Functional differences between myeloid leukemia-initiating and transient leukemia cells in Down's syndrome.

Children with constitutional trisomy 21 or Down's syndrome (DS) are predisposed to develop myeloid leukemia (ML) at a young age. DS-ML is frequently preceded by transient leukemia (TL), a spontaneously resolving accumulation of blasts during the newborn period. Somatic mutations of GATA1 in the blasts of TL and DS-ML likely function as an initiating event. We hypothesized that the phenotypic difference between TL and DS-ML is due to a divergent functional repertoire of the leukemia-initiating cells. Using an NOD/SCID model, we found that cells initiating DS-ML engrafted, disseminated to distant bone marrow sites, and propagated the leukemic clone in secondary recipients. In contrast, TL cells lacked the ability to expand and to migrate, but were able to persist in the recipient bone marrow. We found some evidence of genomic progression with 1 of 9 DS-ML samples and none of 11 TL samples harboring a mutation of N-RAS. The findings of this pilot study provide evidence for the functional impact of second events underlying the transformation of TL into DS-ML and a needed experimental tool for the functional testing of these promoting events.

Telomere maintenance and dysfunction predict recurrence in paediatric ependymoma.

We have recently described the enzymatic subunit of telomerase (hTERT) as an important prognostic marker for paediatric ependymoma. Because of the lack of good, representative pre-clinical models for ependymoma, we took advantage of our large cohort of ependymoma patients, some with multiple recurrences, to investigate telomere biology in these tumours. Our cohort consisted of 133 ependymomas from 83 paediatric patients and included 31 patients with recurrences. Clinical outcome was measured as overall survival, progression-free survival and response to therapy. In all 133 tumours, hTERT expression correlated with proliferative markers, including MIB-1 index (P<0.0001) and mitotic index (P=0.005), as well as overall tumour grade (P=0.001), but not with other markers of anaplasia. There was no correlation between telomere length and hTERT expression or survival. Surprisingly, prior radiation or chemotherapy neither induced sustained DNA damage nor affected telomere maintenance in recurrent tumours. There was an inverse correlation between hTERT expression and telomere dysfunction as measured by gamma H2AX expression (P=0.016). Combining gamma H2AX and hTERT expressions could segregate tumours into three different survival groups (log rank, P<0.0001) such that those patients whose tumours expressed hTERT and showed no evidence of DNA damage had the worst outcome. This study emphasises the importance of telomere biology as a prognostic tool and telomerase inhibition as a therapeutic target for paediatric ependymoma. Furthermore, we have demonstrated that analysing tumours as they progress in vivo is a viable approach to studying tumour biology in humans.

Molecular and clinical characterization of de novo and familial cases with microduplication 3q29: guidelines for copy number variation case reporting.

Microdeletions of 3q29 have previously been reported, but the postulated reciprocal microduplication has only recently been observed. Here, cases from four families, two ascertained in Toronto (Canada) and one each from Edinburgh (UK) and Leiden (Netherlands), carrying microduplications of 3q29 are presented. These families have been characterized by cytogenetic and molecular techniques, and all individuals have been further characterized with genome-wide, high density single nucleotide polymorphism (SNP) arrays run at a single centre (The Centre for Applied Genomics, Toronto). In addition to polymorphic copy-number variants (CNV), all carry duplications of 3q29 ranging in size from 1.9 to 2.4 Mb, encompassing multiple genes and defining a minimum region of overlap of about 1.6 Mb bounded by clusters of segmental duplications that is remarkably similar in location to previously reported 3q29 microdeletions. Consistent with other reports, the phenotype is variable, although developmental delay and significant ophthalmological findings were recurrent, suggesting that dosage sensitivity of genes located within 3q29 is important for eye and CNS development. We also consider CNVs found elsewhere in the genome for their contribution to the phenotype. We conclude by providing preliminary guidelines for management and anticipatory care of families with this microduplication, thereby establishing a standard for CNV reporting.

