I have heard about it for the first time from you! Implementation of tobacco control law by police personnel in India.

Setting and Objetives: Police personnel, alongside other key stakeholders, are responsible for implementing the Cigarettes and Other Tobacco Products Act (COTPA) in India. This study aimed to assess knowledge and attitudes about COTPA among police personnel and explore enablers and barriers in implementing it. Design: This convergent parallel mixed-methods study used a self-administered questionnaire (quantitative) and key informant interviews (qualitative). Of 300 police personnel across all eight police stations in Daman, 155 participated. Quantitative data were analysed using descriptive statistics and the χ2 test. Qualitative data from in-depth interviews of six key informants from all coordinating departments were analysed thematically. Results: Overall, 63.2% of responders were aware of any tobacco control law in India, and only 12.9% were trained in its implementation. One person had conducted inspections for COTPA compliance in the last 12 months. The majority (78.1%) of the police personnel, and significantly more tobacco non-users than users (81.2% vs. 52.9%, P = 0.016), felt that enforcing anti-tobacco regulations is one of their most important functions. Perceived benefits of the act and formal authority to act were the two main enablers of COTPA implementation. Lack of awareness and coordination, competing priorities, concentration of authority with higher-ranking officials and evasion of the law by retailers and the public hampered effective implementation of the law. Conclusion: Knowledge about the COTPA was average and implementation poor. Sensitisation and training of implementers, systematic transparent reporting and creating awareness among public are recommended for effective implementation.

Contexte et objectifs : Le personnel de la police, en collaboration avec d'autres partenaires clés, est responsable de la mise en œuvre de la Loi cigarettes et autres produits dérivés du tabac (COTPA) en Inde. Cette étude a eu pour but d'évaluer les connaissances et l'attitude au sein du personnel de la police en ce qui concerne la COTPA et a exploré les facilitateurs et les entraves à sa mise en œuvre.Schéma : Cette étude convergente parallèle à méthodes mixtes s'est basée sur un questionnaire auto-administré (méthode quantitative) et sur des entretiens avec des informateurs clés (méthode qualitative). Sur 300 personnels de police dans les huit stations de police de Daman, 155 ont participé. Les données quantitatives ont été analysées grâce à des statistiques descriptives et au test du χ2. Les données qualitatives émanant des entretiens approfondis avec six informateurs clés de tous les services de coordination ont été analysées de manière thématique.Résultats : Au total, 63,2% des participants étaient au courant de l'existence d'une loi de lutte contre le tabac en Inde, et seulement 12,9% ont été formés à sa mise en œuvre. Un seul avait réalisé des inspections relatives au respect de la COTPA au cours des 12 derniers mois. La majorité (78,1%) du personnel de police, et significativement plus de non-utilisateurs que d' utilisateurs de tabac (81,2% contre 52,9%, P = 0,016), estimaient que mettre en œuvre la loi anti-tabac était l'une de leurs fonctions importantes. Les bénéfices perçus de cette loi et le pouvoir officiel ont été les deux principaux facilitateurs de la mise en œuvre de la COTPA. Le manque de sensibilisation et de coordination, les priorités concurrentes, la concentration de l'autorité au sein des supérieurs et l'évasion de la loi par les revendeurs et le public a entravé une véritable mise en œuvre de la loi.Conclusion : La connaissance de la loi a été moyenne et sa mise en œuvre médiocre. La sensibilisation et la formation des responsables de la mise en œuvre, des rapports systématiques transparents et la sensibilisation du public sont recommandés pour une mise en œuvre efficace.

