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In the present study, the phenotype of melanoma cells resistant to dabrafenib (a B-RAF inhibitor) was investigated, to shed more light on melanoma resistance to B-RAF inhibition. Melanoma cells resistant to dabrafenib were generated using 3 different cell lines, A375, 397 and 624.38, all carrying B-RAFV600E, and they were characterized by cytofluorometric analysis, Ion Torrent technology, immunofluorescence and biochemistry. All dabrafenib-resistant cells showed, in addition to a re-activation of MAPK signaling, morphological changes compared to their sensitive counterparts, accompanied by an increase in CD90 (mesenchymal marker) expression and a decrease in E-cadherin (epithelial marker) expression, suggesting an epithelial-to-mesenchymal-like phenotypic transition. However, melanoma cells with TGF-β1-induced epithelial-to-mesenchymal transition (EMT) were more sensitive to dabrafenib treatment compared to the sensitivity noted in the non-TGF‑β1‑induced EMT melanoma cells, suggesting that TGF-β1-induced EMT was not associated with dabrafenib resistance. Although dabrafenib-resistant cells exhibited increased cell motility and E-cadherin/vimentin reorganization, as expected in EMT, all of them showed unvaried E-cadherin mRNA and unchanged Snail protein levels, while Twist1 protein expression was decreased with the exception of A375 dabrafenib-resistant melanoma cells, where it was unaffected. These findings suggest a distinct active EMT-like process adopted by melanoma cells under drug exposure. Furthermore, dabrafenib-resistant cells exhibited stem cell-like features, with Oct4 translocation from the cytoplasm to peri-nuclear sites and nuclei, and increased CD20 expression. In conclusion, our data, in addition to confirming that resistance to dabrafenib is dependent on re-activation of MAPK signaling, suggest that this resistance is linked to a distinct active EMT-like process as well as stem-cell features adopted by melanoma cells.

Cutaneous melanoma is an aggressive cancer with a poor prognosis for patients with advanced disease. The identification of several key molecular pathways implicated in the pathogenesis of melanoma has led to the development of novel therapies for this devastating disease. In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms. Targeting various effectors of these pathways with pharmacologic inhibitors may inhibit melanoma cell growth and angiogenesis. Ongoing clinical trials provide hope to improve progression-free survival of patients with advanced melanoma. This review summarizes the most relevant studies focused on the specific action of these new molecular targeted agents. Mechanisms of resistance to therapy are also discussed.

Breast cancer is the worldwide leading cause of cancer incidence among women. Night shift work exposure has been recently considered one of the significant breast cancer risk factors in industrialized countries. The mechanisms by which this work exposure may be responsible for cancer development is still discussed. In the last 15 years, many authors have paid attention to the relationship between night shift work and breast cancer risk. In the current study, eight case-control studies and four prospective epidemiological studies describing such relationship are discussed. A positive correlation between night shift work and breast cancer risk was described in 8 out of 12 studies. However, different reasons suggest that some of these studies have an Achilles heel according to the International Agency of Cancer (IARC) indications. Both the circadian system alteration and the melatonin output reduction, related to the exposure to light-at-night during night shift work, remain the most valid hypotheses on the causal relation of shift work and breast cancer. Overall, the results of the present study suggest that there is an association between night shift work and breast cancer development in western countries. However, further studies are needed to confirm such association and to understand which biomolecular mechanisms may be involved in the pathogenesis of cancer diagnosed in patients with night shift work exposure.

The tumor microenvironment has been largely studied as a dynamic system orchestrated by inflammatory cells, including cancer cells, stroma as well as the extracellular matrix. It is useful to describe and predict the phenotypic characteristics of cancer. Furthermore, a better understanding of its interplay with the various aspects of the tumor cells may be utilized for the discovery of novel molecular targets. Liver cancer is considered a model of the relation occurring between the tumor micro-environment and tumor development. The chronic inflammatory status of the liver, sustained by the infection of hepatitis viruses, as well as the production of cytokines and growth factors within the parenchyma, lead to an intricate microenvironment. The identification of novel molecular therapeutic targets may improve the outcome of patients with liver cancer as it remains the third leading cause of cancer death worldwide. In the present study, the tumor microenvironment in hepatocellular carcinoma (HCC) was explored by a review of the literature. Studies on hepatitis virus infections and the consequent chronic inflammatory status were examined. In this context, immune-mediated and/or virus-related molecular mechanisms have been hypothesized as being responsible for liver cancer development. The interlink among HCC microenvironment components, comprising cellular elements, cytokines, growth factors and several proteins is also described together with the role of matrix metalloproteinases in HCC development. Finally, the rationale for targeting tumor-stromal interface is summarized in the context of new therapeutic opportunities.

