Defects in mTR stability and telomerase activity produced by the Dkc1 A353V mutation in dyskeratosis congenita are rescued by a peptide from the dyskerin TruB domain.

BACKGROUND: The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. MATERIALS AND METHODS: Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. RESULTS: Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. DISCUSSION: These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach.

A role for cancer stem cells in drug resistance and metastasis in non-small-cell lung cancer.

The cancer stem cell (CSC) theory is currently a very important field in cancer research. This theory states that tumours are organised in a hierarchical manner with a subpopulation of limited number called CSCs with the ability to self-renew and undergo asymmetrical divisions, giving rise to a differentiated progeny that represents most of the tumour populations. CSCs are metastatic and chemoresistant, two features that very likely contribute to the poor response of locally advanced lung cancer. CSCs have been identified in non-small-cell lung cancer cell lines as well as those from patient primary samples. A correlation has been found in terms of chemoresistance and bad prognosis in patient-derived samples enriched with CSCs, indicating that these cells are an important target for future therapy combinations. Therefore, understanding the biology and exploring cell markers and signalling pathways specific for CSCs of lung cancer may help in achieving progress in the treatment of the disease.

Telomerase deficiency and cancer susceptibility syndromes.

Telomeres from most eukaryotes are composed of repeats of guanine-rich sequences whose main function is to preserve the end of the chromosomes. Telomeres are synthesised by a reverse transcriptase enzyme, telomerase (TERT), which forms part of a ribonucleoprotein complex containing also a RNA template molecule (TERC) and dyskerin. Exhaustion of telomeres during cell divisions triggers a DNA damage response that induces a senescence phenotype. The DNA damage machinery plays an essential role in maintaining the integrity of the genome and also detecting telomere shortening. However in some syndromes that involved mutations either in the telomerase complex genes or those involved in maintaining DNA secondary structure, such as the recQ helicase WRN, a higher frequency in the development of different types of malignancies is observed. We here describe the biology of some of these diseases, together with the molecular modifications in the telomerase complex genes and the impact of these alterations on the development of particular types of cancer.

Role of CHK2 in cancer development.

DNA repair pathways enable tumour cells to survive DNA damage induced by external agents such as therapeutic treatments. Signalling cascades involved in these pathways comprise the DNA-dependent protein kinase (DNA-PK), Ataxia-telangiectasia mutated (ATM), ATM and Rad3 related (ATR) and checkpoint kinases I and 2 (Chk1/Chk2), among others. ATM and ATR phosphorylate, respectively, Chk2 and Chk1, leading to activation of checkpoints. Chk2 acts as a signal distributor, dispersing checkpoint signal to downstream targets such as p53, Cdc25A, Cdc25C, BRCA1 and E2F1. A role of Chk2 as a candidate tumour suppressor has been suggested based on both mouse genetics and somatic tumour studies. We will discuss here the possible role of this kinase in human carcinogenesis and the possibility to use it as a target to increment DNA damage in cancer cells in response to DNA-damaging therapies.

The role of the NFkappaB signalling pathway in cancer.

The nuclear factor kappa B (NFkappaB) signalling pathway regulates the expression of hundreds of genes that are involved in different cellular processes such as cell proliferation, survival, stress responses, cellular immunity and inflammation. Its aberrant regulation is involved in several pathologies, but its relevance in cellular transformation and cancer development has been extensively studied. Mutations in the core components of NFkappaB as well as in the cellular machinery that regulates its activation have been found in many types of tumours. On the other hand, its role in promoting cell survival is an important obstacle in many cancer therapies. The development of chemical inhibitors that block NFkappaB activation acting either directly on IKKs or on the proteosome machinery has shown antitumour and proapoptotic activity both in preclinical and clinical studies.

Epidermal growth factor receptor and glioblastoma multiforme: molecular basis for a new approach.

High-grade gliomas are the most common primary malignant brain tumours. Surgery, radiotherapy and chemotherapy are the cornerstone of actual treatment. In spite of large therapeutic efforts, overall survival is still poor. New molecular data allow a new molecular classification for high-grade gliomas and open a therapeutic window for targeted therapy. Molecular diagnostic tools may provide a basis for receptor-based therapies and enough information to personalise future treatments. In this regard, epidermal growth factor receptor (EGFR) is a target that will play a critical role in the management of glioma patients. This review summarises basic and preclinical data that support future use of therapies against EGFR.

Molecular biology of malignant gliomas.

Gliomas are the most common primary brain tumours. In keeping with the degree of aggressiveness, gliomas are divided into four grades, with different biological behaviour. Furthermore, as different gliomas share a predominant histological appearance, the final classification includes both, histological features and degree of malignancy. For example, gliomas of astrocytic origin (astrocytomas) are classified into pilocytic astrocytoma (grade I), astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma multiforme (GMB) (grade IV). Tumors derived from oligodendrocytes include grade II (oliogodendrogliomas) and grade III neoplasms (oligoastrocytoma). Each subtype has a specific prognosis that dictates the clinical management. In this regard, a patient diagnosed with an oligodendroglioma totally removed has 10-15 years of potential survival. On the opposite site, patients carrying a glioblastoma multiforme usually die within the first year after the diagnosis is made. Therefore, different approaches are needed in each case. Obviously, prognosis and biological behaviour of malignant gliomas are closely related and supported by the different molecular background that possesses each type of glioma. Furthermore, the ability that allows several low-grade gliomas to progress into more aggressive tumors has allowed cancer researchers to elucidate several pathways implicated in molecular biology of these devastating tumors. In this review, we describe classical pathways involved in human malignant gliomas with special focus with recent advances, such as glioma stem-like cells and expression patterns from microarray studies.

Cell signalling: growth factors and tyrosine kinase receptors.

The mitogenic signaling in mammalian cells is carried out mainly by growth factors that interact with receptors localized at the plasma membrane. Most of these receptors have a tyrosine kinase activity domain that is localized at the cytoplasmic region of the molecule. The interaction of the growth factors with the receptors, besides inducing the kinase activity of the receptor, activate signaling pathways the alter gene expression patterns and induce mitogenesis, or if deregulated are related to cancer. Among these receptors ERBB, VEGF, PDGF and IGF are attractive targets for directed therapies. ERBB receptors are frequently involved in the production of many types of cancers. Both, the over-expression of the growth factor and the receptor, besides mutations at the cytoplasmic tyrosine kinase domain contribute to constitutive signaling in human cancer. VEGF has a pivotal role in maintaining the tumor growth by facilitating growth of new blood vessels. Therefore, inhibition of tumor growth targeting of the tumor vasculature, by interfering with the activity of VEGFr is now a real alternative in combinatorial therapies. PDGF is a growth factor involved in growth of connective tissue and wound healing. Activating mutations of PDGFr have been found in gastrointestinal tumors and the autocrine signaling maintained by this receptor have been described in many tumors. Imatinib, and inhibitor of the tyrosine kinase activity of Bcr-Abl targets also the kinase of the PDGFr. Finally IGF-I an II have an important antiapoptotic and pro-mitogenic role in most tumors. Different inhibitors are now under clinical studies for the use in combination of chemotherapeutic drugs in the treatment of different tumors.