Possible Gender-Related Modulation by the ROCK1 Gene in Colorectal Cancer Susceptibility.

AIM: In view of accumulating evidence supporting a pivotal role of the Rho/ROCK pathway in cancer, we investigated Rho-kinase polymorphisms as potential susceptibility factors in colorectal cancer (CRC) in a representative sample of the Italian population. METHODS: DNA obtained from the peripheral blood samples of 137 CRC patients and 141 healthy controls was genotyped for four ROCK1 (rs35996865; rs73963110; rs2127958; rs288980) and five ROCK2 (rs12692437; rs7563468; rs35768389; rs17463896; rs16857265) selected single nucleotide polymorphisms. RESULTS: None of the allelic variants of the nine selected markers was associated with the occurrence of CRC or with the development of regional lymph node metastasis. By contrast, the ROCK1 rs35996865 G variant allele was significantly more frequent in male patients (p = 0.028) than in the control group. CONCLUSION: This finding is, at present, the first that points to a possible gender-related modulation by the ROCK1 gene in CRC susceptibility.

Human Multidrug Resistance 1 gene polymorphisms and Idiopathic Pulmonary Fibrosis.

BACKGROUND: For the first time we tested an association between the human multidrug resistance gene 1 (MDR1) polymorphisms (SNPs) and idiopathic pulmonary fibrosis (IPF). Several MDR1 polymorphisms are associated with pathologies in which they modify the drug susceptibility and pharmacokinetics. MATERIALS AND METHODS: We genotyped three MDR1 polymorphisms of 48 IPF patients and 100 control subjects with Italian origins. RESULTS: No evidence of association was detected. CONCLUSION: There are 50 known MDR1 SNPs, and their role is explored in terms of the effectiveness of drug therapy. We consider our small-scale preliminary study as a starting point for further research.

Colorectal cancer susceptibility: apparent gender-related modulation by ABCB1 gene polymorphisms.

BACKGROUND: The ATP-binding cassette transporter B1 (ABCB1) gene codes for a membrane efflux pump localized in epithelial cells. Together with other Permeability-glycoproteins in the small and large intestine, its product represents a barrier against xenobiotics, bacterial toxins, drugs and other substances introduced with diet, including carcinogens. The aim of this investigation was to verify the possible contribution of ABCB1 single nucleotide polymorphisms (SNPs) to the genetic risk of colorectal cancer (CRC). RESULTS: DNA obtained from the peripheral blood of 98 CRC patients and 100 healthy controls was genotyped for the three selected SNPs: 1236C > T (rs1128503), 2677G > T/A (rs2032582), and 3435C > T (rs1045642). Molecular data were analyzed to asses allele and haplotype association with CRC. No evidence of an association between ABCB1 alleles and CRC occurrence as a whole was found. However, ABCB1 showed either association with carcinoma of the sigmoid colon, and appeared able to influence the sex ratio among CRC patients. These two effects seemed to act independently based on multivariate analysis. We showed that ABCB1 polymorphisms were able to influence CRC susceptibility related to tumor localization and patient gender. CONCLUSIONS: We suggest that sensitivity to undetermined risk factors could depend on the genetic background of ABCB1 locus, with a mechanism that also depends on patient gender.

A candidate gene study of one-carbon metabolism pathway genes and colorectal cancer risk.

The risk of colorectal cancer (CRC) may be influenced by aberrant DNA methylation and altered nucleotide synthesis and repair, possibly caused by impaired dietary folate intake as well as by polymorphic variants in one-carbon metabolism genes. A case-control study using seventy-one CRC patients and eighty unrelated healthy controls was carried out to assess the genetic association of fifteen SNP and one insertion in nine genes belonging to the folate pathway. Polymorphism selection was based on literature data, and included those which have a known or suspected functional impact on cancer and missense polymorphisms that are most likely to alter protein function. Genotyping was performed by real-time PCR and PCR followed by restriction analysis. The likelihood ratio statistic indicated that most of the polymorphisms were not associated with the risk of CRC. However, an increased risk of CRC was observed for two variant alleles of SNP mapping on the transcobalamin 2 gene (TCN2): C776G (rs1801198) and c.1026-394T>G (rs7286680). Considering the crucial biological function played by one-carbon metabolism genes, further investigations with larger cohorts of CRC patients are needed in order to confirm our preliminary results. These preliminary results indicate that TCN2 polymorphisms can be a susceptibility factor for CRC.

