Modulation of proliferation-specific and differentiation-specific markers in human keratinocytes by SMAD7

We examined the potential role of SMAD7 in human epidermal keratinocyte differentiation. Overexpression of SMAD7 inhibited the activity of the proliferation-specific promoters for the keratin 14 and cdc2 genes and reduced the expression of the mRNA for the proliferation-specific genes cdc2 and E2F1. The ability of SMAD7 to suppress cdc2 promoter activity was lost in transformed keratinocyte cell lines and was mediated by a domain(s) located between aa 195-395 of SMAD7. This domain lies outside the domain required to inhibit TGFbeta1 signaling, suggesting that this activity is mediated by a novel functional domain(s). Examination of AP1, NFkappaB, serum response element, Gli, wnt, and E2F responsive reporters indicated that SMAD7 significantly suppressed the E2F responsive reporter and modestly increased AP1 activity in proliferating keratinocytes. These data Suggest that SMAD7 may have a role in TGFbeta-independent signaling events in proliferating/undifferentiated keratinocytes. The effects of SMAD7 in differentiated keratinocytes indicated a more traditional role for SMAD7 as an inhibitor of TGFbeta action. SMAD7 was unable to initiate the expression of differentiation markers but was able to superinduce/derepress differentiation-specific markers and genes in differentiated keratinocytes. This latter role is consistent with the ability of SMAD7 to inhibit TGFbeta-mediated suppression of keratinocyte differentiation and suggest that the opposing actions of SMAD7 and TGFbeta may serve to modulate squamous differentiation. (C) 2004 Elsevier Inc. All rights reserved.

Impaired sustained attention and executive dysfunction:bipolar disorder versus depression-specific markers of affective disorders

Objective - To identify neurocognitive measures that could be used as objective markers of bipolar disorder. Methods - We examined executive function, sustained attention and short-term memory as neurocognitive domains in 18 participants with bipolar disorder in euthymic state (Beuth), 14 in depressed state (Bdep), 20 with unipolar depression (Udep) and 28 healthy control participants (HC). We conducted four-group comparisons followed by relevant post hoc analyses. Results - Udep and Bdep, but not Beuth showed impaired executive function (p = 0.045 and p = 0.046, respectively). Both Bdep and Beuth, but not Udep, showed impaired sustained attention (p = 0.001 and p = 0.045, respectively). The four groups did not differ significantly on short-term memory. Impaired sustained attention and executive dysfunction were not associated with depression severity, duration of illness and age of illness onset. Only a small number of abnormal neurocognitive measures were associated with medication in Bdep and Beuth. Conclusion - Impaired sustained attention appears specific to bipolar disorder and present in both Beuth and Bdep; it may represent an objective marker of bipolar disorder. Executive dysfunction by contrast, appears to be present in Udep and Bdep and likely represents a marker of depression.

Decreased Expression Of Stem Cell Markers By Simvastatin In 7,12-dimethylbenz(a)anthracene (dmba)-induced Breast Cancer.

Simvastatin, a competitive inhibitor of HMG-CoA reductase widely used in the treatment and prevention of hyperlipidemia-related diseases, has recently been associated to in vitro anticancer stem cell (CSC) actions. However, these effects have not been confirmed in vivo. To assess in vivo anti-CSC effects of simvastatin, female Sprague-Dawley rats with 7,12-dimethyl-benz(a)anthracene (DMBA)-induced mammary cancer and control animals were treated for 14 days with either simvastatin (20 or 40 mg/kg/day) or soybean oil (N = 60). Tumors and normal breast tissues were removed for pathologic examination and immunodetection of CSC markers. At 40 mg/kg/day, simvastatin significantly reduced tumor growth and the expression of most CSC markers. The reduction in tumor growth (80%) could not be explained solely by the decrease in CSCs, since the latter accounted for less than 10% of the neoplasia (differentiated cancer cells were also affected). Stem cells in normal, nonneoplastic breast tissues were not affected by simvastatin. Simvastatin was also associated with a significant decrease in proliferative activity but no increase in cell death. In conclusion, this is the first study to confirm simvastatin anti-CSC actions in vivo, further demonstrating that this effect is specific for neoplastic cells, but not restricted to CSCs, and most likely due to inhibition of cell proliferation.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

