Membranous CD44v6 is upregulated as an early event in colorectal cancer: Downregulation is associated with circulating tumor cells and poor prognosis.

Previous studies have reported that CD44 variant 6 (CD44v6) and metastasis-associated protein 1 (MTA1) are contributing factors to cancer progression. The present study aimed to evaluate the expression profiles for associations with patients' demographic data, clinicopathological characteristics, the presence of partial epithelial-to-mesenchymal transition (pEMT), metastatic potential based on the presence of CK20+ CEA+ CXCR4+ circulating tumor cells (CTCs) and prognosis (median follow-up, 45 months). Thus, frozen tissue samples from 31 patients with stage I-III colorectal cancer (CRC), 15 benign colorectal polyps and seven normal colorectal tissues were analyzed to detect membranous (m)CD44v6 and MTA1 expression via flow cytometry. The results demonstrated that the mCD44v6 and MTA1 expression profiles were significantly correlated (rs=+0.786, P<0.001). Notably, MTA1 expression was not associated with any of the clinicopathological characteristics assessed. The percentage of mCD44v6-positive cells within tumors was higher in the right-sided cancer lesions (P=0.014), suggesting that proximal and distal CRCs are distinct clinicopathological entities. Furthermore, downregulated mCD44v6 expression was significantly associated with the presence of CTCs (P=0.017). This association was stronger for pEMT (co-expression of N- and E-cadherin mRNAs) primary lesions (P=0.009). In addition, patients with CRC with low levels of mCD44v6 had unfavorable survival outcomes (P=0.037). Taken together, these results suggest that targeted analysis of membranous CD44v6 as opposed to membranous-cytoplasmic expression is important in determining the prognosis of patients with CRC. Furthermore, downregulated mCD44v6 expression in malignancies presenting CTCs reinforces the importance of tumor-stroma reciprocal influence during the metastatic process and encourages the assessment of relevant therapeutic strategies.

Distinctive expression profiles of Caveolin-1 and Notch-1 protein in patients with nasal polyps or sinonasal inverted papillomas.

BACKGROUND: Nasal polyposis (NP) and sinonasal inverted papillomas (SIP) are considered benign lesions capable of recurrence or malignant transformation although not with the same prevalence. Since fluctuations of Caveolin-1 and Notch-1 proteins expression have been reported in many pathologies, the current study aimed to investigate their involvement in the epithelial transformation observed in SIPs compared to NP. METHODS: Immunohistochemical expression of Caveolin-1 and Notch-1 proteins was assessed in 104 patients with sinonasal lesions (45 NP, 45 SIP and 14 NP with SIP), semiquantively (percentage times intensity). Proteins expression profiles were evaluated statistically for their correlation with patients demographic and clinicopathological variables (grade of dysplasia, inflammation, recurrence) as well as with markers of proliferation (Ki67) and apoptosis (7-AAD) as determined by flow cytometry analysis. RESULTS: SIP lesions presented increased Caveolin-1 immunopositivity compared to NP (62.2%, vs 40.9%; p = 0.045). Cytoplasmic staining was observed only in epithelium's basal and suprabasal layers. Caveolin-1 positivity was not related to Ki67 expression, apoptosis, inflammation or dysplasia, eventhough 81.8% of highly immunopositive lesions were dysplastic (p = 0.03). Also, smokers presented significantly increased immunopositivy (p = 0.03). In contrast SIP lesions presented reduced Notch-1 expression compared to NP (68.9% vs 100%; p < 0.001). Dysplastic lesions presented low Notch-1 immunopositivity (p < 0.001). Enhancement of Notch-1 gene expression was also associated with inflammation. CONCLUSIONS: The herein presented data suggest that the expression profiles of Caveolin-1 and Notch-1 proteins in sinonasal pathologies are distinctive and that could be explored as potential targets for the development of alternative therapeutic approaches.

An in vitro model for investigating human autologous neuronal-astrocyte and immune cell interactions underlying neurodegenerative and immunosuppressive processes in neuropathy.

