Hypoxia induces the expression of transketolase-like 1 in human colorectal cancer.

BACKGROUND AND AIMS: Transketolase-like (TKTL) 1 is one of the key enzymes for anaerobic sugar degradation even in the presence of oxygen (aerobic glycolysis). Transketolase-dependent reactions supply malignant tumors with ribose and NADPH. Therefore, TKTL1 activity could be crucial for tumor proliferation and survival. The aim of the study was to evaluate the expression of TKTL1 in colorectal cancer (CRC) and its regulation under hypoxic conditions. METHODS: We studied TKTL1 mRNA and protein expression in CRC cell lines and human CRC biopsies by quantitative real-time PCR, Western blotting and immunohistochemistry. Regulation of TKTL1 under oxygen depletion was analyzed by cultivating cells either in a three-dimensional spheroid model or in a hypoxia incubator chamber. RESULTS: TKTL1 mRNA was heterogeneously expressed in monolayers of cells with high levels in HT-29 and SW480. TKTL1 protein was also clearly detectable in HT-29 and SW480. Hypoxia-inducible factor (HIF)-1α protein expression correlated with TKTL1 protein expression in SW480 spheroids over time. On the one hand, induction of hypoxia in T84 spheroids did not induce TKTL1; on the other hand, hypoxia by incubation at 1% O2 in a hypoxia incubator chamber clearly showed an upregulation of TKTL1. In 50% of CRC patients, TKTL1 protein expression was upregulated in tumor compared to non-tumor tissue. The immunohistochemical staining of TKTL1 in CRC patient samples resulted in 14 positive and 30 negative samples. CONCLUSIONS: TKTL1 expression correlated with HIF-1α protein expression and was induced upon hypoxic conditions which could facilitate energy supply to tumors under these circumstances.

Intermediate filament dynamics and breast cancer: aberrant promoter methylation of the Synemin gene is associated with early tumor relapse.

Synemin (SYNM) is a type IV intermediate filament that has recently been shown to interact with the LIM domain protein zyxin, thereby possibly modulating cell adhesion and cell motility. Owing to this multiplicity of potential functions relevant to cancer development, we initiated a study to decipher SYNM expression and regulation in benign human breast tissue and breast cancer. Dot blot array analysis showed significant SYNM mRNA downregulation in 86% (n=100, P<0.001) of breast cancers compared with their normal tissue counterparts, a result that was confirmed by real-time PCR analysis (n=36, P<0.0001). Immunohistochemistry analysis showed abundant SYNM protein expression in healthy myoepithelial breast cells, whereas SYNM expression loss was evident in 57% (n=37, P<0.001) of breast cancer specimens. Next, we analyzed methylation of the SYNM promoter to clarify whether the SYNM gene can be silenced by epigenetic means. Indeed, methylation-specific PCR analysis showed tumor-specific SYNM promoter methylation in 27% (n=195) of breast cancers. As expected, SYNM promoter methylation was tightly associated (P<0.0001) with SYNM expression loss. In-depth analysis of the SYNM promoter by pyrosequencing showed extensive CpG methylation of DNA elements supposed to regulate gene transcription. Demethylating treatment of SYNM methylated breast cancer cell lines with 5-aza-2-deoxycytidine clearly reestablished the SYNM expression. Statistical analysis of the patient cohort showed a close association between SYNM promoter methylation and unfavorable recurrence-free survival (hazard ratio=2.941, P=0.0282). Furthermore, SYNM methylation positively correlated with lymph node metastases (P=0.0177) and advanced tumor grade (P=0.0275), suggesting that SYNM methylation is associated with aggressive forms of breast cancer. This is the first study on the epigenetic regulation of the SYNM gene in a cancer entity. We provide first hints that SYNM could represent a novel putative breast tumor suppressor gene that is prone to epigenetic silencing. SYNM promoter methylation may become a useful predictive biomarker to stratify breast cancer patients' risk for tumor relapse.