The most common therapeutic approach for many cancers is chemotherapy. However, many patients relapse after treatment due to the development of chemoresistance. Recently, targeted therapies represent novel approaches to destroy cancer cells. The PI3K/PTEN/AKT pathway is a key signaling pathway involved in the regulation of cell growth. Dysregulated signaling of this pathway may be associated with activating mutations of PI3K-related genes. Analyses of these mutations reveal that they increase the PI3K signal, stimulate downstream Akt signaling, promote growth factor-independent growth and increase cell invasion and metastasis. In this review, we summarize the PI3K/PTEN/AKT pathway genetic alterations in cancer and their potential clinical applications.

In this study, by using different techniques (i.e. Northern blot hybridization, RT-PCR and Southern blot hybridization) on various normal rat tissues, we were able to identify liver, kidney, heart, small intestine, brain, spleen, stomach and prostate as tissues in which the ApoH gene is transcribed. Moreover, for some of these tissues, by in situ hybridization, we found a specific localization of apoH transcripts. For instance epithelial cells of the bile ducts in liver and of the proximal tubules in kidney are the major sites of apoH synthesis. Our data suggest that some of the different physiological roles proposed for apoH could correlate with its direct expression, while others could correlate with its absorption from bloodstream or adjacent cells.

Partial deletions of the DAZ gene cluster are thought to cause spermatogenesis impairment. The presence of homologous copies of this gene in the Y chromosome does not allow PCR to be used for the identification of this abnormality. Hence, sequence family variants (SFV), following amplification of sY581, sY587 and sY586 and subsequent enzymatic digestion with Sau3A, DraI and TaqI, respectively, and the dual fiber fluorescence in situ hybridization (FISH) have been used to this aim. However, SFV is not always able to identify single DAZ gene copy deletions. We report a quantitative real-time PCR application to evaluate partial deletions of the DAZ gene cluster. To accomplish this, we designed a probe on exon 6 of the DAZ gene which is repeated 3 times in DAZ1, once in DAZ2 and DAZ3 and twice in DAZ4. Five normozoospermic healthy men (C1-C5) having 4 DAZ gene copies by SFV were selected. Fiber-FISH confirmed this outcome in C1-C4, but not in C5 who had an incomplete DAZ gene cluster. The men underwent then quantitative real-time PCR and C1 was arbitrarily selected as calibrator for the calculation of the DAZ gene signals because of the lowest variation in the threshold cycles. Real-time PCR identified 7.2+/-0.05 signals in C2-C4 and 5.4+/-0.05 signals in C5. The overall coefficient of variation was 1.4+/-0.2%. The loss of two signals in this subject may relate to a deletion of both DAZ2 and DAZ3 or of DAZ4 gene. Since SFV showed clearly the presence of DAZ2, it may be hypothesized that C5 lacks DAZ4. In conclusion, these data suggested that quantitative real-time PCR seems to be an effective and reproducible technique that can be used to study the DAZ gene cluster. In addition, the probe chosen for this approach may give indication on the DAZ gene copy deleted.

In this study, we evaluated the expression of genes probably involved in spermatogenesis in the mouse. We examined cytosolic chaperonin theta subunit (CCTtheta), Ngg1 interacting factor 3 like 1 binding protein 1 (NIF3L1 BP1) and apolipoprotein H (ApoH) expression during mouse onto-geny using RT-PCR. Testicular tissue was obtained from mice 3, 6, 8, 10, 12, 14, 18, 20 and 40 (adult) days after birth. For each mouse, one testis was used for histological examination, whereas RNA was extracted from the controlateral testis for expression analysis. RT-PCR analysis showed that CCTtheta gene expression was low until day 10, but increased drastically afterwards. At this age, spermatocytes started to be present in the mouse testis. Therefore, CCT protein could be involved in chromatin packaging and remodeling during spermiogenesis, as also suggested by other studies. NIF3L1 BP1 expression increased steadily during ontogenesis reaching maximum levels in the adult mouse when all germ cell stages are present. This finding suggests that NIF3L1 BP1 is a gene not expressed by a specific germ cell type. ApoH expression was very low or absent during prepuberal stages, whereas it was detectable in the adult testis when spermatogenesis was completed. This suggests that ApoH may be involved in clearing apoptotic bodies during spermatogenesis since apoptotic events increase during spermatogenesis. This study contributes to understanding the role played by genes important for spermatogenesis.