Modulation of the retinoic acid and retinoid X receptor signaling pathways in P19 embryonal carcinoma cells by calreticulin.

Calreticulin is a widely expressed calcium binding protein that can bind to an amino acid sequence motif, KXGFFKR, which is present in the cytoplasmic domain of all integrin alpha-subunits. Closely related sequences, KXFFKR and KXFFRR, are encoded in the DNA-binding domain of all members of the steroid/thyroid/retinoid receptor superfamily and it has recently been demonstrated that calreticulin inhibits their activity both in vitro and in vivo. Here we present novel evidence that calreticulin can interfere directly with the retinoic acid (RARs) and retinoid X (RXRs) receptor pathways. Calreticulin exhibits the ability to inhibit DNA-binding activity of both heterodimeric RAR/RXR and homodimeric RXR complexes in vitro. Inhibition of RXR binding to DNA is achieved with a concentration of calreticulin that is approximately fourfold lower than that required for inhibition of RAR/RXR binding to a cognate binding site. Coprecipitation experiments suggest a direct protein:protein interaction between calreticulin and retinoid receptors. Stable overexpression of calreticulin in P19 embryonal carcinoma cells significantly decreases the rapid activation of the endogenous RA-responsive RARbeta gene, abrogates the ability of endogenous RAR/RXR complexes to bind to DNA, and inhibits the emergence of the RA-induced differentiated phenotype. These data demonstrate that calreticulin can interfere with the two distinct retinoid signaling pathways through a mechanism likely involving direct protein:protein interactions and that disruption of the retinoid signal alters biological processes in vivo.

Isolation of a novel retinoic acid-responsive gene by selection of genomic fragments derived from CpG-island-enriched DNA.

One of the primary goals in transcription factor research is the elucidation of the genetic networks controlled by a factor or by members of a family of closely related factors. The pleiotropic effects of retinoic acid (PA) in the developing and adult animal are mediated by ligand-inducible transcription factors (RA receptors [RARs] and retinoid X receptors [RXRs]) that belong to the superfamily of nuclear receptors. Regulatory regions of PA effector genes contain RAR and RXR binding sites (RAR elements [RAREs] and RXR elements [RXREs]) that generally consist of direct or everted repeats of the core half-site motif, (A/G)G(G/T)TCA. In order to identify novel genes regulated by RA, we devised a selection strategy based on the premise that regulatory regions of a large number of housekeeping and tissue-specific genes are embodied within CpG island DNA. In this method, referred to as CpG-selected and amplified binding, fragments derived from the CpG island fraction of the murine genome are selected by a gel mobility shift assay using in vitro-transcribed and -translated RXR-RAR. Multiple rounds of selection coupled with amplification of the fragments by PCR enabled us to clone a population of CG-rich fragments of which approximately one-fifth contained consensus RAREs or RXREs. Twelve genomic fragments containing novel response elements are described, and the transcription unit associated with one of them, NN-84AG, was characterized in detail. The mouse NN-84AG transcript is upregulated by RA in F9 embryonal carcinoma cells and is homologous to an expressed sequence tag (EST41159) derived from a human infant brain cDNA library. Cloning of the murine NN8-4AG genomic sequence places the RXRE in the proximity of the transcription initiation sites of the gene. Although sequence analysis indicates that the EST41159 gene product is novel, a region of amino acid identity with sequences of a yeast polypeptide of, as yet, unknown function and the Drosophila trithorax protein suggests the presence of an evolutionarily and functionally conserved domain. Our study demonstrates that transcription factor binding sites and corresponding regulated genes can be identified by selecting fragments derived from the CpG island fraction of the genome.

Inhibition of nuclear hormone receptor activity by calreticulin.

