Evangelos Marinos

Enhanced podocalyxin expression alters the structure of podocyte basal surface.

Glomerular basement membrane (GBM) and podocalyxin are essential for podocyte morphology. We provide evidence of functional interconnections between basement membrane components (collagen IV and laminin), the expression of podocalyxin and the morphology of human glomerular epithelial cells (podocytes). We demonstrated that GBM and laminin, but not collagen IV, up-regulated the expression of podocalyxin. Scanning electron microscopy revealed that laminin induced a modified morphology of podocytes with process formation, which was more extensive in the presence of GBM. Under high magnification, podocytes appeared ruffled. Using transmission electron microscopy we observed that raised areas occurred in the basal cell surface. Furthermore, the presence of anti-podocalyxin antibody increased the extent of adhesion and spreading of podocytes to both collagen IV and laminin, thus podocalyxin apparently inhibits cell-matrix interactions. We also performed adhesion and spreading assays on podocytes grown under increased glucose concentration (25 mM). Under these conditions, the expression of podocalyxin was almost totally suppressed. The cells adhered and spread to basement membrane components but there was no increase in the extent of adhesion and spreading in the presence of anti-podocalyxin antibody, or ruffling of the cell edges. Additionally, in podocytes expressing podocalyxin, the presence of anti-podocalyxin antibody partially reversed the inhibition of adhesion to collagen IV provoked by anti-β1 integrin antibody, thus podocalyxin should compete with β1-related cell adhesion. We suggest that the observed podocalyxin-mediated inhibition of binding to the matrix could be in part responsible for the specialized conformation of the basal surface of podocytes.

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer

BackgroundIntegrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.ResultsIn the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.ConclusionA model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

NUCKS overexpression in breast cancer.

BackgroundNUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies.ResultsThe immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures.ConclusionThe results show overexpression of the NUCKS protein in a number of non malignant breast lesions and cancerous tissues. In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.

Mutational Analysis of Circulating Tumor Cells from Colorectal Cancer Patients and Correlation with Primary Tumor Tissue

Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3) mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC) patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.

The number of mitochondria in Xenopus laevis ovulated oocytes

An attempt was made to estimate the total number of mitochondria in Xenopus laevis ovulated oocytes. For this purpose the necessary basic parameters were calculated employing planimetry and simple mathematical formulas. It was found that the number of mitochondria in the ovulated oocyte of Xenopus is of the order of 10(7). The significance of this finding is discussed.

Ultrastructural immunostaining of infiltrating ductal breast carcinomas with the monoclonal antibody H: A comparative study with cytokeratin 8

The monoclonal antibody H (mAbH) detects an epitope consisting of an O-linked N -acetyl glucosamine (O-GlcNAc) and neighboring amino acids. This epitope has been found by using extracts from the MCF-7 human breast carcinoma cell line in immunoblotting experiments, on cytokeratin 8 (CK8) and 5 other polypeptides. In the present study, a double immunogold method was applied for the colocalization of CK8 and mAbH epitope on epoxy thin sections in 18 cases of infiltrating ductal breast carcinomas (IDBC) and in 6 cases of fibroadenomas, to study the accurate subcellular distribution of CK8 in breast cancer cells, as compared to the 5 polypeptides, recognized by mAbH. Furthermore, a detailed quantitative evaluation of the double immunolocalization over the cellular compartments of cancer cells was undertaken with the aid of a computerized image analysis system and the results were assessed statistically. The distribution pattern of CK8 and the mAbH epitope in the neoplastic mammary epithelial cells was similar in IDBC as compared to fibroadenomas, while the gold labeling intensity of these epitopes differed over the cellular compartments between malignant and benign biopsies. The results reveal the significance of the role of CK8 and O-GlcNAc glycosylation in the biology of the neoplastic mammary cells in vivo, determining their malignant potential.

Donor lung pretreatment with surfactant in experimental transplantation preserves graft hemodynamics and alveolar morphology

In experimental lung transplantation, the reduction of endogenous surfactant properties occurs after graft preservation and transplant reperfusion. The aim of this study was to evaluate the efficacy of donor lung pretreatment with exogenous surfactant on graft damage after ischemia and reperfusion. Fourteen (control group A, n = 8; study group B,n= 6) young female white pigs (mean weight 27 ± 3.5 kg) were used in a newly developed autotransplantation model within situcold ischemia. In study group B, before thoracotomy, 1.5 ml/kg surfactant apoprotein-A-free surfactant was administrated into the left main bronchus via flexible bronchoscopy. Belzer UW solution was used for lung preservation. Cold ischemia was achieved for 3 hr with interlobar lung parenchyma temperature at 8 ± 1.3°C, and central temperature maintained at 37.20 ± 0.5°C. Animals were sacrificed after 3 hr of graft reperfusion. At the end of reperfusion, pulmonary vascular resistance index (was 447.80 dyn/sec.cm5.m2(±66.8) in group A vs 249.51 in group B (P< 0.001) and serum nitric oxide was adequately preserved. The mean alveolar surface area estimated by computerized morphometry was 5280.84 (4991.1) μm2(group A) vs 3997.89 (3284.70) μm2(group B;P< 0.005). Histology revealed milder macrophage and lymphocyte infiltration in group B at the end of reperfusion. Pretreatment of donor lung with an surfactant apoprotein-A -free surfactant agent appears to be beneficial in terms of maintaining serum NO and reducing hemodynamic disturbances. Furthermore, alveolar histology and stereomorphology are better preserved.