Marco de Referencia y Objetivos: El personal policial, junto con otros interesados directos, tienen a su cargo la ejecución de la COPTA (del inglés, Cigarettes and Other Tobacco Products Act, por ley sobre el consumo de cigarrillos y otros productos del tabaco) en la India. En el presente estudio se evaluaron los conocimientos y las actitudes de los miembros de la policía con respecto a la COPTA y se exploraron los factores facilitadores y los obstáculos a su aplicación.Método: Fue este un estudio de métodos mixtos convergentes y paralelos que utilizó cuestionarios rellenados por el encuestado (cuantitativos) y entrevistas a informantes clave (cualitativos). De los 300 oficiales de policía de las ocho estaciones de Daman, 155 participaron en la encuesta. Los datos cuantitativos se analizaron mediante métodos estadísticos descriptivos y la prueba del χ2. Los datos cualitativos de las entrevistas exhaustivas de seis informantes clave de todos los departamentos coordinadores se analizaron temáticamente.Resultados: En general, el 63,2% estaba al corriente de una ley de control del tabaco en la India, y solo el 12,9% había recibido capacitación relacionada con su aplicación. Solo un funcionario había realizado inspecciones sobre la conformidad con la COTPA en los últimos 12 meses. La mayor parte del personal de policía (78,1%), y una mayor proporción de no consumidores de tabaco (81,2% contra 52,9%; P = 0,016), consideraba que la aplicación de la reglamentación antitabaco constituía una de sus funciones importantes. Los dos principales factores facilitadores de la aplicación de la COPTA fueron la percepción de los beneficios de la ley y la autoridad oficial para actuar. El desconocimiento y la falta de coordinación, las prioridades concurrentes, la concentración de la autoridad en los funcionarios superiores y la evasión de la ley por parte de los comerciantes al por menor y de la población obstaculizan la aplicación eficaz de la ley.Conclusión: Se observó un conocimiento insuficiente y una escasa aplicación de la COTPA. Con miras a lograr una aplicación eficaz, se recomienda sensibilizar y capacitar al personal encargado de aplicar la ley, practicar una notificación sistemática transparente y trabajar por la concienciación de la población.

Esophageal distensibility measurement: impact on clinical management and procedure length.

Luminal distensibility measurement has demonstrated relevance to various disease processes, though its effects on clinical decision-making have been less well understood. This study aims to characterize the clinical impact of impedance planimetry measurement as well as the learning curve associated with its use in the esophagus. A single provider performed distensibility measurement in conjunction with upper endoscopy for a variety of clinical indications with the functional lumen imaging probe (FLIP) over a period of 21 months. Procedural data were prospectively collected and, along with medical records, retrospectively reviewed. Seventy-three procedures (70 patients) underwent esophageal distensibility measurement over the timeline of this study. The most common procedural indications were known or suspected achalasia (32.9%), dysphagia with connective tissue disease (13.7%), eosinophilic esophagitis (12.3%), and dysphagia with prior fundoplication (9.6%). FLIP results independently led to a change in management in 29 (39.7%) cases and supported a change in management in an additional 15 (20.5%) cases. The most common change in management was a new or amended therapeutic procedure (79.5%). Procedural time added by distensibility measurement was greater among earlier cases than among later cases. The median time added overall was 5 minutes and 46 seconds. Procedural time added varied significantly by procedural indication, but changes in management did not. Distensibility measurement added meaningful diagnostic information that impacted therapeutic decision-making in the majority of cases in which it was performed. Procedural time added by this modality is typically modest and decreases with experience.

The effectiveness of computed tomography-guided biopsy for the diagnosis of spondylodiscitis: an analysis of variables affecting the outcome.

OBJECTIVE: To analyze: (i) the effectiveness of CT-guided biopsy for the diagnosis of suspected spinal infections (spondylodiscitis); (ii) identify common causative microorganisms and assess factors that could affect the diagnostic yield. PATIENTS AND METHODS: Forty-five patients undergoing CT-guided biopsy for suspected spinal infection between November 2012 and October 2014 were analyzed. The time from presentation to diagnosis, administration of antibiotics before biopsy, blood culture results, admission C-reactive protein (CRP)/white cell count, presence of fever or neurological deficits, and soft tissue collections on MRI were analyzed. Multivariable logistic regression was performed to determine variables independently associated with a positive biopsy. RESULTS: Eleven (24.4%) patients had positive blood cultures. The first biopsy was positive in 19 (42.2%) patients. Thirty-eight (84.4%) patients had a single biopsy, while seven (15.5%) patients underwent repeat biopsy with a positive yield in one (14.2%) patient. Overall, causative microorganisms were identified in 26 (57.8%) cases. Admission CRP was significantly associated with isolating the causative pathogen from CT-guided biopsy (p<0.001). A soft tissue collection on MRI was associated with identification of a microorganism in blood cultures (p=0.001). CRP was the only independent variable associated with a positive yield on CT-guided biopsy (p=0.007, OR 1.042) and was more likely in patients with CRP>50 (p<0.001). Administration of empirical antibiotics before biopsy did not affect the yield (p=0.572). CONCLUSIONS: A high CRP was a strong predictor of isolation of the causative organism. Repeat CT-guided biopsy was found to have limited value with a low positive yield (14.2%) in our study.

