Funhaler spacer: improving adherence without compromising delivery.

A novel asthma spacer device, the "Funhaler", incorporates incentive toys which are isolated from the main inspiratory circuit by a valve. Here we show that its use does not compromise drug delivery. Improved adherence combined with satisfactory delivery characteristics suggest that the Funhaler may be useful for management of young asthmatics.

Expression of HOX11 in childhood T-lineage acute lymphoblastic leukaemia can occur in the absence of cytogenetic aberration at 10q24: a study from the Children's Cancer Group (CCG).

Clonal genetic aberrations in tumour cells provide critical information for the development of new diagnostic and therapeutic strategies for patients. In paediatric T-cell acute lymphoblastic leukaemia (T-ALL) chromosomal translocations are present in 30-35% of cases. HOX11 and the closely related HOX11L2 genes play a key role in T-ALL. HOX11 is aberrantly activated by either of the two chromosomal translocations, t(7;10) and t(10;14). In this study, HOX11 expression levels were measured by real-time quantitative reverse-transcriptase polymerase chain reaction. We show that leukaemic blasts from 15/76 (19.7%) paediatric T-ALL patients expressed the HOX11 gene at high level and 22/76 (28.9%) at low level, yet the reported frequency for chromosomal rearrangement of 10q24 is 4-7%. Direct cytogenetic analysis revealed that only 2/16 specimens that showed HOX11 expression exhibited abnor-malities at 10q24. These results confirm and extend our previously published findings, and implicate mechanisms other than gross chromosomal translocations for the deregulation of HOX11. Analysis of clinical outcome for the whole study group showed a trend for better outcome for patients with leukaemic blasts expressing HOX11 at high level. A statistically significant difference in clinical outcome was found in a subgroup of 20 patients treated for high-risk disease on CCG-1901 from the Children's Cancer Group, where HOX11 expression in leukaemic blasts conferred a prognostic advantage (P=0.01).

A complex containing PBX2 contributes to activation of the proto-oncogene HOX11.

Ectopic expression of the homeobox gene HOX11 is associated with a significant proportion of childhood T-cell acute lymphoblastic leukaemias (T-ALLs). We hypothesise that one mechanism of gene deregulation involves overcoming the silencing mechanism(s) of gene expression present in normal cells. Here, we describe a search for trans-acting factors that control transcriptional activity from a distal 5' region of the HOX11 promoter. We have identified a region of this promoter which contributes significantly to HOX11 activation and two distinct regulatory elements are involved. First, a PBX2 Regulatory Element PRE-1048 has been identified which contains a novel DNA-binding sequence and mediates significant activation of the HOX11 gene in K562 cells. This is the first report of a homeobox gene being specifically regulated by PBX2 and the second report of a vertebrate homeobox target gene of a PBX protein. The PREP1 protein was also shown to be part of the PRE-1048-binding complex. The other regulatory element we describe here RE-1019 contains little sequence conservation to known transcription control elements. It appears that this element is a novel sequence that binds an as yet unidentified factor, mediating significant activation of the HOX11 gene in K562 cells. This is the first detailed report of elements that mediate regulation of the proto-oncogene HOX11.

Novel alternative PBX3 isoforms in leukemia cells with distinct interaction specificities.

PBX3 is a member of the PBX family of TALE homeobox genes. The prototypic member, PBX1, was first identified in chromosomal translocations in B-lineage leukemia and is required for normal hematopoiesis. PBX2 and PBX3 were later identified as members of this highly conserved family by their strong homology to PBX1. While the expression pattern of PBX1 is restricted, PBX2 and PBX3 are ubiquitously expressed. Little is known about the functional role of PBX3. Our studies identified two PBX3 transcripts alternative to the canonical forms, PBX3A and PBX3B, resulting from a novel splice in PBX3. These new isoforms, named PBX3C and PBX3D, have been detected in all tissues and cell lines tested. Intriguingly, expression of PBX3D is favored in normal cells, whereas PBX3C expression is favored in leukemia cells. Functional studies showed that PBX3C and PBX3D proteins were unable to interact with the PBX-interacting factor PREP1 and weakly interacted with MEIS proteins. We propose that PBX3C and PBX3D may affect PBX3-mediated transcriptional regulation by acting in opposition to the known PBX proteins through alternative PBX3 complex formation. The identification and characterization of these novel PBX3 isoforms provide a foundation for a better understanding of the biological role of PBX3.

Hemizygous p16(INK4A) deletion in pediatric acute lymphoblastic leukemia predicts independent risk of relapse.

