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D. squalens, a white-rot fungus that efficiently degrades lignocellulose in nature, can be used in various biotechnological applications and has several strains with sequenced and annotated genomes. Here we present a method for the transformation of this basidiomycete fungus, using a recently introduced commercial ascomycete protoplasting enzyme cocktail, Protoplast F. In protoplasting of D. squalens mycelia, Protoplast F outperformed two other cocktails while releasing similar amounts of protoplasts to a third cocktail. The protoplasts released using Protoplast F had a regeneration rate of 12.5% (±6 SE). Using Protoplast F, the D. squalens monokaryon CBS464.89 was conferred with resistance to the antibiotics hygromycin and G418 via polyethylene glycol mediated protoplast transformation with resistance cassettes expressing the hygromycin phosphotransferase (hph) and neomycin phosphotransferase (nptII) genes, respectively. The hph gene was expressed in D. squalens using heterologous promoters from genes encoding β-tubulin or glyceraldehyde 3-phosphate dehydrogenase. A Southern blot confirmed integration of a resistance cassette into the D. squalens genome. An average of six transformants (±2 SE) were obtained when at least several million protoplasts were used (a transformation efficiency of 0.8 (±0.3 SE) transformants per μg DNA). Transformation of D. squalens demonstrates the suitability of the Protoplast F cocktail for basidiomycete transformation and furthermore can facilitate understanding of basidiomycete gene function and development of improved strains for biotechnological applications.

Background: Despite recent advances in cancer therapy, the 5-year survival rate for Ewing's sarcoma is still very low, and new therapeutic approaches are necessary. It was found previously that melatonin induces cell death in the Ewing's sarcoma cell line, SK-N-MC, by activating the extrinsic apoptotic pathway. Methods: Melatonin actions were analysed by metabolic viability/survival cell assays, flow cytometry, quantitative PCR for mRNA expression, western blot for protein activation/expression and electrophoretic mobility shift assay for transcription factor activation. Results: Melatonin increases the expression of Fas and its ligand Fas L, this increase being responsible for cell death induced by the indolamine. Melatonin also produces a transient increase in intracellular oxidants and activation of the redox-regulated transcription factor Nuclear factor-kappaB. Inhibition of such activation prevents cell death and Fas/Fas L upregulation. Cytotoxic effect and Fas/Fas L regulation occur in all Ewing's cell lines studied, and do not occur in the other tumour cell lines studied where melatonin does not induce cell death. Conclusion: Our data offers new insights in the study of alternative therapeutic strategies in the treatment of Ewing's sarcoma. Further attention deserves to be given to the differences in the cellular biology of sensitive tumours that could explain the cytotoxic effect of melatonin and the increase in the level of free radicals caused by this molecule, in particular cancer types.

Melatonin is an endogenous indolamine, classically known as a light/dark regulator. Besides classical functions, melatonin has also showed to have a wide range of antitumoral effects in numerous cancer experimental models. However, no definite mechanism has been described to explain the whole range of antineoplasic effects. Here we describe a dual effect of melatonin on intracellular redox state in relation to its antiproliferative vs cytotoxic actions in cancer cells. Thus, inhibition of proliferation correlates with a decrease on intracellular reactive oxygen species (ROS) and increase of antioxidant defences (antioxidant enzymes and intracellular gluthation,GSH levels), while induction of cell death correlates with an increase on intracellular ROS and decrease of antioxidant defences. Moreover, cell death can be prevented by other well-known antioxidants or can be increased by hydrogen peroxide. Thus, tumour cell fate will depend on the ability of melatonin to induce either an antioxidant environment--related to the antiproliferative effect or a prooxidant environment related to the cytotoxic effect.

The mechanisms underlying microRNA (miRNA) disruption in human disease are poorly understood. In cancer cells, the transcriptional silencing of tumor suppressor genes by CpG island promoter hypermethylation has emerged as a common hallmark. We wondered if the same epigenetic disruption can “hit” miRNAs in transformed cells. To address this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in combination with a miRNA expression profiling. We have observed that DNA hypomethylation induces a release of miRNA silencing in cancer cells. One of the main targets is miRNA-124a, which undergoes transcriptional inactivation by CpG island hypermethylation in human tumors from different cell types. Interestingly, we functionally link the epigenetic loss of miRNA-124a with the activation of cyclin D kinase 6, a bona fide oncogenic factor, and the phosphorylation of the retinoblastoma, a tumor suppressor gene. [Cancer Res 2007;67(4):1424–9]