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in the gene coding for the low density lipoprotein receptor (LDL-R). It is characterized by a high concentration of low density lipoprotein (LDL), which frequently gives rise to premature coronary artery disease. We studied the probands of five FH Sicilian families with 'definite' FH and one proband of Paraguayan descent with homozygous FH who has been treated with an effective living-donor liver transplantation. In order to seek the molecular defect in these six families, we used direct sequencing to define the molecular defects of the LDL-R gene responsible for the disease. We described three novel missense mutations (C100Y, C183Y and G440C), two frameshift mutations (g.1162delC in exon 8 and g.2051delC in exon 14) and one mutation (g.2390-1Gright curved arrow A) at splicing acceptor consensus sequences located in intron 16 of the LDL-R gene; the analysis of cDNA of this splicing mutation showed the activation of a cryptic splice site in intron 16 and the binding studies showed a reduction in internalisation of LDL-DIL in the proband's cultured fibroblasts. Moreover, a g.2051delC in exon 14 was identified in the proband of Paraguayan ancestry with clinical features of homozygous FH. The mutation identified in the South American patient represents the first description of a variant in South American patients other than Brazilian FH patients. The 5 mutations identified in the Sicilian patients confirm the heterogeneity of LDL-R gene mutations in Sicily.

Bovine seminal-ribonuclease (BS-RNase) is a member of the 'ribonucleases with special biological actions' family since it possesses specific anti-tumour, anti-spermatogenic and embryotoxic activities and exerts an immunosuppressive effect on T lymphocytes. In previous studies it was demonstrated that BS-RNase induced apoptosis in proliferating, malignant and normal cells and that telomerase activity loss also caused apoptotic death in neoplastic cells. Since an obvious relationship between cell proliferation and telomerase activity exists, the aim of this work was to study if the pro-apoptotic cytotoxic action exerted by BS-RNase on proliferating malignant cells (HT29) and proliferating normal cells (PHA-stimulated lymphocytes) could be linked to a possible BS-RNase effect on telomerase activity. In BS-RNase-treated HT29 cells (Na-butyrate-differentiated or not) and human lymphocytes (proliferating or not), we investigated cell vitality (MTT method) and morphology (SEM), BS-RNase localization (immunofluorescence), telomerase activity (TRAP-ELISA method), hTR mRNA expression (RT-PCR), and hTERT levels (western blot). While no BS-RNase effect was detectable on not proliferating cells, a clear relationship was noticed between the diminished number of vital elements of both proliferating cell populations after treatment (48 h and 72 h for HT29 and PHA-stimulated lymphocytes, respectively) with 50 microg/ml BS-RNase and the decrease of their telomerase activity. At the same time, we found that hTR levels, the RNA subunit of telomerase, in proliferation-inhibited BS-RNase-treated cells were diminished. Moreover, by immunofluorescence technique, we detected BS-RNase in the HT29 cell nucleolus after 3-h treatment. Therefore, as hTR has been recently proven to co-fractionate with nucleoli, we hypothesize that a BS-RNase direct action on the telomerase hTR subunit could be a possible mechanism of action by which BS-RNase exerts its pro-apoptotic effects only on proliferating cells.

The effects of all trans retinoic acid and hyperthermia were studied in the human colon adenocarcinoma cell line HT29. Cell cytotoxicity after exposure to ATRA or heat-shock, alone or in association, was evaluated by the MTT assay while cell surface and ultrastructure modifications and actin fibre assembly changes were investigated by electron microscopy and by the FITC-phalloidin method. Apoptosis was evaluated by flow cytofluorimetry and electron microscopy. Reverse transcriptase-polymerase chain reaction was employed to study mRNA expression of genes involved in apoptosis, differentiation and growth arrest. Joint treatments were more effective in reducing the vital cell yield, being this effect only partially due to apoptosis. A marked up-regulation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 expression, not followed by any differentiation process, was responsible for growth arrest. Modulation of Hsp-70 expression, involved in cell response to treatments, was considered. Our results demonstrate that cell treatment with ATRA followed by heat-shock may elicit useful effects to treat tumours, which are responsive to retinoids, as well as those malignant cells which may be constitutively thermotolerant.