Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues.

Retinoblastoma (Rb) is a pediatric intraocular malignancy and probably the most robust clinical model on which genetic predisposition to develop cancer has been demonstrated. Since deletions in chromosome 13 have been described in this tumor, we performed next generation sequencing to test whether recurrent losses could be detected in low coverage data. We used Illumina platform for 13 tumor tissue samples: two pools of 4 retinoblastoma cases each and one pool of 5 medulloblastoma cases (raw data can be found at http://www.ebi.ac.uk/ena/data/view/PRJEB6630). We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5). From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at http://www.ncbi.nlm.nih.gov/geo/accession number GSE11488). The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press) [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma.

Overview of recurrent chromosomal losses in retinoblastoma detected by low coverage next generation sequencing.

Genes are frequently lost or gained in malignant tumors and the analysis of these changes can be informative about the underlying tumor biology. Retinoblastoma is a pediatric intraocular malignancy, and since deletions in chromosome 13 have been described in this tumor, we performed genome wide sequencing with the Illumina platform to test whether recurrent losses could be detected in low coverage data from DNA pools of Rb cases. An in silico reference profile for each pool was created from the human genome sequence GRCh37p5; a chromosome integrity score and a graphics 40 Kb window analysis approach, allowed us to identify with high resolution previously reported non random recurrent losses in all chromosomes of these tumors. We also found a pattern of gains and losses associated to clear and dark cytogenetic bands respectively. We further analyze a pool of medulloblastoma and found a more stable genomic profile and previously reported losses in this tumor. This approach facilitates identification of recurrent deletions from many patients that may be biological relevant for tumor development.

Alterations of cell cycle regulators affecting the RB pathway in nonfamilial retinoblastoma.

We undertook the present study to examine alterations affecting the RB pathway in the G1 checkpoint and to determine their potential clinical significance in children affected with nonfamilial retinoblastoma. Using immunohistochemistry, patterns of expression of pRB, p16/INK4A, and E2F1 were analyzed in tissue from a cohort of 86 well-characterized patients with nonfamilial retinoblastoma diagnosed at the "Instituto Nacional de Pediatria" in Mexico City. The relationship of these phenotypes to proliferative index was assessed by analysis of Ki67 antigen expression. pRB expression was found in 11 (13%) cases. Using a hypophosphorylated specific pRB antibody, we observed low levels of underphosphorylated pRB expression in only 1 of 9 evaluable positive cases. These data suggest that the detected pRB products were hyperphosphorylated and thus had decreased functional activity. Increased p16 nuclear expression was found in only 6 tumors. No tumors showed deletions or mobility shifts of the INK4A gene. Undetectable pRB levels were significantly associated with undetectable p16 expression (odds ratio, 10.8; 95% confidence interval, 1.4-81.3; P =.03). All tumors showed nuclear immunoreactivities for E2F1 and Ki67. Increased Ki67 proliferative index was associated with increased staining for E2F1 (r =.44; P =.008) and increasing clinical stage (P =.03). Among children with unilateral disease, the mean Ki67 proliferative index was significantly higher in children with advanced clinical disease (stages 3 and 4) (mean 81.25; SD 6.78) than in those with earlier stage disease (mean 69.50; SD 9.45) (P = 0.001). Among children with bilateral disease, however, the mean proliferative index was not significantly higher for children with advanced clinical stage. When examining all cases together, there was a significant trend toward increasing proliferative index with increasing clinical stage (P =.03). In unilateral tumors, we also found that presence of detectable pRB was associated with a lower percentage of cells expressing E2F1 (46.7% v 70.8%) (P = 0.05), whereas there was no association between presence of pRB and E2F1 among bilateral tumors. We have found that expression of some of the cell cycle markers examined varies according to laterality, suggesting underlying differences in the capacity for cell cycle regulation between these 2 forms of the disease. Differences in capacities for cell cycle regulation may account for some differences in clinical behavior. Thus, the inclusion of molecular markers may become useful adjuncts to clinicopathological staging and subsequent determination of therapy.

Murine betaglycan primary structure, expression and glycosaminoglycan attachment sites.

The primary structure of murine betaglycan, also known as transforming growth factor beta (TGF-beta) type III receptor, was deduced from the nucleotide sequence of a cDNA clone isolated from a heart library. Murine betaglycan is a single spanning membrane polypeptide of 850 amino acids which is highly similar to betaglycan of other species. Transfection of this cDNA into COS1 cells resulted in the expression of a membrane proteoglycan that binds TGF-beta and is recognized by antibodies raised against rat betaglycan. COS1 cells transfected with the double mutant Ser533Ala; Ser544Ala of the murine betaglycan cDNA produced a TGF-beta type III receptor devoid of glycosaminoglycan chains.

p27Kip1: chromosomal mapping to 12p12-12p13.1 and absence of mutations in human tumors.

The p27Kip1 gene codes for a cyclin-dependent kinase inhibitor implicated in G1 arrest by transforming growth factor beta, cell-cell contact, agents that elevate cyclic AMP, and the growth-inhibitory drug rapamycin. p27 binds to and inhibits complexes formed by cyclin E-cdk2, cyclin A-cdk2, and cyclin D-cdk4. The involvement of p27 in the negative regulation of cell proliferation suggests that it may also function as a tumor suppressor gene. Using a combination of somatic cell hybrid panels and fluorescence in situ hybridization p27Kip1 has been mapped to the short arm of chromosome 12 at the 12p12-12p13.1 boundary, reported to harbor deletions and rearrangements in leukemia and mesotheliomas. In order to assess potential p27Kip1 gene alterations, we have screened a total of 147 human primary solid tumors and found no detectable cancer-specific mutations. These results argue that the often observed loss of antimitogenic transforming growth factor beta responsiveness in human cancer cells is not due to structural defects in p27Kip1.

