Potential role of Shh-Gli1-BMI1 signaling pathway nexus in glioma chemoresistance.

Chemoresistance is a common hurdle for the proper treatment of gliomas. The role of Shh-Gli1 signaling in glioma progression has been reported. However, its role in glioma chemoresistance has not been well studied yet. In this work, we found that Shh-Gli1 signaling regulates the expression of one stem cell marker, BMI1 (B cell-specific Moloney murine leukemia virus), in glioma. Interestingly, we also demonstrated high expression of MRP1 (multi-drug resistance protein 1) in glioma. MRP1 expression was decreased by BMI1 siRNA and Shh-Gli1 cell signaling specific inhibitor GANT61 in our experiments. GANT61 very efficiently inhibited cell colony growth in glioma cell lines, compared to temozolomide. Moreover, a synergic effect of GANT61 and temozolomide drastically decreased the LD50 of temozolomide in the cell colony experiments. Therefore, our results suggest that there is a potential nexus of Shh-Gli1-BMI1 cell signaling to regulate MRP1 and to promote chemoresistance in glioma. Henceforth, our study opens the possibility of facing new targets, Gli1 and BMI1, for the effective treatment of glioma suppression of chemoresistance with adjuvant therapy of GANT61 and temozolomide.

Patterns of mammalian diversification in recent evolutionary times: global tendencies and methodological issues.

Changes in diversification patterns estimated from phylogenetic trees are an important source of information about the dynamics of evolution. To study the diversification of mammals, we reconstructed phylogenetic trees of 29 families and fitted both constant-rate and variable-rate models of diversification. In addition, we investigated the effect of clock models and phylogenetic reconstruction problems on diversification analyses. We observed, first, that none of the families increased its diversification rate during the last few million years, including the Pleistocene. Furthermore, we detected a decrease in diversification that, after application of different tests, was significant only for a minority of families. However, when diversification variation was analysed in a combined tree of all families, a global decline in diversification became significant. Therefore, although distorted by some methodological artefacts, we found an underlying signal of gradually decreasing diversification that suggests that ecological factors may have shaped the recent diversification of mammals.

Study of the phenotypic relationship in the IMR-32 human neuroblastoma cell line by sedimentation field flow fractionation.

Neuroblastoma (NB) is the most common childhood solid tumor. Although spontaneous regression can occur in patients <1-year old, 70% of patients over the age of 1 year have a high-risk and difficult-to-treat NB. Cell type heterogeneity is observed either in the morphological appearance of NB tumors or in cell lines isolated from tumor specimens. NB consists of two principal neoplastic cell types: i) neuroblastic or N-type (undifferentiated cells); and ii) stromal or S-type (differentiated cells). As NB cells seem to have the capacity to differentiate spontaneously in vivo and in vitro, their heterogeneity could affect treatment outcome, in particular the response to apoptosis induced by chemotherapy. Therefore, it is important to understand the underlying process governing changes in differentiation in order to improve treatment response and NB patient outcome and the neoplastic population in IMR-32 represented a good model for such a study. Results showed that this cell line was extremely heterogeneous and highly variable in its stage of differentiation and we demonstrated that sedimentation field flow fractionation (SdFFF) permitted the isolation of 2 N-phenotypes and contributed to the understanding of the IMR-32 cell population dynamics. The first N-phenotype forms a pool of quiescent undifferentiated cells while the second one was able to proliferate (incorporation of BrdU) and also give rise to adherent S-type cells (PSA-N-CAM+ and N-CAM+). The results could also suggest a close interaction between these different cellular phenotypes to create the IMR-32 cell lineage.

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma.

AIMS: Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. METHODS AND RESULTS: We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. CONCLUSIONS: Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.

Silencing of DMBT1 in neuroblastoma cell lines is not due to methylation of CCWGG motifs on its promoter.

DMBT1 is one of the putative suppressor genes on 10q25-qter, which frequently lacks expression in many different kind of tumors, such as glioblastoma, and lung, esophageal and colorectal cancer. However, little is known about the reasons for this lack of expression in neoplasia. In a previous report, our group demonstrated how MC-IXC, a neuroblastoma cell line which lacked DMBT1 expression, restored it after a 5-Aza-2'-deoxycitidine treatment. So, we wondered whether DMBT1 aberrant promoter methylation could be responsible for DMBT1 silencing in several tumor cell lines, in spite of the fact that there is no CpG island near the 5' end of the gene. We studied the possibility that methylation in CCWGG sequences of the DMBT1 promoter (where "W" means "A" or "T") is able to silence the gene, as had previously been reported for TP53 in leukemia. We digested genomic DNA by the methylation sensitive restriction enzyme EcoR II (C|CWGG), and made two PCRs to amplify the three CCWGG domains placed in the 1 kb upstream DMBT1 5' end. After the PCRs, we could not find correlation between methylation in CCWGG domains and DMBT1 lack of expression. A positive regulator of DMBT1 might be silenced by aberrant methylation.