No-match ORESTES explored as tumor markers

Sequencing technologies and new bioinformatics tools have led to the complete sequencing of various genomes. However, information regarding the human transcriptome and its annotation is yet to be completed. The Human Cancer Genome Project, using ORESTES (open reading frame EST sequences) methodology, contributed to this objective by generating data from about 1.2 million expressed sequence tags. Approximately 30 of these sequences did not align to ESTs in the public databases and were considered no-match ORESTES. On the basis that a set of these ESTs could represent new transcripts, we constructed a cDNA microarray. This platform was used to hybridize against 12 different normal or tumor tissues. We identified 3421 transcribed regions not associated with annotated transcripts, representing 83.3 of the platform. The total number of differentially expressed sequences was 1007. Also, 28 of analyzed sequences could represent noncoding RNAs. Our data reinforces the knowledge of the human genome being pervasively transcribed, and point out molecular marker candidates for different cancers. To reinforce our data, we confirmed, by real-time PCR, the differential expression of three out of eight potentially tumor markers in prostate tissues. Lists of 1007 differentially expressed sequences, and the 291 potentially noncoding tumor markers were provided.

Molecular and morphological markers for rapid distinction between 2 Colletotrichum species

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia (espinheira-santa) in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense. Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Coll and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.

The origin of a Robertsonian chromosomal translocation in house mice inferred from linked microsatellite markers

The Western European house mouse, Mus domesticus, includes many distinct Robertsonian (Rb) chromosomal races. Two competing hypotheses may explain the distribution of Rb translocations found in different populations: they may have arisen independently multiple times or they may have arisen once and been spread through long distance dispersal. We investigated the origin of the Rb 5.15 translocation using 6 microsatellite loci linked to the centromeres of chromosomes 5 and 15 in 84 individuals from 3 Rb populations and 4 neighboring standard-karyotype populations. Microsatellite variation on the 5.15 metacentric chromosomes was significantly reduced relative to the amount of variation found on acrocentric chromosomes 5 and 15, suggesting that linked microsatellite loci can track specific mutational events. Phylogenetic analyses resulted in trees which are consistent with multiple origins of the 5.15 metacentric found in the three Rb populations. These results suggest that cytologically indistinguishable mutations have arisen independently in natural populations of house mice.

E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis, To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes, Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes, Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas.

Inactive free : total prostate specific antigen ratios in ejaculate from men with suspected and known prostate cancer, compared with young control men

Objective To measure free:total prostate specific antigen (PSA) ratios in ejaculate from men with suspected and known prostate cancer, and in young control men, to determine if this ratio might be useful in discriminating benign from malignant prostatic conditions. Patients, subjects and methods Forty-seven men with prostate cancer (positive biopsies), 52 men with suspected prostate cancer but who had negative biopsies and 28 young men (< 30 years old) and with no family history of cancer, provided either a single ejaculate specimen (total 59) or multiple specimens (total 193) on subsequent occasions. Free and total PSA were measured using appropriate assays. All specimens were diluted in a PSA-negative female serum pool. Results The median free:total PSA ratios were 0.76-0.81 among the patient groups and control men, and there was no statistical difference between the groups. These data presumably only reflect the inactive component of free PSA, given that any alpha(2)-macroglobulin or alpha(1)-antichymotrypsin in the assay serum diluent was likely to have bound the active free PSA component in these samples. Similar results were obtained from those providing single and multiple samples, suggesting that a single specimen is sufficient to reflect the seminal plasma free:total PSA ratio over that period. There was no relationship between seminal plasma free:total PSA ratio and age for the controls or the positive biopsy group, although there was a negative relationship (i.e. a decline with age) that almost reached significance in those with negative biopsies (P = 0.058, R-2 = 0.07). Conclusions This is the first report of free:total PSA ratios in the ejaculate of men with suspected and known prostate cancer compared with young control men. Although no significant changes were detected in the free:total PSA ratios in ejaculate, these results may be confounded by differences in ratios with age, as is the case for serum PSA or different molecular forms of PSA. Indeed, these data suggest that a large proportion of free PSA in seminal plasma may be inactive. Further studies are needed to determine the potential utility of measuring free:total PSA, or other candidate markers, in ejaculate to better discriminate benign from malignant prostate disease.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman(TM) RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P < 0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P < 0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the-codon:reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more Widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin, secretion. (C) 2003 Elsevier Science B.V. All rights reserved.