Gene expression profile of human colon cancer cells treated with cross-reacting material 197, a diphtheria toxin non-toxic mutant.

Cross-Reacting Material 197 (CRM197) is a diphtheria toxin non-toxic mutant that has shown antitumor activity in mice and humans. It is still unclear whether this anti-tumorigenic effect depends on its strong inflammatory-immunological property, its ability to inhibit heparin-binding epidermal growth factor (HB-EGF), or even its possible weak toxicity. CRM197 is utilized as a specific inhibitor of HB-EGF that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. In this study we evaluate the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible influence on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment (MTT modified assay), or changes in cell cycle distribution (flow cytometry), in EGFR localization, phospho-EGFR detected signals (immunohistochemistry) or in morphology (scanning electron microscopy, SEM) they show a change in the gene expression profile by microarray analysis (cDNA microarray SS-H19k8). The overexpression of genes like protein phosphatase 2, catalytic subunit, alpha isozyme (PPP2CA), guanine nucleotide-binding protein G subunit alpha-1(GNAI1) and butyrophilin, subfamily 2, member A1 (BTN2A1) has been confirmed with real-time-qPCR. This is the first study where the CRM197 treatment on HT-29 shows a possible scarce implication of endogenous HB-EGF on EGFR expression and cancer cell development. At the same time, our results show the alteration of a specific and selected number of genes.

A role for epidermal growth factor receptor in idiopathic pulmonary fibrosis onset.

In idiopathic pulmonary fibrosis (IPF) patients the presence of missense polymorphisms (SNP) in members of the epidermal growth factor receptor (EGFR) family or their genetic association could influence the binding affinity of natural ligands, modifying the expression and the behavior of the correlated genes. EGFR family members are particularly involved in the epithelial injury and fibrotic process in IPF. Genetic variations in HER family of receptors may alter the possible therapeutic efficacy of EGFR inhibitors. This study aimed to analyze the relationships between IPF and specific EGF receptor family functional polymorphisms. We tested the presence of common EGFR, HER2 and HER3 non-synonymous SNPs in the peripheral blood of 20 Italian IPF patients and their association with the disease. Our data indicated that the HER2 variant allele frequency was significantly lower in patients than in controls, with an odds ratio of 0.31 (95% CI 0.080, 0.98). Our finding suggests that HER2 variant could be a protective factor against IPF onset.

The Roadmap of Colorectal Cancer Screening.

Colorectal cancer (CRC) is the third most common form of cancer in terms of incidence and the second in terms of mortality worldwide. CRC develops over several years, thus highlighting the importance of early diagnosis. National screening programs based on fecal occult blood tests and subsequent colonoscopy have reduced the incidence and mortality, however improvements are needed since the participation rate remains low and the tests present a high number of false positive results. This review provides an overview of the CRC screening globally and the state of the art in approaches aimed at improving accuracy and participation in CRC screening, also considering the need for gender and age differentiation. New fecal tests and biomarkers such as DNA methylation, mutation or integrity, proteins and microRNAs are explored, including recent investigations into fecal microbiota. Liquid biopsy approaches, involving novel biomarkers and panels, such as circulating mRNA, micro- and long-non-coding RNA, DNA, proteins and extracellular vesicles are discussed. The approaches reported are based on quantitative PCR methods that could be easily applied to routine screening, or arrays and sequencing assays that should be better exploited to describe and identify candidate biomarkers in blood samples.

Colorectal cancer screening: Assessment of CEACAM6, LGALS4, TSPAN8 and COL1A2 as blood markers in faecal immunochemical test negative subjects.