[Novel prognostic marker in invasive breast cancer. ITIH5 expression is abrogated by aberrant promoter methylation]. / Neuer Prognosemarker beim invasiven Mammakarzinom. ITIH5-Expression wird über aberrante Promotormethylierung inaktiviert.

We have recently characterized ITIH5 as a new extracellular matrix protein that exhibits clear expression loss in a variety of human tumour entities, including breast cancer. The aim of the present study was to decipher the molecular cause of ITIH5 expression loss in breast cancer and to learn more about the possible role of this molecule in cancer diseases. ITIH5 protein expression was found to be strongly reduced in 42% of invasive breast carcinomas-interestingly, with significant association with poor patient outcome. ITIH5 promoter methylation was frequently detected in breast cell lines and in primary carcinomas (40%), and it was functionally correlated with loss of ITIH5 mRNA expression. Moreover, ITIH5 promoter methylation was also significantly associated with poor clinical patient outcome and also with the occurrence of lymph node and distant metastases. In conclusion, we propose that ITIH5 may represent a novel metastasis repressor in human breast cancer. Both ITIH5 protein expression and ITIH5 promoter methylation may serve as prognostic biomarkers, thereby helping improve clinical patient outcome.

[Persistent diarrhea and loss of weight during therapy with leflunomide]. / Persistierende Diarrhoe und Gewichtsverlust unter Leflunomid-Therapie.

HISTORY AND CLINICAL FINDINGS: A 55-year-old woman had suffered from diarrhea and a weight loss of 15 kg over the previous six months. Neither the search for a causative pathogen nor coloscopy had provided a diagnosis. She was known to have type 1 diabetes mellitus, previous Hashimoto's thyroiditis, rheumatoid arthritis treated with leflunomide and drug- treated arterial hypertension. She was in a reduced general condition. INVESTIGATIONS: : The results of extensive initial diagnostic tests were unremarkable. Biopsies obtained at colonoscopy revealed marked lymphocytic colitis (LC). TREATMENT AND COURSE: When leflunomide was discontinued and replaced by salazosulfapyridine (sulfasalazine) and the steroid budesonide, the diarrhea ceased within a few days and the LC was no longer evident histologically after three months. CONCLUSIONS: Leflunomide can in rare cases cause a LC. that can quickly regress once the drug has been stopped. If the findings on colonoscopy in a case of otherwise unexplained diarrhea are grossly normal, a biopsy should be taken to rule out microscopic colitis.

The extracellular matrix protein ITIH5 is a novel prognostic marker in invasive node-negative breast cancer and its aberrant expression is caused by promoter hypermethylation.

Inter-alpha-trypsin inhibitors (ITIs) are protease inhibitors stabilizing the extracellular matrix. ITIs consist of one light (bikunin) and two heavy chains (ITIHs). We have recently characterized ITIH5, a novel member of the ITIH gene family, and showed that its messenger RNA is lost in a high proportion of breast tumours. In the present study, an ITIH5-specific polyclonal antibody was generated, validated with western blot and used for immunohistochemical analysis on a tissue microarray; ITIH5 was strongly expressed in epithelial cells of normal breast (n=11/15), while it was lost or strongly reduced in 42% (92/217) of invasive breast cancers. ITIH5 expression in invasive carcinomas was associated with positive expression of oestrogen receptor (P=0.008) and histological grade (P=0.024). Correlation of ITIH5 expression with clinical outcome revealed that patients with primary tumours retaining abundant ITIH5 expression had longer recurrence-free survival (RFS; P=0.037) and overall survival (OS; P=0.044), compared to those with reduced expression (mean RFS: 102 vs 78 months; mean OS: 120 vs 105 months). Methylation-specific PCR analysis frequently showed strong methylation of the ITIH5 promoter in primary breast tumours (41%, n=109) and breast cancer cell lines (n=6). Methylation was significantly associated with mRNA loss (P<0.001; n=39), and ITIH5 expression was induced after treatment of tumour cell lines with the demethylating agent 5-aza-2'-deoxycytidine. Moreover, ITIH5 promoter methylation was significantly associated with reduced OS (P=0.008). The cellular function of ITIH5 was evaluated by forced expression of a full-length ITIH5 complementary DNA in the breast cancer cell line MDA-MB-231, which does not endogenously express ITIH5. ITIH5-expressing clones showed a 40% reduced proliferation rate compared to mock-transfected cells. Overall, these data show that promoter methylation-mediated loss of ITIH5 expression is associated with unfavourable outcome in breast cancer patients, and thus ITIH5 could be used as a prognostic marker, although this marker is not multivariate independent due to its close association with ER expression. Our data indicate that ITIH5 is a candidate class II tumour suppressor gene and could be involved in tumour progression, invasion and metastasis, as its absence is associated with increased proliferation rates and a prognostic value indicating poor clinical outcome.