Genomovar status, virulence markers and genotyping of Burkholderia cepacia complex strains isolated from Brazilian cystic fibrosis patients

Burkholderia cepacia complex isolates obtained by microbiological culture of respiratory samples from Brazilian CF patients were studied by recA based PCR, screened by specific PCR for virulence markers and genotyped by RAPD. Forty-one isolates of B. cepacia complex were identified by culture and confirmation of identity and genomovar determination obtained in 32 isolates, with predominance of B. cenocepacia (53.1%). Virulence markers were not consistently found among isolates. Genotyping did not identify identical patterns among different patients. B. cenocepacia was the most prevalent B. cepacia complex member among our patients, and cross-infection does not seem to occur among them. V 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Immunohistochemical detection of polyductin and co-localization with liver progenitor cell markers during normal and abnormal development of the intrahepatic biliary system and in adult hepatobiliary carcinomas

The longest open reading frame of PKHD1 (polycystic kidney and hepatic disease 1), the autosomal recessive polycystic kidney disease (ARPKD) gene, encodes a single-pass, integral membrane protein named polyductin or fibrocystin. A fusion protein comprising its intracellular C-terminus, FP2, was previously used to raise a polyclonal antiserum shown to detect polyductin in several human tissues, including liver. In the current study, we aimed to investigate by immunohistochemistry the detailed polyductin localization pattern in normal (ductal plate [DP], remodelling ductal plate [RDP], remodelled bile ducts) and abnormal development of the primitive intrahepatic biliary system, known as ductal plate malformation (DPM). This work also included the characterization of polyductin expression profile in various histological forms of neonatal and infantile cholestasis, and in cholangiocellular carcinoma (CCC) and hepatocellular carcinoma (HCC). We detected polyductin expression in the intrahepatic biliary system during the DP and the RDP stages as well as in DPM. No specific staining was found at the stage of remodelled bile ducts. Polyductin was also detected in liver biopsies with neonatal cholestasis, including mainly biliary atresia and neonatal hepatitis with ductular reaction as well as congenital hepatic fibrosis. In addition, polyductin was present in CCC, whereas it was absent in HCC. Polyductin was also co-localized in some DP cells together with oval stem cell markers. These results represent the first systematic study of polyductin expression in human pathologies associated with abnormal development of intrahepatic biliary tree, and support the following conclusions: (i) polyductin expression mirrors developmental properties of the primitive intrahepatic biliary system; (ii) polyductin is re-expressed in pathological conditions associated with DPM and (iii) polyductin might be a potential marker to distinguish CCC from HCC.