We have shown that a polypeptide of M(r) 60,000 (60K) that shares N-terminal homology with a calcium-binding protein, calreticulin, can bind to an amino-acid sequence motif, KXGFFKR, found in the cytoplasmic domains of all integrin alpha-subunits. The homologous amino-acid sequence, KXFFKR (where X is either G, A or V), is also present in the DNA-binding domain of all known members of the steroid hormone receptor family; amino acids in this sequence make direct contact with nucleotides in their DNA-responsive elements and are crucial for DNA binding. Here we show that both the 60K protein (p60), purified on a KLGFFKR-Sepharose affinity matrix, and recombinant calreticulin can inhibit the binding of androgen receptor to its hormone-responsive DNA element in a KXFFKR-sequence-specific manner. Calreticulin can also inhibit androgen receptor and retinoic acid receptor transcriptional activities in vivo, as well as retinoic acid-induced neuronal differentiation. Our results indicate that calreticulin can act as an important modulator of the regulation of gene transcription by nuclear hormone receptors.

Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis.

Treatment with retinoic acid (RA) is known to produce complex teratogenic effects in vertebrates, and its presence in the developing embryo as an endogenous substance has led to the suggestion that RA might be a natural morphogenetic agent. Although our understanding of the molecular mechanism of RA action has improved considerably with the identification of nuclear receptors for RA (RARs) and RA-responsive genes, the exact relationship between the proposed morphogenetic activity of RA and its teratogenic effects remains to be characterized. Here, we show that a RA response element (RARE) present in the RAR beta gene can direct specific spatial and temporal expression of an hsplacZ transgene during mouse embryogenesis. In the early embryo, the transgene is expressed in a specific anterior-posterior domain that is completely obliterated by treatment of pregnant mice with teratogenic doses of RA. The expression of the transgene becomes more restricted as organogenesis progresses and mimics closely the reported expression of the RAR beta gene. These results suggest that, in vivo, some of the morphogenetic effects of RA could be mediated through localized transcriptional activity controlled by the various RARs. The specific pattern of expression of the RAREhsplacZ transgene does not correlate with the proposed sites of action of RA as defined by its teratogenic effects but does support a role for RA in early anterior-posterior patterning along the body axis.

Translation of the Saccharomyces cerevisiae tcm1 gene in the absence of a 5'-untranslated leader.

The role of eukaryotic 5'-untranslated messenger RNA leaders is not entirely clear, since they share little sequence similarity among each other. The importance of the leader in determining the efficiency of translation initiation was addressed here by examining the polyribosome distribution of several leader-deletion alleles of the yeast tcm1 gene (coding for ribosomal protein L3). Shortening of this 22-nucleotide leader, or complete removal of it (the first nucleotide of the mRNA becoming the A of the translation initiation codon AUG) permitted translation, albeit reduced. Further deletion of as few as the first two nucleotides of the initiation codon leads to a substantial reduction in ribosome loading, which is compatible with inefficient initiation at the next downstream, out-of-frame, AUG triplet. A second measure of translation initiation was obtained by assaying qualitatively for the production of biologically active L3 protein using growth-resistance to trichodermin. This experiment indicates that ribosomes can recognize the correct initiation codon even in the complete absence of a leader. We conclude that the 5'-untranslated leader of the yeast tcm1 gene is not essential for accurate translation initiation, but enhances its efficiency.

Identification of a novel isoform of the retinoic acid receptor gamma expressed in the mouse embryo.

Retinoic acid is known to have profound effects on developmental processes. It has been implicated as a putative morphogen in the developing chick limb bud and regenerating amphibian limb blastema and has been demonstrated to have powerful teratogenic effects in mammals, including humans. Recently, three specific retinoic acid receptors (RARs), RAR alpha, -beta, and -gamma, were identified and shown to be members of the steroid receptor superfamily. We report the identification of a novel RAR gamma isoform, mRAR gamma B, which differs from the previously described mouse RAR gamma at its amino terminus. In addition, we show that both RAR gamma isoforms are expressed maximally at midgestation in structures known to be affected adversely by retinoic acid administration to pregnant mice. Multiple RAR isoforms, each of which may play a unique or combinatorial role as a regulator of mammalian development, are thus expressed in the mouse embryo.