[Direct evidence of cytomegalovirus in coronary atheromas of patients with advance coronary heart artery disease]. / Direkter Nachweis von Zytomegalievirus in koronaren Atheromen von Patienten mit fortgeschrittener koronarer Herzerkrankung.

BACKGROUND AND AIM: Experimental and clinical data support an infectious cause of atherosclerosis and thereby coronary artery disease. This study was intended to assess the prevalence and possible clinical associations of the presence of cytomegalovirus DNA within coronary samples from patients undergoing coronary artery bypass grafting. PATIENTS AND METHODS: A coronary thrombendatherectomy was performed in 53 patients with advanced coronary artery disease. Two samples of each atheroma were used for further analysis and pathogen detection. RESULT: In 30% of patients with advanced coronary artery disease cytomegalovirus DNA was detected in coronary samples as assessed by highly sensitive PCR methods. The occurrence of the virus within the vessels was characterized by an inhomogeneous distribution pattern. CONCLUSION: Due to an increased proportion of restenotic lesions and a higher degree of calcification in cytomegalovirus-positive lesions, a causative association between the virus presence and mechanisms of restenosis post angioplasty is further supported. Antiviral pharmacological interventions to prevent restenosis in high-risk patients, however, seem not to be justified by the data currently available.

Infectious agents in coronary lesions obtained by endatherectomy: pattern of distribution, coinfection, and clinical findings.

BACKGROUND: Cytomegalovirus (CMV), Chlamydia pneumoniae (C. pneumoniae), and Helicobacter pylori (H. pylori) have been implicated in atherosclerosis and restenosis after angioplasty. The patterns of distribution within coronary lesions and possible coinfections of these pathogens in the coronary vasculature had not previously been evaluated. DESIGN: A prospective, observational clinical study. METHODS: Large coronary specimens (9-105 mm long) were obtained by endatherectomy of 53 patients undergoing aortocoronary bypass surgery. Samples were taken from two different sites of every lesion, resulting in a total of 106 probes. Presence of each pathogen was determined by polymerase chain reaction, subsequent hybridization, and DNA sequencing. RESULTS: Cytomegalovirus and C. pneumoniae were detected in 30 and 32% of the samples, respectively; H. pylori was not detectable. The pathogens were not homogeneously distributed. A concurrent infection with both pathogens was observed in five of 106 (5%) lesions and five of 53 (9%) patients. Restenotic lesions were more often found in specimens in which cytomegalovirus was detected (five of 16 versus two of 37). Patients with C. pneumoniae-positive coronary lesions more commonly presented with unstable angina. CONCLUSIONS: Inhomogeneous infections with cytomegalovirus and C. pneumoniae of coronary atherosclerotic lesions are found to be prevalent when serial analysis is performed. Concurrent infection with both pathogens occurs coincidentally; however, possible clinical implications of this new observation and the pathogenic impact on atherosclerosis need further investigation.