Differential expression of hENT1 and hENT2 in colon cancer cell lines.

Human equilibrative nucleoside transporters (hENT) 1 and 2, encoded by SLC29A1 and SLC29A2, permit the bidirectional passage of nucleoside analogues into cells and may correlate with clinical responses to chemotherapy in patients with colorectal cancer (CRC). The purpose of this study was to evaluate the expression profiles of SLC29A1 and SLC29A2 in human cancer cell lines. Using quantitative real-time polymerase chain reaction, we comprehensively profiled the transcription levels of SLC29A1 and SLC29A2 in 16 colon cancer cell lines. We validated the ubiquitous and heterogeneous distribution of SLC29A1 and SLC29A2 in human colon cancer cell lines and demonstrated that SLC29A1 was highly expressed in 25% of metastatic cell lines (Colo201 and Colo205) and 62.5% of primary cell lines (Caco2, Colo320, HCT116, RKO, and SW48). For the first time, we showed that both SLC29A1 and SLC29A2 were expressed at lower levels in colon cancer cell lines originating from metastatic sites than from primary sites. These findings indicate that most patients with metastatic CRC (mCRC) may have low hENT1 expression, and treatment with nucleoside analogues may be inefficient. However, some patients still show high hENT1 expression and have a high probability of benefiting from these drugs. Therefore, evaluating transporter expression profiles and different drug responses between primary and metastatic tumors in patients with mCRC is important. Further assessment of the association between hENTs and drug-based treatment of mCRC is required to elucidate the mechanisms of chemotherapy resistance.

Prognostic value of CpG island methylator phenotype among colorectal cancer patients: a systematic review and meta-analysis.

BACKGROUND: Divergent findings regarding the prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) patients exist in current literature. We aim to review data from published studies in order to examine the association between CIMP and CRC prognosis. MATERIALS AND METHODS: A comprehensive search for studies reporting disease-free survival (DFS), overall survival (OS), or cancer-specific mortality of CRC patients stratified by CIMP is carried out. Study findings are summarized descriptively and quantitatively, using adjusted hazard ratios (HRs) as summary statistics. RESULTS: Thirty-three studies reporting survival in 10 635 patients are included for review. Nineteen studies provide data suitable for meta-analysis. The definition of CIMP regarding gene panel, marker threshold, and laboratory method varies across studies. Pooled analysis shows that CIMP is significantly associated with shorter DFS (pooled HR estimate 1.45; 95% confidence interval (CI) 1.07-1.97, Q = 3.95, I(2) = 0%) and OS (pooled HR estimate 1.43; 95% CI 1.18-1.73, Q = 4.03, I(2) = 0%) among CRC patients irrespective of microsatellite instability (MSI) status. Subgroup analysis of microsatellite stable (MSS) CRC patients also shows significant association between shorter OS (pooled HR estimate 1.37; 95% CI 1.12-1.68, Q = 4.45, I(2) = 33%) and CIMP. Seven studies have explored CIMP's value as a predictive factor on stage II and III CRC patient's DFS after receiving adjuvant 5-fluorouracil (5-FU) therapy: of these, four studies showed that adjuvant chemotherapy conferred a DFS benefit among CIMP(+) patients, one concluded to the contrary, and two found no significant correlation. Insufficient data was present for statistical synthesis of CIMP's predictive value among CRC patients receiving adjuvant 5-FU therapy. CONCLUSION: CIMP is independently associated with significantly worse prognosis in CRC patients. However, CIMP's value as a predictive factor in assessing whether adjuvant 5-FU therapy will confer additional survival benefit to CRC patients remained to be determined through future prospective randomized studies.

