Antonio Elipe Sánchez

Cytogenetic peculiarities in the Algerian hedgehog: silver stains not only NORs but also heterochromatic blocks

Hedgehogs belong to one of the several mammalian taxa in which karyotype differences are based on variations in heterochromatin content. Furthermore, the number and location of nucleolar organizer regions (NORs) can also vary widely. In the present study these cytogenetic features were investigated in the Algerian hedgehog, Erinaceus (Aethechinus) algirus. The heterochromatin and NOR distribution patterns in the karyotype of this species are new among hedgehogs, whereas the euchromatic regions, including their G-band pattern, are similar to those reported by others. In addition, silver staining revealed a cytogenetic feature exclusive to the heterochromatic blocks of E. algirus: their silver staining with standard cytogenetic procedures. Because no similar phenomenon has been described previously in a mammalian species, several hypotheses about the significance and specificity of silver staining to NOR sites are discussed. Finally, the existence of different types of heterochromatin in the species analysed here, lead us to propose that what hedgehogs have inherited from their common ancestor is a mechanism which permits the accumulation of heterochromatin on specific chromosomes, rather than the heterochromatin itself.

X chromosome painting in Microtus: Origin and evolution of the giant sex chromosomes

Sex chromosomes in species of the genus Microtus present some characteristic features that make them a very interesting group to study sex chromosome composition and evolution. M. cabrerae and M. agrestis have enlarged sex chromosomes (known as ‘giant sex chromosomes’) due to the presence of large heterochromatic blocks. By chromosome microdissection, we have generated probes from the X chromosome of both species and hybridized on chromosomes from six Microtus and one Arvicola species. Our results demonstrated that euchromatic regions of X chromosomes in Microtus are highly conserved, as occurs in other mammalian groups. The sex chromosomes heterochromatic blocks are probably originated by fast amplification of different sequences, each with an independent origin and evolution in each species. For this reason, the sex heterochromatin in Microtus species is highly heterogeneous within species (with different composition for the Y and X heterochromatic regions in M. cabrerae) and between species (as the composition of M. agrestis and M. cabrerae sex heterochromatin is different). In addition, the X chromosome painting results on autosomes of several species suggest that, during karyotypic evolution of the genus Microtus, some rearrangements have probably occurred between sex chromosomes and autosomes.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2010-31 January 2011: PERMANENT GENETIC RESOURCES NOTE

This article documents the addition of 238 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alytes dickhilleni, Arapaima gigas, Austropotamobius italicus, Blumeria graminis f. sp. tritici, Cobitis lutheri, Dendroctonus ponderosae, Glossina morsitans morsitans, Haplophilus subterraneus, Kirengeshoma palmata, Lysimachia japonica, Macrolophus pygmaeus, Microtus cabrerae, Mytilus galloprovincialis, Pallisentis (Neosentis) celatus, Pulmonaria officinalis, Salminus franciscanus, Thais chocolata and Zootoca vivipara. These loci were cross‐tested on the following species: Acanthina monodon, Alytes cisternasii, Alytes maurus, Alytes muletensis, Alytes obstetricans almogavarii, Alytes obstetricans boscai, Alytes obstetricans obstetricans, Alytes obstetricans pertinax, Cambarellus montezumae, Cambarellus zempoalensis, Chorus giganteus, Cobitis tetralineata, Glossina fuscipes fuscipes, Glossina pallidipes, Lysimachia japonica var. japonica, Lysimachia japonica var. minutissima, Orconectes virilis, Pacifastacus leniusculus, Procambarus clarkii, Salminus brasiliensis and Salminus hilarii.

Rapid, independent, and extensive amplification of telomeric repeats in pericentromeric regions in karyotypes of arvicoline rodents

The distribution of telomeric repeats was analyzed by fluorescence in situ hybridization in 15 species of arvicoline rodents, included in three different genera: Chionomys, Arvicola, and Microtus. The results demonstrated that in most or the analyzed species, telomeric sequences are present, in addition to normal telomeres localization, as large blocks in pericentromeric regions. The number, localization, and degree of amplification of telomeric sequences blocks varied with the karyotype and the morphology of the chromosomes. Also, in some cases telomeric amplification at non-pericentromeric regions is described. The interstitial telomeric sequences are evolutionary modern and have rapidly colonized and spread in pericentromeric regions of chromosomes by different mechanisms and probably independently in each species. Additionally, we colocalized telomeric repeats and the satellite DNA Msat-160 (also located in pericentromeric regions) in three species and cloned telomeric repeats in one of them. Finally, we discuss about the possible origin and implication of telomeric repeats in the high rate of karyotypic evolution reported for this rodent group.

Molecular and cytogenetic characterization of highly repeated DNA sequences in the vole Microtus cabrerae.