Glioblastoma multiforme (GBM) is an incurable malignancy with inherent tendency to recur. In this study, we have comparatively analyzed the epigenetic profile of 32 paired tumor samples of relapsed GBM and their corresponding primary neoplasms with special attention to genes involved in the mitochondria-independent apoptotic pathway. The CpG island promoter hypermethylation status was assessed by methylation-specific polymerase chain reaction and selected samples were double checked by bisulfite genomic sequencing. Thirteen genes were analyzed for DNA methylation: the pro-apoptotic CASP8, CASP3, CASP9, DcR1, DR4, DR5 and TMS1; the cell adherence CDH1 and CDH13; the candidate tumor suppressor RASSF1A and BLU; the cell cycle regulator CHFR and the DNA repair MGMT. The CpG island promoter hypermethylation profile of relapsed GBM in comparison with their corresponding primary tumors was identical in 37.5% of the cases, whereas in 62.5% of patients, differences in the DNA methylation patterns of the 13 genes were observed. The most prominent distinction was the presence of previously undetected CASP8 hypermethylation in the GBM relapses (P = 0.031). This finding was also linked to the observation that an unmethylated CASP8 CpG island together with methylated BLU promoter in the primary GBM was associated with prolonged time to tumor progression (P = 0.0035). Our data strongly suggest that hypermethylation of the pro-apoptotic CASP8 is a differential feature of GBM relapses. These remarkable findings may foster the development of therapeutic approaches using DNA demethylating drugs and activators of the extrinsic apoptotic pathway to improve the dismal prognosis of GBM.

It is well established that melatonin exerts antitumoral effects in many cancer types, mostly decreasing cell proliferation at low concentrations. On the other hand, induction of apoptosis by melatonin has been described in the last few years in some particular cancer types. The cytotoxic effect occurs after its administration at high concentrations, and the molecular pathways involved have been only partially determined. Moreover, a synergistic effect has been found in several cancer types when it is administered in combination with chemotherapeutic agents. In the present review, we will summarize published work on the pro-apoptotic effect of melatonin in cancer cells and the reported mechanisms involved in such action. We will also construct a hypothesis on how different cell signaling pathways may relate each other on account for such effect.

Incorporation of new therapeutic agents remains as a major challenge for treatment of patients with malignant haematological disorders. Melatonin is an indolamine without relevant side effects. It has been shown previously to exhibit synergism with several chemotherapeutic drugs in Ewing sarcoma cells by potentiating the extrinsic pathway of apoptosis. It also sensitizes human glioma cells against TRAIL by increasing DR5 expression. Here, we report the induction of cell death by melatonin in several human malignant haematological cell lines through the activation of the extrinsic pathway of apoptosis. Such activation was mediated by the increase in the expression of the death receptors Fas, DR4 and DR5 and their ligands Fas L and TRAIL, with a remarkable rise in the expression of Fas and Fas L. The cytotoxic effect and the increase in Fas and Fas L were dependent on Akt activation. Results were corroborated in blasts from bone marrow and peripheral blood of acute myeloid leukaemia patients, where melatonin induced cell death and increased both Fas and Fas L expressions. We conclude that melatonin may be considered as a potential antileukaemic agent and its therapeutic use, either alone or in combination with current chemotherapeutic drugs, should be taken into consideration for further research.

The concept of ion channels as membrane therapeutic targets and diagnostic/prognostic biomarkers has attracted growing attention. We therefore investigated the expression pattern and clinical significance of the Kv3.4 potassium channel subunit during the development and progression of head and neck squamous cell carcinomas (HNSCCs). KCNC4 mRNA levels were determined by real-time RT-PCR in both HNSCC tissue specimens and derived cell lines. Kv3.4 protein expression was evaluated by immunohistochemistry in paraffin-embedded tissue specimens from 84 patients with laryngeal/pharyngeal squamous cell carcinomas and 67 patients with laryngeal dysplasias. Molecular alterations were correlated with clinicopathological parameters and patient outcome. Increased KCNC4 mRNA levels were found in 15 (54%) of 28 tumours, compared to the corresponding normal epithelia and varied mRNA levels were detected in 12 HNSCC-derived cell lines analysed. Increased Kv3.4 protein expression was observed in 34 (40%) of 84 carcinomas and also at early stages of HNSCC tumourigenesis. Thus, 35 (52%) of 67 laryngeal lesions displayed Kv3.4-positive staining in the dysplastic areas, whereas both stromal cells and normal adjacent epithelia exhibited negligible expression. No significant correlations were found between Kv3.4-positive expression in HNSCC and clinical data; however, Kv3.4 expression tended to diminish in advanced-stage tumours. Interestingly, patients carrying Kv3.4-positive dysplasias experienced a significantly higher laryngeal cancer incidence than did those with negative lesions (p = 0.0209). In addition, functional studies using HNSCC cells revealed that inhibition of Kv3.4 expression by siRNA leads to the inhibition of cell proliferation via selective cell cycle arrest at the G2/M phase without affecting apoptosis. Collectively, these data demonstrate for the first time that Kv3.4 expression is frequently increased during HNSCC tumourigenesis and correlated significantly with a higher cancer risk. Our findings support a role for Kv3.4 in malignant transformation and provide original evidence for the potential clinical utility of Kv3.4 expression as a biomarker for cancer risk assessment.