Acetyl-coenzyme A carboxylase mRNA metabolism in the rat liver.

The acetyl-coenzyme A carboxylase (ACC) gene contains two promoters (PI and PII), both of which are active in the liver. Various physiological stimuli affect one, or both of the promoters of the ACC gene, and result in the generation of two classes of ACC mRNAs which differ in the composition of their 5' untranslated regions (5' UTR). We have analyzed the amounts of the two major mRNAs species that are generated from each of these promoters in order to examine the regulation of ACC gene activity in the liver under different physiological conditions. Our findings can be summarized as follows: (1) In liver from normal animals, fed a complete laboratory chow ad libitum, the level of class 2 ACC mRNA species generated by PII is very low. These mRNA species disappear on starvation. Refeeding starved animals with a fat-free diet stimulates both PI and PII with different time courses of induction: PII responds quickly and PII gene products accumulate to maximum levels within 18 hours, while the PI response, as measured by the accumulation of class 1 mRNAs, shows a lag period of 6 hours before reaching maximal levels at the end of a 24-hour refeeding period. The half-lives estimated from the induction kinetics were 4.4 hours for class 2 mRNAs and 11.8 hours for class 1 mRNAs. Reinstatement of starvation causes an almost instantaneous disappearance of class 1 mRNA species, as compared with class 2 mRNA species. This rapid decay of PI transcripts suggests that factors stabilizing this class of ACC mRNAs contribute to the steady-state levels reached after the dietary induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl-coenzyme A carboxylase messenger ribonucleic acid metabolism in liver, adipose tissues, and mammary glands during pregnancy and lactation.

In the rat, the acetyl-coenzyme A carboxylase gene exists as a single copy per haploid chromosome set. However, multiple forms of acetyl-coenzyme A carboxylase mRNA exist, the relative abundance of which varies in a tissue-specific manner under different physiological conditions. In the mammary gland, the major acetyl-coenzyme A carboxylase mRNA species are of the class 2 type, which are products of promoter II. In parametrial white adipose tissue, the main form of species of acetyl-coenzyme A carboxylase is of the class 1 type, which are produced by promoter I. Pregnancy and lactation affect the amounts of these acetyl-coenzyme A carboxylase mRNA. Although the mammary gland acetyl-coenzyme A carboxylase mRNA species increase dramatically upon parturition, the parametrial white adipose tissue forms decrease precipitously at the same time and are not expressed at all during the lactation period. In the liver of these animals, the only form of acetyl-coenzyme A carboxylase mRNA that is expressed is the FL56 form; this form shows a modest decrease during pregnancy that is slowly reversed during lactation. These observations indicate that the changes in lipogenesis that occur during pregnancy and lactation are determined by the transcriptional activity of the acetyl-coenzyme A carboxylase gene. In order to analyze the complex transcriptional activity of this gene in a meaningful way, it is necessary to examine the metabolism of individual isoforms of acetyl-coenzyme A carboxylase mRNA.

In vivo regulation of the activity of the two promoters of the rat acetyl coenzyme-A carboxylase gene.

The acetyl-coenzyme-A carboxylase (ACC) gene contains two promoters: promoter I (PI) and promoter II (PII). Depending upon which promoter is active, two classes of ACC mRNA are formed. The physiological significance of the presence of two promoters in the gene is not clear at this time. However, this question can be indirectly approached by examination of their expression patterns under different physiological conditions. We have examined the activities of these two promoters under different physiological conditions by means of primer extension analysis. Under normal conditions, the Wistar rat, fed standard chow ad libitum, expresses only basal levels of PI in white adipose tissue and PII in the liver. Starvation leads to the virtual disappearance of transcriptional products from these promoters. When fatty acid synthesis is stimulated by refeeding a fat-free diet to starved rats, both PI and PII are activated in the liver; however, in white adipose tissue, only PI, not PII, is responsive to this nutritional induction. On the other hand, in streptozotocin-diabetic rats, in which the activity of both promoters in both tissues is depressed, the administration of insulin quickly induces PI in adipose tissues, but has no significant effect on either of the promoters in the liver. During the weaning transition, the increase in hepatic lipogenesis is accompanied by activation of PI and PII when the pups are weaned onto a fat-free diet. Weaning onto a standard chow causes only a slight increase in PII. During the lactation period, profound alterations occur in the metabolism of the lipogenic tissues. In the lactating rat mammary gland only PII is active, and its activity is increased throughout lactation, reaching a plateau by day 7. Concomitantly, all ACC gene activity is completely shut off in the adipose tissue, while in the liver, PII, the only promoter active, is affected only minimally. The fat accumulation of the genetically obese Zucker rats is largely due to an abnormally high hepatic lipogenesis. In the obese Zucker rat (fa/fa), the level of expression of PII is similar to that in its lean siblings; however, PI is constitutively expressed at high levels, comparable to those in the Wistar rat that has been subjected to the starvation/refeeding induction. These studies demonstrate that the in vivo transcriptional control of the dual promoter rat ACC gene is a highly regulated and tissue-specific process.