Genetic heterogeneity in supratentorial and infratentorial primitive neuroectodermal tumours of the central nervous system.

AIMS: Medulloblastoma (MB), a kind of infratentorial primitive neuroectodermal tumour (PNET), is the most frequent malignant brain tumour in childhood. In contrast, supratentorial PNET (sPNET) are very infrequent tumours, but they are histologically similar to MB, although they present a worse clinical outcome. We investigated the differences in genetic abnormalities between sPNET and MB. METHODS AND RESULTS: We analysed 20 central PNET (14 MB and six sPNET) by conventional comparative genomic hybridization (CGH) in order to determine whether a different genetic profile for each tumour exists. Isochromosome 17q was detected in four of the 14 MB cases, but not in any sPNET. Gains at 17q and 7 happened more frequently in MB, and those at 1q in sPNET. Losses at chromosome 10 were detected only in MB, while losses at 16p and 19p happened more frequently in sPNET. A new amplification site, on 4q12, was detected in two MB. CONCLUSIONS: Central PNET are a heterogeneous group of tumours from the genetic point of view. The present and previous data, together with further results from larger series, might contribute to the establishment of specific treatments for supratentorial and infratentorial PNET.

Improvement of the methylation specific PCR technical conditions for the detection of p16 promoter hypermethylation in small amounts of tumor DNA.

Methylation specific PCR (MSP) is a technique that enables the detection of hypermethylation at a CpG island. The objective of this study is the introduction of small modifications to the MSP technique to make it more suitable for the study of promoter hypermethylation at tumor suppressor genes whenever there is a shortage of material available for study. This commonly happens in the case of using archival material from the Pathology departments. Tumor DNA was extracted from a collection of 40 fresh-frozen soft tissue sarcomas and 19 paraffin-embedded PNETs (primitive neuroectodermal tumors). The MSP technique was performed to detect hypermethylation at the p16 promoter. Also, blood genomic DNA was mixed with herry sperm genomic DNA as a carrier, in nine different combinations, in order to test for the best conditions that could produce MSP bands even when low amounts of genomic tumor DNA is available for study. We demonstrate the benefit of using herry sperm carrier DNA up to 10 microg together with small quantities of tumor DNA. This result will facilitate the incorporation of paraffin-embedded samples for study of promoter hypermethylation at tumor suppressor genes. Other technical conditions for the MSP technique are also studied.

Gain of chromosome 3 and loss of 13q are frequent alterations in pituitary adenomas.

Genetic changes underlying the tumorigenesis of pituitary adenomas (PA) are poorly characterized. To search for characteristic genomic imbalances involved in PA, we examined 38 cases: 12 hormone-secreting (HS) and 26 non-functioning (NF) PA, by comparative genomic hybridization. The most frequent DNA copy number change in both kinds of tumors was loss of 13q. Gains of chromosomes 3, 7 and 14, 6p, and 20q were more frequent in HSPA than in NFPA. These data indicate that the 13q region may harbor tumor suppressor genes determining the tumorigenesis of PA and gain in chromosome 3 may be related to hormone secretion. These findings provide a basis to search for candidate diagnostic markers of HSPA.

Non-isotopic silver-stained SSCP is more sensitive than automated direct sequencing for the detection of PTEN mutations in a mixture of DNA extracted from normal and tumor cells.

The sensitivity of non-isotopic PCR-SSCP was compared to direct sequencing of PTEN exons. DNA from leukocytes derived from healthy donors, and from the glioblastoma cell line LN319 was extracted and mixed in different proportions from 0 to 100%. The LN319 cell line contains a point mutation at codon 15 exon 1 of the PTEN gene. The PCR-SSCP experiments demonstrated mutations in samples containing as little as 10% tumor DNA. In contrast, direct DNA sequencing experiments were less sensitive, requiring 30-70% of tumor DNA in the sample, depending on the DNA strand sequenced. In conclusion, PCR-SSCP, in our hands, is more sensitive than automated sequencing for detecting PTEN point mutations. We recommend to always sequence both strands, and take into account that samples containing less than 30% tumor cells should not only be sequenced, but also studied by PCR-SSCP in order to discriminate false negative results.

Cytochrome b phylogeny and the taxonomy of great apes and mammals.