Combination of Multiple Molecular Markers Can Improve Prognostication in Patients With Locally Advanced and Lymph Node Positive Bladder Cancer

Purpose: We tested whether the combination of 4 established cell cycle regulators (p53, pRB, p21 and p27) could improve the ability to predict clinical outcomes in a large multi-institutional collaboration of patients with pT3-4N0 or pTany Npositive urothelial carcinoma of the bladder. We also assessed whether the combination of molecular markers is superior to any individual biomarker. Materials and Methods: The study comprised 692 patients with pT3-4N0 or pTany Npositive urothelial carcinoma of the bladder treated with radical cystectomy and bilateral lymphadenectomy (median followup 5.3 years). Scoring was performed using advanced cell imaging and color detection software. The base model incorporated patient age, gender, stage, grade, lymphovascular invasion, number of lymph nodes removed, number of positive lymph nodes, concomitant carcinoma in situ and adjuvant chemotherapy. Results: Individual molecular markers did not improve the predictive accuracy for disease recurrence and cancer specific mortality. Combination of all 4 molecular markers into number of altered molecular markers resulted in significantly 1 higher predictive accuracy than any single biomarker (p < 0.001.). Moreover addition of number of altered molecular markers to the base model significantly improved the predictive accuracy for disease recurrence (3.9%, p < 0.001) and cancer specific mortality (4.3%, p < 0.001). Addition of number of altered molecular markers retained statistical significance for improving the prediction of clinical outcomes in the subgroup of patients with pT3N0 (280), pT4N0 (83) and pTany Npositive (329) disease (p < 0.001). Conclusions: While the status of individual molecular markers does not add sufficient value to outcome prediction in patients with advanced urothelial carcinoma of the bladder, combinations of molecular markers may improve molecular staging, prognostication and possibly prediction of response to therapy.

Biological markers and prognosis in recurrent oral cancer after salvage surgery

Objective: To analyze the prognostic effect of epidermal growth factor receptor (EGFR), matrix metalloproteinases 2 and 9, and vascular endothelial growth factor expression in patients with locally recurrent oral carcinoma after salvage surgery. Design: Retrospective cohort study. Settings: Tertiary center cancer hospital. Patients: The charts of 111 patients with local recurrence of oral carcinomas were retrospectively analyzed. The previous treatment consisted of surgery in 33 patients (30.0%), radiotherapy with or without chemotherapy in 46 patients (41.0%), and surgery with adjuvant radiotherapy in 32 patients (29.0%). The expression of EGFR, matrix metalloproteinases 2 and 9, and vascular endothelial growth factor was analyzed with a tissue microarray immunohistochemical technique. Main Outcome Measures: Overall survival and cancer-specific survival (CSS). Results: The recurrences were diagnosed in less than 1 year in 69 patients (62.2%) and in more than 1 year in 42 patients (37.8%). The prognosis was worse in the group with the disease-free interval of less than 1 year (P=.01). Patients with more advanced disease (clinical stage of recurrence, III/IV) had worse rates of CSS (P=.04). Cases that were positive for EGFR had a 3-year CSS of 27.2%, while EGFR-negative cases had a 3-year CSS of 64.3% (P=.001). The expression of matrix metalloproteinases 2 (P=.83) and 9 (P=.15) and vascular endothelial growth factor (P=.86) was not significant in this group. In multivariate analysis, only the disease-free interval and the overexpression of EGFR were associated with a higher risk of cancer death. Conclusions: Local recurrence in oral carcinomas carries a poor prognosis. A disease-free interval of more than 1 year and a EGFR-negative expression are the main prognostic factors related to better CSS in patients treated with salvage surgery.

Application of recombinant antibody library for screening specific antigens in a bovine sperm cell subpopulation

Antibody phage display libraries are a useful tool in proteomic analyses. This study evaluated an antibody recombinant library for identification of sex-specific proteins on the sperm cell surface. The Griffin.1 library was used to produce phage antibodies capable of recognizing membrane proteins from Nelore sperm cells. After producing soluble monoclonal scFv, clones were screened on Simental sperm cells by flow cytometry and those that bound to 40-60% of cells were selected. These clones were re-analyzed using Nelore sperm cells and all clones bound to 40-60% of cells. Positive clones were submitted to a binding assay against male and female bovine leukocytes by flow cytometry and one clone preferentially bound to male cells. The results indicate that phage display antibodies are an alternative method for identification of molecules markers on sperm cells. (C) 2007 Elsevier B.V. All rights reserved.