Prevention is essential to reduce Colorectal Cancer (CRC) mortality. We previously reported a panel of four genes: CEACAM6, LGALS4, TSPAN8, COL1A2 (CELTiC) able to discriminate patients with CRC. Here, we assessed the CELTiC panel by quantitative polymerase chain reaction, in the blood of 174 healthy subjects, who resulted negative to the faecal immunochemical test (FITN). Using non-parametric statistic and multinomial logistic models, the FITN were compared to previously analysed subjects: 36 false positive FIT (NFIT), who were negative at colonoscopy, 36 patients with low risk lesions (LR) and 92 patients with high risk lesions or CRC (HR/CRC). FITN showed a significantly lower expression of the four genes when compared to HR/CRC. Moreover, FITN showed a significantly lower expression of TSPAN8 and COL1A2 compared to NFIT and LR patients. The multinomial logistic model confirmed that TSPAN8 alone specifically discriminated FITN from NFIT, LR and HR/CRC, while LGALS4 was able to differentiate FITN from false positive FIT. Finally, ROC curves analysis of the comparisons between FITN and HR/CRC, LR or NFIT reported AUC greater than 0.87, with a sensitivity and specificity of 83% and 76%, respectively. The CELTiC panel was confirmed a useful tool to identify CRC patients and to discriminate false FIT positive subjects.

Glucocorticoid Receptor Modulates EGFR Feedback upon Acquisition of Resistance to Monoclonal Antibodies.

Evidences of a crosstalk between Epidermal Growth Factor Receptor (EGFR) and Glucocorticoid Receptor (GR) has been reported, ranging from the modulation of receptor levels or GR mediated transcriptional repression of EGFR target genes, with modifications of epigenetic markers. The present study focuses on the involvement of EGFR positive and negative feedback genes in the establishment of cetuximab (CTX) resistance in metastatic Colorectal Cancer (CRC) patients. We evaluated the expression profile of the EGFR ligands TGFA and HBEGF, along with the pro-inflammatory cytokines IL-1B and IL-8, which were previously reported to be negatively associated with monoclonal antibody response, both in mice and patient specimens. Among EGFR negative feedback loops, we focused on ERRFI1, DUSP1, LRIG3, and LRIG1. We observed that EGFR positive feedback genes are increased in CTX-resistant cells, whereas negative feedback genes are reduced. Next, we tested the expression of these genes in CTX-resistant cells upon GR modulation. We unveiled that GR activation leads to an increase in ERRFI1, DUSP1, and LRIG1, which were shown to restrict EGFR activity, along with a decrease in the EGFR activators (TGFA and IL-8). Finally, in a cohort of xenopatients, stratified for response to cetuximab, we observed an inverse association between the expression level of LRIG1 and CRC progression upon CTX treatment. Our model implies that combining GR modulation to EGFR inhibition may yield an effective treatment strategy in halting cancer progression.

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.

BACKGROUND: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. METHODS: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. RESULTS: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. CONCLUSION: This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.

LGALS4, CEACAM6, TSPAN8, and COL1A2: Blood Markers for Colorectal Cancer-Validation in a Cohort of Subjects With Positive Fecal Immunochemical Test Result.

BACKGROUND: A noninvasive blood test for the early detection of colorectal cancer (CRC) is highly required. We evaluated a panel of 4 mRNAs as putative markers of CRC. MATERIALS AND METHODS: We tested LGALS4, CEACAM6, TSPAN8, and COL1A2, referred to as the CELTiC panel, using quantitative reverse transcription polymerase chain reaction, on subjects with positive fecal immunochemical test (FIT) results and undergoing colonoscopy. Using a nonparametric test and multinomial logistic model, FIT-positive subjects were compared with CRC patients and healthy individuals. RESULTS: All the genes of the CELTiC panel displayed statistically significant differences between the healthy subjects (n = 67), both low-risk (n = 36) and high-risk/CRC (n = 92) subjects, and those in the negative-colonoscopy, FIT-positive group (n = 36). The multinomial logistic model revealed LGALS4 was the most powerful marker discriminating the 4 groups. When assessing the diagnostic values by analysis of the areas under the receiver operating characteristic curves (AUCs), the CELTiC panel reached an AUC of 0.91 (sensitivity, 79%; specificity, 94%) comparing normal subjects to low-risk subjects, and 0.88 (sensitivity, 75%; specificity, 87%) comparing normal and high-risk/CRC subjects. The comparison between the normal subjects and the negative-colonoscopy, FIT-positive group revealed an AUC of 0.93 (sensitivity, 82%; specificity, 97%). CONCLUSION: The CELTiC panel could represent a useful tool for discriminating subjects with positive FIT findings and for the early detection of precancerous adenomatous lesions and CRC.

Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer.

BACKGROUND: The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. METHODS: In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22) that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. CONCLUSION: The design of new approaches to identify such markers is warranted.

Systematic large-scale meta-analysis identifies a panel of two mRNAs as blood biomarkers for colorectal cancer detection.

Colorectal cancer (CRC) is the third most common cancer in the world. A significant survival rate is achieved if it is detected at an early stage. A whole blood screening test, without any attempt to isolate blood fractions, could be an important tool to improve early detection of colorectal cancer. We searched for candidate markers with a novel approach based on the Transcriptome Mapper (TRAM), aimed at identifying specific RNAs with the highest differential expression ratio between colorectal cancer tissue and normal blood samples. This tool permits a large-scale systematic meta-analysis of all available data obtained by microarray experiments. The targeting of RNA took into consideration that tumour phenotypic variation is associated with changes in the mRNA levels of genes regulating or affecting this variation.A real time quantitative reverse transcription polymerase chain reaction (qRT- PCR) was applied to the validation of candidate markers in the blood of 67 patients and 67 healthy controls. The expression of genes: TSPAN8, LGALS4, COL1A2 and CEACAM6 resulted as being statistically different.In particular ROC curves attested for TSPAN8 an AUC of 0.751 with a sensitivity of 83.6% and a specificity of 58.2% at a cut off of 10.85, while the panel of the two best genes showed an AUC of 0.861 and a sensitivity of 92.5% with a specificity of 67.2%.Our preliminary study on a total of 134 subjects showed promising results for a blood screening test to be validated in a larger cohort with the staging stratification and in patients with other gastrointestinal diseases.

Adenomatous polyposis coli (APC) regulates miR17-92 cluster through ß-catenin pathway in colorectal cancer.

Adenomatous polyposis coli (APC) mutation is the most common genetic change in sporadic colorectal cancer (CRC). Although deregulations of miRNAs have been frequently reported in this malignancy, APC-regulated miRNAs have not been extensively documented. Here, by using an APC-inducible cell line and array analysis, we identified a total of 26 deregulated miRNAs. Among them, members of miR-17-92 cluster were dramatically inhibited by APC and induced by enforced expression of ß-catenin. Furthermore, we demonstrate that activated ß-catenin resulted from APC loss binds to and activates the miR-17-92 promoter. Notably, enforced expression of miR-19a overrides APC tumor suppressor activity, and knockdown of miR-19a in cancer cells with compromised APC function reduced their aggressive features in vitro. Finally, we observed that expression of miR-19a significantly correlates with ß-catenin levels in colorectal cancer specimens, and it is associated to the aggressive stage of tumor progression. Thus, our study reveals that miR-17-92 cluster is directly regulated by APC/ß-catenin pathway and could be a potential therapeutic target in colon cancers with aberrant APC/ß-catenin signaling.

A module of inflammatory cytokines defines resistance of colorectal cancer to EGFR inhibitors.