Primary mixed neuronal-astrocytic cultures were established from human brain tissues from elective surgical procedures and maintained in vitro for over 21 days. The majority of cells (a) expressed morphological and cytoskeletal markers of differentiated neurons (MAP2a&b; Tau) or astrocytes (GFAP) in anticipated proportion (1:2), and (b) regenerated synaptic connections and neural-astrocytic associations. Co-cultures with autologous blood leukocytes established that alterations in the viability (by Annexin V/PI) of brain and immune cells over 3 days were indicative of neurodegenerative or immunosuppressive processes. During co-culture, B-cells (CD19+) remained largely unaffected while T-lymphocytes (CD3+) and monocytes (CD14+) declined, consistent with immunosuppressive process. Indications of immunosuppression were not observed when immune cells were maintained in free of neural cells medium collected from neuro-cultures. Decline in brain cell viability in neuro-immune co-cultures may be associated with density of activated monocytes (HLA-DR+/CD14+), consistent with neurodegenerative process. Our findings, though preliminary and associated with significant variability between individuals, establish an approach to investigate neuroimmune pathology in humans.

Breast cancer nodal metastasis correlates with tumour and lymph node methylation profiles of Caveolin-1 and CXCR4.

DNA methylation is the best characterised epigenetic change so far. However, its role in breast cancer metastasis has not as yet been elucidated. The aim of this study was to investigate the differences between the methylation profiles characterising primary tumours and their corresponding positive or negative for metastasis lymph nodes (LN) and correlate these with tumour metastatic potential. Methylation signatures of Caveolin-1, CXCR4, RAR-ß, Cyclin D2 and Twist gene promoters were studied in 30 breast cancer primary lesions and their corresponding metastasis-free and tumour-infiltrated LN with Methylation-Specific PCR. CXCR4 and Caveolin-1 expression was further studied by immunohistochemistry. Tumours were typified by methylation of RAR-ß and hypermethylation of Cyclin-D2 and Twist gene promoters. Tumour patterns were highly conserved in tumour-infiltrated LN. CXCR4 and Caveolin-1 promoter methylation patterns differentiated between node-negative and metastatic tumours. Nodal metastasis was associated with tumour and lymph node profiles of extended methylation of Caveolin-1 and lack of CXCR4 hypermethylation. Immunodetection studies verified CXCR4 and Caveolin-1 hypermethylation as gene silencing mechanism. Absence of Caveolin-1 expression in stromal cells associated with tumour aggressiveness while strong Caveolin-1 expression in tumour cells correlated with decreased 7-year disease-free survival. Methylation-mediated activation of CXCR4 and inactivation of Caveolin-1 was linked with nodal metastasis while intratumoral Caveolin-1 expression heterogeneity correlated with disease progression. This evidence contributes to the better understanding and, thereby, therapeutic management of breast cancer metastasis process.

14-3-3 gene expression exerts isoform-dependent functions in sinonasal pathophysiology.

The expression profiles of 14-3-3ß and θ isoforms, known to exert both oncogenic and antiapoptotic effects, were assessed in different entities of nasal pathophysiology. Flow cytometry and immunohistochemistry were used on paraffin-embedded sections of 51 inverted papillomas (IP), 26 nasal polyps (NP), 9 polyps with IP (NPIP) and 10 specimens of normal epithelium (NE). 14-3-3ß expression was significantly upregulated in IP as compared with both NP (p=0.015) and NE (p=0.002). 14-3-3ß was also increased in NPIP as compared with NE (p=0.008). 14-3-3ß cytoplasmic staining was more pronounced in basal cells of the respiratory epithelium although serous glands and the vascular system were often positive as well. High 14-3-3ß immunopositivity in IP patients concurred with increased proliferative activity shown by PCNA immunostaining (p=0.04). Expression of 14-3-3θ was also found increased in IP and NPIP patients, compared to NP (p=0.005, p=0.002 respectively) and NE (p=0.004 and p=0.001 respectively). 14-3-3θ cytoplasmic immunopositivity was detected in columnar epithelium, particularly in basal and subluminal cells, whereas no immunoreactivity was observed in NP and NE. Our results demonstrate differential expression of 14-3-3ß and θ isoforms in sinonasal pathophysiology, supporting their implication, respectively, in the proliferative and inflammatory process engaged in the formation of IP.

Detection of epithelial cells by RT-PCR targeting CEA, CK20, and TEM-8 in colorectal carcinoma patients using OncoQuick density gradient centrifugation system.