Olanzapine-induced hyperglycaemic coma and neuroleptic malignant syndrome: case report and review of literature.

Although the relationship between antipsychotic medication, particularly second-generation antipsychotics (SGAs), and metabolic disturbance is increasingly accepted, there is an important, but little recognised, potential interaction between this and the other important serious adverse effect of neuroleptic malignant syndrome (NMS). We report a case of a 35-year old female who developed new onset type II diabetes mellitus with hyperosmolar hyperglycaemic coma and acute renal failure following treatment with a SGA for a first manic episode. The history is strongly suggestive of concurrent NMS. This case raises important questions about non-ketotic, hyperosmolar diabetic coma with antipsychotics, the possible association between hyperglycaemia and hyperthermia, and the direction of causality in this, the recognition of either syndrome when they co-exist and management issues in such patients. These questions are considered in the context of currently available literature.

Long-term function after restorative proctocolectomy.

PURPOSE: Early functional outcome after restorative proctocolectomy and formation of an ileoanal pouch is known to be good, but there are minimal data on the long-term function of the pouch. The aim of this study was to look at the long-term functional outcome in patients who had undergone restorative proctocolectomy and formation of an ileoanal pouch. METHODS: A total of 151 consecutive patients (96 males, 55 females) who underwent ileoanal pouch surgery between April 1983 and May 1993 were identified. Functional outcomes from the previous 12 months were appraised by a standardized questionnaire. RESULTS: The median age at surgery was 31 years (range, 6-63 years), with a median follow-up of 142 months (range, 100-221 months). Eighteen patients have had their pouches excised, with another patient being defunctioned. Therefore 19 patients (13 percent) had suffered pouch failure. Altogether, 115 patients were available for follow-up, and 98 patients (85 percent) returned questionnaires. The median pouch-emptying frequency was five times (range, 1-17) during the day and one time (range, 0-6) at night. A total of 74 percent of patients had perfect continence during the day. Most of the patients had no life-style restrictions related to the pouch, and 98 percent of patients would recommend a pouch to others. CONCLUSIONS: Long-term functional outcome after ileoanal pouch surgery is good in most patients. For patients requiring proctocolectomy, ileoanal pouch surgery can now be recommended as an excellent long-term option.

Purification of anthrax edema factor from Escherichia coli and identification of residues required for binding to anthrax protective antigen.

The structural gene for anthrax edema factor (EF) was expressed in Escherichia coli under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from E. coli. EF purified from E. coli was biologically and functionally as active as its Bacillus anthracis counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.

Hydrophobic residues Phe552, Phe554, Ile562, Leu566, and Ile574 are required for oligomerization of anthrax protective antigen.

Anthrax protective antigen (PA) plays a central role in facilitating the entry of active toxin components, namely, lethal factor and edema factor, into the cells. PA is also the main immunogen of both human and veterinary vaccine against anthrax. During host cell intoxication, protective antigen binds to the receptors on cell surface, gets proteolytically activated, oligomerizes to form a heptamer and binds to lethal factor or edema factor. The complex, formed by binding of lethal factor or edema factor to oligomerized PA, is internalized by receptor-mediated endocytosis. Acidification of the endosome results in the insertion of the heptamer into the membrane, thereby forming a pore through which lethal factor or edema factor can translocate into the cytosol. In this study we have identified hydrophobic residues, Phe552, Phe554, Ile562, Leu566, and Ile574, which are required for oligomerization of anthrax protective antigen. Mutation of these conserved residues to alanine impaired the oligomerization of protective antigen. Consequently, these mutants became nontoxic in combination with lethal factor and edema factor. Therapeutic importance of these mutants and their potential as vaccine candidates is discussed.