In the Linnaean system of classification, the generic status of a species is part of its binomial name, and it is therefore important that the classification at the level of genus is consistent at least in related groups of organisms. Using maximum-likelihood phylogenetic trees constructed from a large number of complete or nearly complete mammalian cytochrome b sequences, I show that the distributions of intrageneric and intergeneric distances derived from these trees are clearly separated, which allows the limits for a more rational generic classification of mammals to be established. The analysis of genetic distances among hominids in this context provides strong support for the inclusion of humans and chimpanzees in the same genus. It is also of interest to decipher the main reasons for the possible biases in the mammalian classification. I found by correlation analysis that the classification of mammals of large body size tends to be oversplit, whereas that of small mammals has an excess of lumping, which may be a manifestation of the larger difficulty in finding diagnostic characters in the classification of small animals. In addition, and contrary to some previous observations, there is no correlation between body size and rate of cytochrome b evolution in mammals, which excludes the difference in evolutionary rates as the cause of the observed body size taxonomic bias.

A molecular phylogeny of two extinct sloths.

Xenarthra (Edentata) is an extremely diverse mammalian order whose modern representatives are the armadillos, anteaters, and sloths. The phylogeny of these groups is poorly resolved. This is particularly true for the sloths (phyllophagans), originally a large and diverse group now reduced to two genera in two different families. Both morphological analyses and molecular analyses of rDNA genes of living and extinct sloths have been used with limited success to elucidate their phylogeny. In an attempt to clarify relationships among the sloths, DNA was extracted and mitochondrial cytochrome b gene sequences were determined from representatives of two extinct groups of sloths (Mylodontidae and Megatheriidae), their two living relatives (two-toed sloths [Megalonychidae], three-toed sloths [Bradypodidae]), anteaters and armadillos. A consistent feature of the latter two species was the nuclear copies of cytochrome b gene sequences. Several methods of phylogenetic reconstruction were applied to the sequences determined, and the results were compared with 12S rDNA sequences obtained in previous studies. The cytochrome b gene exhibited a phylogenetic resolving power similar to that of the 12S rDNA sequences. When both data sets were combined, they tended to support the grouping of two-toed sloths with mylodontids and three-toed sloths with megatheriids. The results strengthen the view that the two families of living sloths adapted independently to an arboreal life-style.

Comparative genomics and bioenergetics.

Bacterial and archaeal complete genome sequences have been obtained from a wide range of evolutionary lines, which allows some general conclusions about the phylogenetic distribution and evolution of bioenergetic pathways to be drawn. In particular, I searched in the complete genomes for key enzymes involved in aerobic and anaerobic respiratory pathways and in photosynthesis, and mapped them into an rRNA tree of sequenced species. The phylogenetic distribution of these enzymes is very irregular, and clearly shows the diverse strategies of energy conservation used by prokaryotes. In addition, a thorough phylogenetic analysis of other bioenergetic protein families of wide distribution reveals a complex evolutionary history for the respective genes. A parsimonious explanation for these complex phylogenetic patterns and for the irregular distribution of metabolic pathways is that the last common ancestor of Bacteria and Archaea contained several members of every gene family as a consequence of previous gene or genome duplications, while different patterns of gene loss occurred during the evolution of every gene family. This would imply that the last universal ancestor was a bioenergetically sophisticated organism. Finally, important steps that occurred during the evolution of energetic machineries, such as the early evolution of aerobic respiration and the acquisition of eukaryotic mitochondria from a proteobacterium ancestor, are supported by the analysis of the complete genome sequences.

Nitric oxide reductases in bacteria.

Nitric oxide reductases (NORs) that are found in bacteria belong to the large enzyme family which includes cytochrome oxidases. Two types of bacterial NORs have been characterised. One is a cytochrome bc-type complex (cNOR) that receives electrons from soluble redox protein donors, whereas the other type (qNOR) lacks the cytochrome c component and uses quinol as the electron donor. The latter enzyme is present in several pathogens that are not denitrifiers. We summarise the current knowledge on bacterial NORs, and discuss the evolutionary relationship between them and cytochrome oxidases in this review.

Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis.

The use of some multiple-sequence alignments in phylogenetic analysis, particularly those that are not very well conserved, requires the elimination of poorly aligned positions and divergent regions, since they may not be homologous or may have been saturated by multiple substitutions. A computerized method that eliminates such positions and at the same time tries to minimize the loss of informative sites is presented here. The method is based on the selection of blocks of positions that fulfill a simple set of requirements with respect to the number of contiguous conserved positions, lack of gaps, and high conservation of flanking positions, making the final alignment more suitable for phylogenetic analysis. To illustrate the efficiency of this method, alignments of 10 mitochondrial proteins from several completely sequenced mitochondrial genomes belonging to diverse eukaryotes were used as examples. The percentages of removed positions were higher in the most divergent alignments. After removing divergent segments, the amino acid composition of the different sequences was more uniform, and pairwise distances became much smaller. Phylogenetic trees show that topologies can be different after removing conserved blocks, particularly when there are several poorly resolved nodes. Strong support was found for the grouping of animals and fungi but not for the position of more basal eukaryotes. The use of a computerized method such as the one presented here reduces to a certain extent the necessity of manually editing multiple alignments, makes the automation of phylogenetic analysis of large data sets feasible, and facilitates the reproduction of the final alignment by other researchers.