Ewing sarcoma, the second most frequent bone cancer type, affects mainly adolescents, who have a survival of 50% 5 yr after diagnosis. Current treatments include a combination of surgery, radiotherapy and chemotherapy, which present potential serious side effects. Melatonin, a natural molecule without relevant side effects, has been previously shown to induce cytotoxicity in SK-N-MC cells, a Ewing sarcoma cell line. Here, we found that there is a synergy in the antitumor effect when melatonin (50 mum-1 mm) is combined with vincristine at the concentration of 5-10 nm or with ifosfamide at the range of 100 mum-1 mm. This synergism is due to the potentiation of cell death, particularly to the potentiation of apoptosis, i.e., mainly the extrinsic apoptotic pathway. There is a significant increase in the activation of caspase-3, -8, -9 and Bid when melatonin is combined with vincristine or ifosfamide compared to the individual treatments. Finally, there is also a potentiation of the early free radical production, likely dependent on the extrinsic apoptosis pathway activation, when the drugs are combined with melatonin. Other proteins which are related to this pathway including mitogen-activated protein kinase or protein kinase B/Akt are not involved in apoptosis induced by these agents separately or when combined. The results shown here together with the facts that: (i) no relevant side effects have been reported for melatonin and (ii) melatonin has a cytoprotective effect on noncancer cells, opens the door for a new approach in the treatment of the Ewing sarcoma family of tumors.

An undifferentiated status and the epigenetic inactivation of tumor-suppressor genes are hallmarks of transformed cells. Promoter CpG island hypermethylation of differentiating genes, however, has rarely been reported. The Groucho homologue Transducin-like Enhancer of Split 1 (TLE1) is a multitasked transcriptional corepressor that acts through the acute myelogenous leukemia 1, Wnt, and Notch signaling pathways. We have found that TLE1 undergoes promoter CpG island hypermethylation–associated inactivation in hematologic malignancies, such as diffuse large B-cell lymphoma and AML. We also observed a mutual exclusivity of the epigenetic alteration of TLE1 and the cytogenetic alteration of AML1. TLE1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas TLE1-short hairpin RNA depletion in unmethylated cells enhances tumor growth. We also show that these effects are mediated by TLE1 transcriptional repressor activity on its target genes, such as Cyclin D1, Colony-Stimulating Factor 1 receptor, and Hairy/Enhancer of Split 1. These data suggest that TLE1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting critical differentiation and growth-suppressing pathways. [Cancer Res 2008;68(11):4116–22]

Vicenzo Calvanese et al..., Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation., This work was primarily supported by the European Union (LSHG-CT-2006-018739; ESTOOLS). MFF is funded by the Spanish Ramon & Cajal Programme and the Health Department of the Spanish Government (PI061267). The Cancer Epigenetics group at the CNIO is supported by the Health (FIS01-04) and Education and Science (I+D+I MCYT08-03, FU2004-02073/BMC and Consolider MEC09-05) Departments of the Spanish Government, the European Grant TRANSFOG LSHC-CT-2004-503438, and the Spanish Association Against Cancer (AECC). VC is a recipient of a Fellowship from the FPU Spanish Research Programme. CLL and BSA are supported by the Health Department of the Spanish Government (PI051707). The BACM is supported by the Consejería de Salud de la Junta de Andalucía (0029 and, 0030/2006 to PM) and, the Spanish Ministry of Health to PM (FIS PI070026). CB is supported by the International Jose Carreras Foundation against Leukemia (EDThomas-05) and the ISCIII (FIS 3+3 contract). The Institute of Reconstructive Neurobiology received additional funding from the DFG and the Hertie Foundation. BS, AbH and AnH are supported by the Fundación Progreso y Salud and Instituto de Salud Carlos III-Red Española de Terapia Celular (RD06/0010/0025 ). PWA, NH and HDM are also supported by the MRC.