Rapid purification of recombinant anthrax-protective antigen under nondenaturing conditions.

Anthrax-protective antigen is the central moiety of the anthrax toxin complex that mediates the entry of the other two toxin components, lethal factor and edema factor into the cells. It is also the main immunogen of the cell-free vaccine against anthrax. However, in addition to PA, the vaccine contains trace amounts of other culture-derived proteins that contribute to the side effects of the vaccine like pain, edema, erythrema, etc. Thus there is a need to develop high-resolution purification methods to purify PA to homogeneity. In this study we have presented a purification strategy for rapid purification of recombinant protective antigen under nondenaturing conditions, which ensures that not only biological activity but also the conformational integrity of immunological epitopes is well-preserved. The protein was purified to homogeneity in a two-step purification procedure that takes just 6 h for completion. Three milligrams of recombinant protective antigen obtained from 1-liter culture was comparable to B. anthracis protective antigen in terms of functional and biological activity. Moreover, the immunogenicity elicited by the purified protein in mice was also studied. The studies reported here are part of continuing research that aims to provide a safe and efficacious alternative to the current vaccine against anthrax.

Accelerated age-related CpG island methylation in ulcerative colitis.

CpG island hypermethylation is a mechanism of gene silencing that can be usurped by neoplastic cells to inactivate undesirable genes. In the colon, hypermethylation often starts in normal mucosa as a function of age and is markedly increased in cancer. To test the hypothesis that subjects at increased risk of colon cancer have higher levels of methylation in their nonneoplastic mucosa, we studied methylation patterns of five genes in the normal and dysplastic mucosa of patients with ulcerative colitis (UC), a condition associated with a marked increased risk of colon cancer. One gene (Mlh1) was unmethylated in all tissues examined. All four remaining genes had low but detectable levels of methylation in the epithelium of UC patients without evidence of dysplasia, and this methylation was not different from non-UC controls. By contrast, all four genes were highly methylated in dysplastic epithelium from high-grade dysplasia (HGD)/cancer patients with UC; methylation in HGD versus controls averaged 40.0% versus 7.4% (P = 0.00003) for ER, 44.0% versus 3.0% (P < 0.00003) for MYOD, 9.4% versus 2.4% (P = 0.03) for p16 exon 1, and 57.5% versus 30.6% (P = 0.01) for CSPG2. Importantly, three of the four genes were also highly methylated in the normal appearing (nondysplastic) epithelium from these same HGD/cancer patients, indicating that methylation precedes dysplasia and is widespread in these patients. Compared with controls, methylation averaged 20.1% versus 7.2% (P = 0.07) for ER, 18.4% versus 3.0% (P < 0.008) for MYOD, and 7.9% versus 2.4% (P = 0.007) for p16 exon 1. These results are consistent with the hypothesis that age-related methylation marks (and may lead to) the field defect that reflects acquired predisposition to colorectal neoplasia. Furthermore, the data suggest that chronic inflammation is associated with high levels of methylation, perhaps as a result of increased cell turnover, and that UC can be viewed as resulting in premature aging of colorectal epithelial cells.

Aging, methylation and cancer.

Alterations in methylation are widespread in cancers. DNA methylation of promoter-associated CpG islands is an alternate mechanism to mutation in silencing gene function, and affects tumor-suppressor genes such as p16 and RBI, growth and differentiation controlling genes such as ER and many others. Evidence is now accumulating that some of these methylation changes may initiate in subpopulations of normal cells as a function of age and progressively increase during carcinogenesis. Age-related methylation appears to be widespread and is one of the earliest changes marking the risk for neoplasia. In colon cancer, we have shown a pattern of age-related methylation for several genes, including ER, IGF2, N33 and MyoD, which progresses to full methylation in adenomas and neoplasms. Hypermethylation of these genes is associated with gene silencing. Age-related methylation involves at least 50% of the genes which are hypermethylated in colon cancer, and we propose that such age-related methylation may partly account for the fact that most cancers occur as a function of old age. Age-related methylation, then, may be a fundamental mark of the field defect in patients with neoplasia. The causes of age-related methylation are still unknown at this point, but evidence points to an interplay between local predisposing factors in DNA (methylation centers), levels of gene expression and environmental exposure. The concept that age-related methylation is a predisposing factor for neoplasia implies that it may serve as a diagnostic risk marker in cancer, and as a novel target for chemoprevention. Studies in animal models support this hypothesis and should lead to novel approaches to risk-assessment and chemoprevention in humans.

Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype.

Colorectal cancers (CRCs) are characterized by multiple genetic (mutations) and epigenetic (CpG island methylation) alterations, but it is not known whether these evolve independently through stochastic processes. We have recently described a novel pathway termed CpG island methylator phenotype (CIMP) in CRC, which is characterized by the simultaneous methylation of multiple CpG islands, including several known genes, such as p16, hMLH1, and THBS1. We have now studied mutations in K-RAS, p53, DPC4, and TGFbetaRII in a panel of colorectal tumors with or without CIMP. We find that CIMP defines two groups of tumors with significantly different genetic lesions: frequent K-RAS mutations were found in CIMP(+) CRCs (28/41, 68%) compared with CIMP(-) cases (14/47, 30%, P = 0.0005). By contrast, p53 mutations were found in 24% (10/41) of CIMP(+) CRCs vs. 60% (30/46) of CIMP(-) cases (P = 0.002). Both of these differences were independent of microsatellite instability. These interactions between CIMP, K-RAS mutations, and p53 mutations were preserved in colorectal adenomas, suggesting that they occur early in carcinogenesis. The distinct combinations of epigenetic and genetic alterations in each group suggest that activation of oncogenes and inactivation of tumor suppressor genes is related to the underlying mechanism of generating molecular diversity in cancer, rather than simply accumulate stochastically during cancer development.

Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype.

Aberrant methylation of 5' CpG islands is thought to play an important role in the inactivation of tumor suppressor genes in cancer. In colorectal cancer, a group of tumors is characterized by a hypermethylator phenotype termed CpG island methylator phenotype (CIMP), which includes methylation of such genes as p16 and hMLH1. To study whether CIMP is present in gastric cancer, the methylation status of five newly cloned CpG islands was examined in 56 gastric cancers using bisulfite-PCR. Simultaneous methylation of three loci or more was observed in 23 (41%) of 56 cancers, which suggests that these tumors have the hypermethylator phenotype CIMP. There was a significant concordance between CIMP and the methylation of known genes including p16, and hMLH1; methylation of p16 was detected in 16 (70%) of 23 CIMP+ tumors, 1 (8%) of 12 CIMP intermediate tumors, and 1 (5%) of 21 CIMP- tumors (P<0.0001). Methylation of the hMLH1 gene was detected in three of five tumors that showed microsatellite instability, and all three of the cases were CIMP+. The CIMP phenotype is an early event in gastric cancer, being present in the normal tissue adjacent to cancer in 5 of 56 cases. These results suggest that CIMP may be one of the major pathways that contribute to tumorigenesis in gastric cancers.

CpG island methylator phenotype in colorectal cancer.

Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP+ tumors also have a high incidence of p16 and THBS1 methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to hMLH1 methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency.

Methylation and silencing of the Thrombospondin-1 promoter in human cancer.

Neovascularization is a common feature of many human cancers, but relatively few molecular defects have been demonstrated in genes regulating angiogenesis. Decreased expression of Thrombospondin-1 (THBS1), a P53 and Rb regulated angiogenesis inhibitor, has been observed in some human tumors, including glioblastoma multiforme (GBM). To study whether methylation-associated inactivation is involved in down-regulating THBS1 expression in cancer, we analysed the methylation status of THBS1 in several cell lines and primary tumors. Three cell lines (RKO, CEM and RAJI) were completely methylated at several CpG sites within the THBS1 5' CpG island, and had no detectable expression by RT-PCR. THBS1 expression was readily reactivated using the methylation-inhibitor 5-deoxy-azacytidine in all three lines. Furthermore, THBS1 methylation was present in 33% (14/42) of primary GBMs. Thus, de novo methylation may serve as a potential way to inactivate THBS1 expression in human neoplasms.

Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification.

CpG island methylation has been linked to tumor suppressor gene inactivation in neoplasia and may serve as a useful marker to clone novel cancer-related genes. We have developed a novel PCR-based method, methylated CpG island amplification (MCA), which is useful for both methylation analysis and cloning differentially methylated genes. Using restriction enzymes that have differential sensitivity to 5-methyl-cytosine, followed by adaptor ligation and PCR amplification, methylated CpG rich sequences can be preferentially amplified. In a model experiment using a probe from exon 1 of the p16 gene, signal was detected from MCA products of a colorectal cancer cell line but not in normal colon mucosa. To identify novel CpG islands differentially methylated in colorectal cancer, we have applied MCA coupled with representational difference analysis to the colon cancer cell line Caco2 as a tester and normal colon mucosa as a driver. Using this strategy, we isolated 33 differentially methylated DNA sequences, including fragments identical to several known genes (PAX6, Versican, alpha-tubulin, CSX, OPT, and rRNA gene). The association of hypermethylation of the clones obtained and transcriptional suppression in colorectal cancer was confirmed by examining the Versican gene, which we found to be silenced in methylated cell lines and reactivated by the methylation inhibitor 5-aza-2'-deoxycytidine. We therefore propose that MCA is a useful technique to study methylation and to isolate CpG islands differentially methylated in cancer.

Activation of phospholipase C and protein kinase C is required for expression of anthrax lethal toxin cytotoxicity in J774A.1 cells.

Anthrax lethal toxin (LT) comprises two proteins: the protective antigen (PA) and the lethal factor (LF). The LT is cytotoxic to macrophage-like cell line J774A.1. Pre-treatment of these cells with neomycin, a phospholipase C inhibitor, protected them against anthrax LT cytotoxicity. Protection obtained with neomycin indicated that LT stimulates phospholipase C in these cells. It was found that levels of inositol 1,4,5-triphosphate (IP3) dramatically increased in toxin-treated cells. The rise in IP3 levels was proportional to the dose of LF that was allowed to bind to receptor-bound PA. By using protein kinase C (PKC) inhibitors, we found that the activation of PKC is required for mediating anthrax LT cytotoxicity. Activation of phospholipase C or PKC is not required for the binding of PA to the cell surface receptors or for the uptake or internalisation of the toxin. In this study, we demonstrate that the IP3 signalling cascade is initiated by anthrax lethal toxin in J774A.1 cells. The second messengers generated during the cascade aid LF in mediating lethality only after its translocation into the cytosol.

Aging and DNA methylation in colorectal mucosa and cancer.

DNA methylation of promoter-associated CpG islands may function as an alternate mechanism of silencing tumor suppressor genes in multiple neoplasias including colorectal cancer. De novo methylation of genes appears to be an early and frequent event in most neoplasias. For the ER and IGF2 genes, we have previously shown that methylation actually begins in the normal colon mucosa as an age-related event and progresses to hypermethylation in cancer. In this study, we have determined the frequency of age-related methylation in normal colonic mucosa among the genes hypermethylated in colorectal cancer. We studied six genes, including N33, MYOD, p16, HIC-1, THBS1, and CALCA. The N33 gene showed partial methylation in normal colon mucosa, which was age-related (r = 0.7; P = 0.003 using regression analysis). Adenomas and cancers showed further hypermethylation at this locus. Similarly, the MYOD gene showed age-related methylation in normal colon mucosa (r = 0.7; P < 0.00001 using regression analysis) and hypermethylation in cancers. Age-related methylation seems to be gene specific, because p16, THBS1, HIC-1, and CALCA were not affected. Furthermore, this process may also be modulated by tissue-specific factors. Our study suggests that aging is a major contributing factor to hypermethylation in cancer.