[Molecular genetics of astrocytomas]. / Genética molecular de los astrocitomas.

Astrocytomas are the most frequent group of intracranial tumors. Among them, glioblastoma is the most aggressive one. In this review we will describe the most common genetic abnormalities found out in astrocytomas. We will refer to the epidermal growth factor receptor (EGFR), p53, p16, PTEN and DMBT1 genes. We will also present certain genetic aspects that influence the progression to glioblastoma.

Respiratory chains in the last common ancestor of living organisms.

Sequences in current databases show that a number of proteins involved in respiratory processes are homologous in archaeal and bacterial species. In particular, terminal oxidases belonging to oxygen, nitrate, sulfate, and sulfur respiratory pathways have been sequenced in members of both domains. They include cytochrome oxidase, nitrate reductase, adenylylsulfate reductase, sulfite reductase, and polysulfide reductase. These proteins can be assigned to the last common ancestor of living organisms assuming that the deepest split of the three domains of life occurred between Archaea and Bacteria and that they were not acquired through lateral gene transfer by one of these domains. These molecular data indicate that several of the most important respiratory pathways arose early in evolution and that the last common ancestor of living organisms was not a simple organism in its energetic metabolism. Rather, it may have been able to gain energy by means of at least four electron transport chains, and therefore it may have been prepared to face a wide range of environmental conditions.

Lack of gene amplification as a mechanism of CDK4 activation in human neuroblastoma.

Neuroblastoma is the most frequent solid tumor in childhood. We have analysed 48 neuroblastomas of different stages and degrees of cellular differentiation for CDK4 amplification by a fluorescent differential PCR assay. We explored the relative densitometric measure of a 119 bp fragment of the CDK4 gene versus an 82 bp fragment of the IFNG gene. We were not able to detect any case of CDK4 gene amplification in the neuroblastomas. In conclusion, CDK4 activation does not seem to be relevant to the development of neuroblastomas, at least through gene amplification.

The mitochondrial genome of the hemichordate Balanoglossus carnosus and the evolution of deuterostome mitochondria.

The complete nucleotide sequence of the mitochondrial genome of the hemichordate Balanoglossus carnosus (acorn worm) was determined. The arrangement of the genes encoding 13 protein, 22 tRNA, and 2 rRNA genes is essentially the same as in vertebrates, indicating that the vertebrate and hemichordate mitochondrial gene arrangement is close to that of their common ancestor, and, thus, that it has been conserved for more than 600 million years, whereas that of echinoderms has been rearranged extensively. The genetic code of hemichordate mitochondria is similar to that of echinoderms in that ATA encodes isoleucine and AGA serine, whereas the codons AAA and AGG, whose amino acid assignments also differ between echinoderms and vertebrates, are absent from the B. carnosus mitochondrial genome. There are three noncoding regions of length 277, 41, and 32 bp: the larger one is likely to be equivalent to the control region of other deuterostomes, while the two others may contain transcriptional promoters for genes encoded on the minor coding strand. Phylogenetic trees estimated from the inferred protein sequences indicate that hemichordates are a sister group of echinoderms.

Differential polymerase chain reaction: a technical comparison of three methods for the detection of CDK4 gene amplification in glioblastomas.

Among the different techniques used to detect oncogene amplification in tumor DNA, Southern blot and differential PCR have been the most frequently used. We report on a technical comparison of three different methods to detect gene amplification by differential PCR: ethidium bromide staining, silver staining (both after standard differential PCR), and fluorescent differential PCR. We explored the relative densitometric measure of a 119 bp fragment of the CDK4 gene versus an 82 bp fragment of the IFNG gene. In total agreement with previous studies carried out by Southern blot and differential PCR by other authors, we were able to detect CDK4 amplification in 3 of the 21 glioblastomas (14%), but only by the fluorescent differential PCR method. In conclusion, fluorescent differential PCR is more sensitive than standard differential PCR for detection oncogene amplification in tumor DNAs.

Codon reassignment and amino acid composition in hemichordate mitochondria.

In the mitochondrial genome of the hemichordate Balanoglossus carnosus, the codon AAA, which is assigned to lysine in most metazoans but to asparagine in echinoderms, is absent. Furthermore, the lysine tRNA gene carries an anticodon substitution that renders its gene product unable to decode AAA codons, whereas the asparagine tRNA gene has not changed to encode a tRNA with the ability to recognize AAA codons. Thus, the hemichordate mitochondrial genome can be regarded as an intermediate in the process of reassignment of mitochondrial AAA codons, where most metazoans represent the ancestral situation and the echinoderms the derived situation. This lends support to the codon capture hypothesis. We also show that the reassignment of the AAA codon is associated with a reduction in the relative abundance of lysine residues in mitochondrial proteins.