Epidermal Growth Factor Receptor (EGFR) activates a robust signalling network to which colon cancer tumours often become addicted. Cetuximab, one of the monoclonal antibodies targeting this pathway, is employed to treat patients with colorectal cancer. However, many patients are intrinsically refractory to this treatment, and those who respond develop secondary resistance along time. Mechanisms of cancer cell resistance include either acquisition of new mutations or non genomic activation of alternative signalling routes. In this study, we employed a colon cancer model to assess potential mechanisms driving resistance to cetuximab. Resistant cells displayed increased ability to grow in suspension as colonspheres and this phenotype was associated with poorly organized structures. Factors secreted from resistant cells were causally involved in sustaining resistance, indeed administration to parental cells of conditioned medium collected from resistant cells was sufficient to reduce cetuximab efficacy. Among secreted factors, we report herein that a signature of inflammatory cytokines, including IL1A, IL1B and IL8, which are produced following EGFR pathway activation, was associated with the acquisition of an unresponsive phenotype to cetuximab in vitro. This signature correlated with lack of response to EGFR targeting also in patient-derived tumour xenografts. Collectively, these results highlight the contribution of inflammatory cytokines to reduced sensitivity to EGFR blockade and suggest that inhibition of this panel of cytokines in combination with cetuximab might yield an effective treatment strategy for CRC patients refractory to anti-EGFR targeting.

Identification of candidate genes involved in the reversal of malignant phenotype of osteosarcoma cells transfected with the liver/bone/kidney alkaline phosphatase gene.

Alkaline phosphatases (ALPs) are a family of cell surface glycoproteins that catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate. Liver/bone/kidney (L/B/K) ALP participates in bone mineralization, but its other physiological and pathological functions remain obscure. In human osteosarcoma, an inverse relationship has been found between cellular L/B/K ALP expression and aggressiveness. To explore this relationship, we employed cDNA microarray technology to characterize and compare the gene expression profile of two U-2 OS osteosarcoma clones with high L/B/K ALP activity (U-2/ALP28 and U-2/ALP40) and one with contrasting characteristics (U-2/ALP23). We identified 79 differentially expressed genes (58 upregulated in U-2/ALP28 and U-2/ALP40 compared to U-2/ALP23). Using GenMAPP/MAPPFinder, we highlighted nine functional groups strictly related to high L/B/K ALP activity, including microtubule-based movement and cell adhesion groups, two functions well related to tumor invasiveness. Notably, cadherin 13 (CDH13) and caveolin 1 (CAV1) genes were upregulated in our cells. Since these two genes are involved in cell-cell adhesion and cell growth, their co-expression with L/B/K ALP could help explain the lower levels of malignancy found in osteosarcoma cells with high L/B/K ALP activity. Although functional studies are needed to better define the role of CDH13 and CAV1 in the malignant behavior of osteosarcoma cells, the data presented here provide an aid to understanding the biological functions of L/B/K ALP in bone tumors.

Search for epithelial-specific mRNAs in peripheral blood of patients with colon cancer by RT-PCR.

Research has widely supported the efficacy of screening for colorectal cancer in reducing mortality. A blood-based assay potentially represents a more accessible early detection tool for the identification of solid tumor cells originating from a primary tumor site in the body. We demonstrate a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as markers of malignancy in blood samples of patients with colon cancer. The present study aims to identify a set of specific mRNAs expressed in epithelial cells but not in blood cells, which may be useful as markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay following semi-automated RNA extraction from peripheral blood samples. Our approach includes a systematic search for candidate markers using digital differential display, search on UniGene colon EST libraries and analysis of published data on colon cancer gene expression. A final list included the following genes: bone morphogenetic protein 4 (BMP4), cyclin D (CycD), family with sequence similarity 3, member D (FAM3D), gastrin (GAS), glycoprotein A33 transmembrane (GPA33), glutathione peroxidase 2 gastrointestinal (GPX2), galactoside-binding, soluble, 4 (galectin 4) (LGALS4), non-SMC, structural maintenance of chromosomes, element 1 protein (NSE1), tumor-associated calcium signal transducer 1 (TACSTD1), telomerase reverse transcriptase (hTERT), trefoil factor 3 intestinal (TFF3), transmembrane 4 superfamily member 3 (TM4SF3), UDP glycosyltransferase 1 family, polypeptide A9 (UGT1A9), villin 1 (VIL1), and the novel gene FLJ20127. The mRNA expression of these genes was evaluated in a pool of 16 samples from subjects diagnosed with colon cancer and from 16 normal-controls. We observed expression in 13 of the 15 investigated genes from the blood samples of the vast majority of patients considered, but also in a certain percentage of the controls (from 14.3 to 100%). This finding confirms that the extreme sensitivity of RT-PCR is able to detect minimal amounts of mRNA expressed in a non tissue-specific manner ('illegitimate transcription'). On the contrary, NSE1 and GAS mRNAs were not detected either in patient or in control blood samples; however, they were abundantly expressed in normal and cancerous colon mucosa, encouraging further search for useful markers able to detect epithelial cells in peripheral blood.

