleukin 8 secretion in undifferentiated Caco-2 cells, whereas differentiated Caco-2 cells were essentially resistant to these effects. Consistently, butyrate selectively induced histone hyperacetylation in undifferentiated Caco-2 cells. This resistance was also observed during HT29cl.19A cell differentiation, but not in the nondifferentiating SW620 cell line. Finally, the rate of butyrate use significantly increased as Caco-2 cells underwent spontaneous differentiation. CONCLUSIONS: Colonic epithelial cells become progressively more refractory to the effects of butyrate during absorptive cell differentiation. We postulate that this resistance is caused by the rapid use of butyrate by differentiated Caco-2 cells, which likely results in low intracellular concentrations and subsequently in its inability to inhibit histone deacetylase.

Dissociation of staurosporine-induced apoptosis from G2-M arrest in SW620 human colonic carcinoma cells: initiation of the apoptotic cascade is associated with elevation of the mitochondrial membrane potential (deltapsim).

We have identified an alternative apoptotic cascade induced in SW620 human colonic carcinoma cells by the protein kinase antagonist staurosporine (stsp). Consistent with its effect in other colonic epithelial cells, stsp induced G2-M arrest and apoptosis of SW620 cells. However, despite the paradigm that growth arrest triggers apoptotic cascades, apoptosis was detected before G2-M arrest. Reports have linked dissipation of the mitochondrial membrane potential (deltapsim) to the initiation of apoptosis and have linked elevation of the deltapsim to the escape from apoptosis However, neither apoptosis nor cell cycle arrest were altered by the collapse of the deltapsim, and increased deltapsim enhanced the initiation of apoptosis but blocked G2-M arrest. Although reactive oxygen species (ROS) have been implicated in some colonic epithelial cell and stsp-induced cascades, neither antioxidants nor the inhibition of RNA or protein synthesis altered apoptosis of SW620 cells. Finally, cytosolic cytochrome c has been linked to activation of caspase-3 and dissipation of the deltapsim. However, caspase-3 activation preceded the accumulation of cytochrome c in the cytosol and was accompanied by transient elevations in both the deltapsim and mitochondria-associated cytochrome c. Therefore, we have identified a distinct apoptotic cascade in SW620 cells that was induced independently of growth arrest, dissipation of the deltapsim, ROS production, or synthesis of de novo RNA or protein, and we have linked its efficient initiation to early elevation of the deltapsim.

Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer.

The short-chain fatty acid butyrate, produced by microbial fermentation of dietary fiber in the large intestine, is a physiological regulator of major pathways of colonic epithelial cell maturation: cell cycle arrest, lineage-specific differentiation, and apoptosis. Microarray analysis of 8,063 sequences demonstrated a complex cascade of reprogramming of SW620 colonic epithelial cells upon treatment with butyrate characterized by the progressive recruitment of gene sets as a function of time. Comparison with the effects of trichostatin A, in conjunction with differences in the kinetics of alteration of histone acetylation induced by butyrate and trichostatin A, identified subsets of induced and repressed genes likely coordinately regulated by altered histone acetylation. The butyrate response was also compared in detail with that of sulindac, a nonsteroidal anti-inflammatory drug with significant chemopreventive activity for colon cancer, and curcumin, a component of mustard and curry structurally and functionally related to sulindac that also has chemopreventive activity. Although gene clusters were identified that showed similar responses to butyrate and sulindac, the data were characterized by the extensive differences in the effects of the two agents. This was striking for functional classes of genes involved in signaling pathways and in cell cycle progression, although butyrate and sulindac induce a similar G0-G1 arrest, elevation of beta-catenin-Tcf signaling, and apoptotic cascade. As regards cell cycle arrest, the underlying mechanism in response to butyrate was most similar to that of the Caco-2 cell line that had spontaneously undergone a G0-G1 arrest and least similar to the G2-M arrest stimulated by curcumin. Thus, high-throughput microarray analysis of gene expression profiles can be used to characterize and distinguish the mechanisms of response of colonic epithelial cells to physiological and pharmacological inducers of cell maturation. This has important implications for characterization of chemopreventive agents and recognition of potential toxicity and synergies. The data bases, gene clusters, and analyses are available at http:// sequence.aecom.yu.edu/genome/.

Divergent phenotypic patterns and commitment to apoptosis of Caco-2 cells during spontaneous and butyrate-induced differentiation.

Caco-2 cells differentiate spontaneously when cultured in confluence and on exposure to the physiologically relevant short-chain fatty acid, butyrate. This study aimed to compare the phenotype induced by these pathways and their relations to cell turnover. Caco-2 cells were treated with butyrate at a nontoxic concentration of 2 mM for 3 days, or allowed to spontaneously differentiate for 0-21 days. Brush border hydrolase activities and carcinoembryonic antigen (CEA) expression, transepithelial resistance and dome formation, expression of components of the urokinase system, and cell turnover by flow cytometry, and the degree of DNA fragmentation were quantified. Butyrate induced increases in alkaline phosphatase activity and CEA expression but not the activities of other hydrolases, while culture alone induced progressive increases in the activities/expression of all markers. Butyrate induced a significantly greater increase in transepithelial resistance (TER) than occurred during culture alone but the densities of domes were similar. Butyrate induced a ninefold increase in urokinase receptor expression and twofold increase in urokinase activity, while culture alone induced a significantly smaller increase in receptor expression, an increase in plasminogen activator inhibitor-1 but no change in activity. While both stimuli induced cell cycle arrest, only butyrate increased the proportion of cells undergoing apoptosis. In conclusion, differentiation of Caco-2 cells can proceed along multiple pathways but does not necessarily lead to apoptosis. The phenotypic changes during spontaneous differentiation mimic those that occur in normal colonic epithelial cells in vivo during their migration from the crypt base to neck, while butyrate-induced effects more closely follow those occurring when normal colonic epithelial cells migrate from crypt neck to the surface compartment.

Short-chain fatty acids reduce expression of specific protein kinase C isoforms in human colonic epithelial cells.

LIM1215 colon cancer cells were used as a model of human colonic epithelium to examine the effects of butyrate on protein kinase C (PKC) activity and isoform expression. On Western blot analysis, LIM1215 cells express the PKC isoforms alpha, beta, epsilon, zeta, and lambda, but not gamma, straight theta, or micro. Treatment with 2 mM butyrate for 48 h reduced cellular PKC activity up to 50% and specifically reduced the expression of PKC alpha and PKC epsilon. Similar results were obtained using Caco-2 colon cancer cells. These effects were neither a consequence of the induction of differentiation itself nor the result of direct or indirect activation of PKC. Although dependent on gene transcription and protein synthesis, the effect was not due to a reduction in the synthesis of PKC protein. Butyrate's effect was independent of its beta-oxidation but was mimicked, at least in part, by trichostatin A, an inhibitor of histone deacetylase.