Dynamic expression of the vertebrate-specific protein Nucks during rodent embryonic development.

The nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) is a highly phosphorylated nuclear protein that is overexpressed in many types of cancer. The flexibility of NUCKS and its extensive posttranslational modifications indicate that it is multifunctional, and its expression in most cell types suggests a housekeeping function. However, spatiotemporal expression of the Nucks protein during rodent development has not been reported. Thus, we investigated the expression of both the Nucks mRNA and protein during rat and mouse development by immunohistochemistry, in situ hybridization, Western immunoblotting, and reverse-transcription PCR analysis. We also used BLAST analysis against expressed sequence tag databases to determine whether a NUCKS homologue is expressed in invertebrate organisms. We found that Nucks expression increased during the initial stages of embryonic development, and then gradually decreased until birth in all tissues except the nervous tissue and muscle fibers. Interestingly, the expression of Nucks was very strong in migrating neural crest cells at E13.5 and ectoderm-derived tissues. In most tissues analyzed, the levels of Nucks correlated with the levels of Bax and activated caspase-3, which are indicative of apoptosis. Moreover, Nucks was upregulated very early during neuronal apoptosis in vitro. Expression analysis revealed that no transcript with close homology to the Nucks gene was present in invertebrates. The expression of Nucks in both proliferating and quiescent cells and its correlation with Bax levels and apoptosis strongly suggest that Nucks plays complex roles in cell homeostasis. Furthermore, the lack of homology in invertebrate organisms indicates a specific role for Nucks in vertebrate embryogenesis.

RGD Binding to Integrin Alphavbeta3 Affects Cell Motility and Adhesion in Primary Human Breast Cancer Cultures

Integrins mediate cell adhesion to the extracellular matrix. Integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site and has been associated with high malignant potential in breast cancer cells, signaling the onset of widespread metastasis. In recent years, several antagonists of integrin alphavbeta3, including RGD peptides, have been used as potential anti-cancer agents. In the present work, the effect of the linear RGD hexapeptide GRGDSP was studied, for the first time, on breast tumor explants, as well as on well-spread human breast cancer cells from primary cultures, using the explant technique, to clarify the role of this peptide in the suppression of breast cancer cell migration. The results showed that incubation of breast tumor explants with RGD peptide at the beginning of culture development inhibited completely the migration of cancer cells out of the tissue fragment as revealed by electron microscopy. RGD incubation of well-spread breast cancer cells from primary culture resulted in rounding and shrinkage of the cells accompanied by altered distribution of integrin alphavbeta3 and concomitant F-actin cytoskeletal disorganization, as revealed by immunofluorescence. Electron immunocytochemistry showed aggregation of integrin alphavbeta3 at the cell periphery and its detection in noncoated vesicles. However, Western immunoblotting showed no change in beta3 subunit expression, despite the altered distribution of the integrin alphavbeta3. In light of the above, it appears that the RGD peptide plays an important role in the modulation of cell motility and in the perturbation of cell attachment affecting the malignant potential of breast cancer cells in primary cultures.

Nuclear Localization of Cytokeratin 8 and the O-linked N-Acetylglucosamine-containing Epitope H in Epithelial Cells of Infiltrating Ductal Breast Carcinomas: A Combination of Immunogold and EDTA Regressive Staining Methods

In a previous study, the authors have shown cytokeratin 8 (CK8) and epitope H ultrastructural localization in breast cancer cell nuclei. Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H. In this study, double immunogold labeling of CK8 and epitope H combined with the EDTA regressive staining method was applied in biopsy material from infiltrating ductal breast carcinomas and fibroadenomas, to localize both antigens in correlation to RNPs distribution in the nuclear subcompartments of cancer cells. CK8 and epitope H were localized mostly over condensed chromatin, whereas staining was weaker over interchromatin granule clusters and perichromatin fibers. These results revealed, the distribution of CK8 in the nucleus as MAR-binding protein, contributing in the organization of the nuclear DNA in the neoplastic cell, as well as the distribution of O-GlcNAc glycosylated polypeptides bearing the epitope H. The latter finding indicates that these polypeptides might play a significant role in the neoplastic behavior of breast cancer cells because they colocalize in the same nuclear subcompartments with proteins modified by O-GlcNAc, such as hnRNPs G and A1, RNA polymerase II, its transcription factors, and the oncogene product of c-myc. These proteins are known to participate in coordinated transcription/RNA processing events, contributing in the neoplastic behavior of breast cancer cells.