BACKGROUND: The detection of epithelial-specific mRNA correlates well with the presence of cancer cells in the peripheral blood and provides a rational explanation for subsequent metastasis. MATERIAL AND METHODS: Forty-two, patients with colorectal cancer and 14 controls were included in our study. Peripheral blood samples were acquired at 24 h before and 48 h after laparotomy. Tissue samples were also acquired from the primary lesion. All samples were examined for the expression profile of CEA, CK20, and TEM-8. RESULTS: Tissue samples expressed CEA in every specimen, CK20 in 30, and TEM-8 in 41. CEA and CK20 were not identified in the control blood samples while TEM-8 was detected in 4. CEA was detected in 17, CK20 in 28 and TEM-8 in 23, of the preoperative blood samples. CEA mRNA expression in preoperative blood sample and TNM stage were found independently associated with increased tumor size. Positive CEA, CK20, and TEM-8 signals were found in 25, 25, and 23 of the postoperative blood samples respectively. CONCLUSIONS: CK20 and CEA are significantly more frequently detected in colon cancer patients than in healthy controls and can serve as markers. Cancer cell mRNA is commonly detected in the preoperative and postoperative peripheral blood samples. Tumor size was independently associated with the preoperative detection of CEA mRNA. Although TEM-8 mRNA detection in the peripheral blood showed no specificity for cancer patients or correlation with clinical stage, identification and validation of genes and proteins implicated in metastatic process needs to be further investigated.

Long-term primary cultures of human adult atrial cardiac myocytes: cell viability, structural properties and BNP secretion in vitro.

BACKGROUND: Human adult cardiomyocytes (CM) have been used in short-term cultures for in vitro studies of the adult myocardium. However, little information is available regarding human adult CMs cultured for long term (>2 weeks). METHODS: Human adult CMs were isolated from atrial specimens of 43 patients undergoing cardiopulmonary bypass surgery. Cell viability, cytoskeletal properties, intercellular junctional mediators and responsiveness to extracellular stimuli were monitored in CM cultures for 8 weeks. RESULTS: Absolute numbers of CMs decreased through the first 2 weeks, with substantially lower rates of cell loss thereafter. Apoptosis predominated over necrosis as the principal mode of cell death, affecting 4.1+/-1.6% of freshly dissociated cells, that declined in culture (3.6+/-1.0% week 1, 1.3+/-0.5% week 2). CMs maintained rod-shaped morphology and cross-striated expression pattern of sarcomeric proteins desmin and beta-myosin heavy chain for the first 4 weeks. Levels of desmin remained stable on first 3 weeks, but declined thereafter. CMs expressed cardiac-specific adherence molecule N-cadherin throughout the culture duration, indicating conserved contractile potential. CMs remained functional early in culture, as indicated by BNP secretion, with maximal levels on 1st week that declined gradually by week 4. Cell responsiveness to metabolic stresses (serum deprivation) was detected, inducing an early (6 h) 1.8-fold increase in levels of BNP. CONCLUSION: Long-term cultured human adult CMs maintain morphological integrity, adult-type cytoskeletal protein expression, cell-cell communication potential and functionality for 3-4 weeks in vitro.

A model system for analyzing somatic mutations in Drosophila melanogaster.

Presently there are no good assays for comparing somatic mutation frequencies and spectra between different vertebrate and invertebrate organisms. Here we describe a new lacZ mutation reporter system in D. melanogaster, which complements existing systems in the mouse. The results obtained with the new model indicate two-to threefold higher frequencies of spontaneous mutations than in the mouse, with most of the mutations characterized as large genome rearrangements.

A misquoted mutation in exon16 of the BRCA2 gene.

A pathogenic mutation in the BRCA2 gene, nt7602del16, has been misquoted as a mutation, possibly due to the incorrect inclusion of the last 16 nucleotides of exon15 of the BRCA2 gene as part of the intron15-exon16 BRCA2 gene sequence in publicly available databases. This was concluded following mutational screening by sequencing and enzymatic mapping of the BRCA2 gene exon15-exon16 region in DNA from peripheral blood samples from a total of 74 breast cancer and non-breast cancer patients as well as healthy individuals and the cell line MCF7. Careful interpretation of genetic variants and direct feedback to the corresponding sequence databases prevent systemic errors by integrating updated data into broadly referenced sources.