The human TruB family of pseudouridine synthase genes, including the Dyskeratosis Congenita 1 gene and the novel member TRUB1.

A novel human gene denominated TruB pseudouridine (psi) synthase homolog 1 (E. coli) (approved symbol, TRUB1) has been identified and characterized. Spanning approximately 40 kb on chromosome 10 and including 8 exons, TRUB1 is the first described human ortholog of bacterial TruB/psi55, a gene involved in tRNA pseudouridinilation. TRUB1 gene encodes a 349-amino acid product, with a VFAVHKPKGPTSA box in positions 71-83 corresponding to motif I of the TruB family (probably involved in conserving protein structure). The TruB domain of TRUB1 lies between W104 and I255, and contains another short motif, GGTLDS AARGVLVV, including the highly conserved D residue that characterizes motif II (involved in uridine recognition and in catalytic function of psi synthases). Northern blot analysis revealed that TRUB1 mRNA is widely expressed in various human tissues (especially heart, skeletal muscle and liver). Phylogenetic analysis of the TruB domain revealed another human gene (approved symbol TRUB2) encoding a conserved TruB domain, located on human chromosome 9. Thus, the human TruB family includes at least three members: i.e. DKC1 (previously identified), TRUB1 and TRUB2. The TRUB1 and TRUB2 products could be the hitherto unidentified human tRNA psi synthases. Although TRUB1 is not highly similar to DKC1/dyskerin (whose mutations cause X-linked dyskeratosis congenita) and putatively affects tRNA rather than rRNA modification, it is the most similar human protein to dyskerin. Study of TRUB1 (and TRUB2) should facilitate understanding of the molecular mechanisms of RNA modification and the involvement of psi synthases in human pathology, including dyskeratosis-like diseases.

Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment.

Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR's positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR's feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy.

Idiopathic pulmonary fibrosis and polymorphisms of the folate pathway genes.

OBJECTIVES: This study aims to determine the possible association between folate pathway gene polymorphisms and idiopathic pulmonary fibrosis. This represents the first study carried out on folate pathway gene polymorphisms as possible risk factors in this kind of pathology. The premise is that several polymorphisms mapping on genes responsible for folate uptake are associated with the risk of numerous diseases occurring between pregnancy and old age, and that too little is currently known about idiopathic pulmonary fibrosis. DESIGN AND METHODS: We genotyped 9 single nucleotide polymorphisms and 1 polymorphic insertion in 7 essential genes belonging to the folate pathway in 32 Italian idiopathic pulmonary fibrosis patients and 81 control subjects. This was done by PCR and restriction analysis. RESULTS: Allelic and genotypic association tests indicated that for all the analysed polymorphisms there were no significant differences between patients and controls. Nevertheless, the haplotype association analysis revealed a significant association between idiopathic pulmonary fibrosis and transcobalamin II gene polymorphisms: specifically the haplotype 776G (rs1801198)-c.1026-394G (rs7286680)-444C (rs10418) (OR=2.84; 95% C.I. 1.36-5.93, P value=0.004). CONCLUSIONS: This small-scale preliminary study would suggest the importance of further research focusing on the role of folate in the onset of idiopathic pulmonary fibrosis.