The genus Microtus presents several species with extremely large sex chromosomes that contain large blocks of constitutive heterochromatin. Several cytogenetic and molecular studies of the repetitive sequences in species of the genus Microtus have demonstrated that the heterochromatin is highly heterogeneous. We have cloned and characterized a family of repetitive DNA sequences from M. cabrerae, a species with large heterochromatic blocks on the giant sex chromosomes. These repetitive sequences are 65.84% A–T rich, organized in tandem, with a 161-bp unit and are located on the centromeric region of autosomes and the X chromosome. In addition, this repetitive DNA is located throughout the entire heterochromatic block of the X chromosome and on three interstitial bands in the heterochromatic block of the Y chromosome. Comparative analysis of this family of repetitive sequences from three Microtus species revealed that the development of these sequences has occurred by concerted evolution. Our results support the hypothesis that the heterochromatic blocks from the sex chromosomes of different species are evolving independently and they probably have the genetic capacity to amplify and retain different satellite DNAs. For a topic related to the location of these repetitive DNA sequences on the Y chromosome of M. cabrerae, we propose a model to explain the origin of a length polymorphism previously described for this chromosome.

Distribution of L1-retroposons on the giant sex chromosomes of Microtus cabrerae (Arvicolidae, Rodentia): functional and evolutionary implications

Long interspersed nuclear elements (L1 or LINE-1) are the most abundant and active retroposons in the mammalian genome. Traditionally, the bulk of L1 sequences have been explained by the ‘selfish DNA’ hypothesis; however, recently it has been also argued that L1s could play an important role in genome and gene organizations. The non-random chromosomal distribution of these retroelements is a striking feature considered to reflect this functionality. In the present study we have cloned and analyzed three different L1 fragments from the genome of the rodent Microtus cabrerae. In addition, we have examined the chromosomal distribution of this L1 in several species of Microtus, a very interesting group owing to the presence in some species of enlarged (‘giant’) sex chromosomes. Interestingly, in all species analyzed, L1-retroposons have preferentially accumulated on both the giant- and the normal-sized sex chromosomes compared with the autosomes. Also we have demonstrated that L1-retroposons are not similarly distributed among the heterochromatic blocks of the giant sex chromosomes in M. cabrerae and M. agrestis, which suggest that L1 retroposition and amplification over the sex heterochromatin have been different and independent processes in each species. Finally, we proposed that the main factors responsible for the L1 distribution on the mammalian sex chromosomes are the heterochromatic nature of the Y chromosome and the possible role of L1 sequences during the X-inactivation process.

Interchromosomal, intercellular and interindividual variability of NORs studied with silver staining and in situ hybridization.

We investigated the relationship between transcriptional activity and the quantity of ribosomal cistrons of the nucleolar organizing regions (NORs) in chromosomal pair 3 of the Spanish mole Talpa occidentalis. Transcriptional activity was estimated by the size of conventional silver-staining signals. The number of ribosomal cistrons was estimated by the size of the in situ hybridization signals obtained with a ribosomal DNA probe. A sample of cells was studied with each method in four different individuals, and the results obtained with the two techniques were compared. Furthermore, sequential analysis on the same cells was carried out to study the association of interstitial nucleolar constrictions with silver staining and in situ hybridization. Interchromosomal, intercellular and interindividual variability were found with both silver staining and in situ hybridization methods. Our results show that transcriptional activity of NORs does not depend exclusively on the number of ribosomal cistrons they have as a high percentage of cells had two NORs with abundant ribosomal cistrons, only one of which was active. Additional factors, probably responsible for the activation of transcription, may be involved in this variability.

Exosomes enriched in stemness/metastatic-related mRNAS promote oncogenic potential in breast cancer

Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a “stemness and metastatic” signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients.

Action and resistance of monoclonal CD20 antibodies therapy in B-cell Non-Hodgkin Lymphomas

Anti-CD20 monoclonal antibodies (mAbs) have improved patients survival with Non-Hodgkin Lymphoma, when combined with chemotherapy. Several mechanisms of action have been reported, including antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis. Despite the large amount of studies and published data, the role each mechanism played in vivo is not fully understood. Furthermore, the reason why a significant percentage of patients are refractory or resistant remains unknown. Several activated intracellular signaling pathways have been implicated in the mechanisms of resistance of rituximab. In the present manuscript, we review those mechanisms and new anti-CD20 mAbs, as well as the efforts being accomplished to overcome it, focusing on new drugs targeting pathways implicated in resistance to rituximab.

Cancer treatments: can we find treasures at the bottom of the sea?

Because of the poor results observed after platinum-based first-line chemotherapy, research on new strategies for second-line treatment of advanced non-small-cell lung cancer (NSCLC) is warranted. Current research focuses on the development of new agents and the assessment of a combination of therapies, especially those with different mechanisms of action. PM02734 (elisidepsin, Irvalec) is a compound related to Kahalalide F (KF), a moderately soluble marine product that belongs to a family of dehydro aminobutyric acid-containing peptides isolated from the herbivorous marine mollusk Elysia rufescens. Preclinical and clinical studies showed that KF induces strong cytotoxic activity against different solid tumors, including NSCLC, particularly in patients with squamous histology; in fact, almost 40% of patients treated in the second line were still alive at 1 year after beginning treatment with KF. Analysis of data collected during clinical development has revealed that KF has a predictable and manageable toxicity profile. The toxicities most commonly associated with KF are generally transient and mild or moderate. The absence of hematologic toxicity and cumulative toxic effects suggests that KF may be suitable for combination trials with other anticancer agents. The development of KF could stopped because of the unavailability of a natural source of the compound. PM02734 is a closely related derivative of KF with similar activity and characteristics. Herein, we summarize the studies of PM02734 and future clinical perspectives.