The genes at the INK4A/ARF locus at 9p21 are frequently involved in human cancer. Virtually all p16(INK4A) exon 2 (henceforth called p16) inactivation in pediatric acute lymphoblastic leukemia (ALL) occurs by gene deletion. The results of this study illustrate that real-time quantitative polymerase chain reaction is capable of detecting gene deletion in primary patient specimens with a precision not previously achieved by conventional methods. Importantly, this assay includes the detection of hemizygous deletions. The study revealed, strikingly, that the risk ratio for relapse for hemizygous deletion compared with no deletion was 6.558 (P =.00687) and for homozygous deletion was 11.558 (P =.000539). These results confirm and extend the authors' previous findings that homozygous deletion of p16 in pediatric ALL patients is an independent prognostic indicator of outcome from therapy.

Promoter demethylation accompanies reactivation of the HOX11 proto-oncogene in leukemia.

Despite considerable work on the epigenetic control of tumor suppressor genes, little is known about the potential role of promoter CpG demethylation in the activation of oncogenes in lymphoid tumors. The HOX11 proto-oncogene is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL). HOX11 activation can occur in the absence of translocation of the gene to the T-cell receptor locus (Salvati et al., 1995), implying that activation mechanisms must be involved other than the juxtaposition of the gene to adjacent enhancing sequences. We tested whether the methylation status of the proximal promoter was correlated with expression status in T-ALL and found that, in all cases, expression of HOX11 in T-ALL was associated with extensive demethylation of the proximal HOX11 promoter, regardless of whether or not translocation was involved. In contrast, cells that did not express HOX11 showed a more methylated pattern of CpG residues in the proximal promoter. Methylation of this sequence in vitro was sufficient to silence the proximal promoter. We propose a model in which the selection of leukemia clones via a pathway involving HOX11 expression requires the demethylation of its promoter as a prerequisite for additional gene activation mechanisms.

The topoisomerase II-associated protein, Pat1p, is required for maintenance of rDNA locus stability in Saccharomyces cerevisiae.

The Pat1 protein of Saccharomyces cerevisiae was identified during a screen for proteins that interact with topoisomerase II. Previously, we have shown that pat1 delta mutants exhibit a slow-growth phenotype and an elevated frequency of both mitotic and meiotic chromosome mis-segregation. Here, we have studied the effects of deleting the PAT1 gene on chromosomal stability, with particular reference to rates of homologous recombination within the rDNA locus. This locus was analyzed because rDNA-specific hyperrecombination is known to occur in conditional top2 mutants. We show that pat1 delta strains mimic top2 mutants in displaying an elevated rate of intrachromosomal excision recombination at the rDNA locus, but not elsewhere in the genome. The elevated rate of recombination is dependent upon Rad52p, but not upon Rad51p or Rad54p. However, pat1 delta strains display additional manifestations of more general genomic instability, in that they show mild sensitivity to UV light and an increased incidence of interchromosomal recombination between heteroalleles.

Sequence of 10q24 locus surrounding the HOX11 oncogene reveals a new gene HUG1 expressed in a T-ALL cell line.

HOX11 is a gene encoding a homeobox protein which is found to be deregulated in T-cell acute lymphoblastic leukaemia (T-ALL). As a basis for studying the mechanism of deregulation of HOX11 expression in leukaemia, the locus containing the HOX11 proto-oncogene at 10q24 was cloned from a genomic P1 Artificial Chromosome (PAC) library. The PAC clone with an insert size of 120kb was isolated and mapped by restriction analysis. A series of contiguous subclones were then obtained which span 20kb surrounding the HOX11 gene. These subclones were used to sequence across the entire 20kb region to the 3' boundary of the PAC insert. This work provides for the first time the full intron and 5' non-coding sequences of the HOX11 gene which will aid the identification of novel transcriptional control elements which may be involved in silencing HOX11 expression in normal cells. The sequence information was also used to search for novel large open reading frames (ORFs). One such ORF (1.1kb) would encode a protein of at least 39kDa. This basic protein (pI, 12.5) would be very proline rich and could potentially encode a novel transcription factor. In order to establish if this ORF corresponds to a bona fide transcribed gene, RT-PCR analysis was performed. The mRNA for this protein is expressed in the T-ALL cell line Jurkat and has been designated HUG1, for HOX11 Upstream Gene.

Molecular characterization of a complex chromosomal translocation breakpoint t(10;14) including the HOX11 oncogene locus.

Based on cytogenetic studies, non-random chromosomal translocations which involve the HOX11 gene at locus 10q24 and the TCR genes at loci 7q35 or 14q11 have been reported to occur in 5% of T-ALL. HOX11, a member of the homeobox family of genes, has been shown to play a role in T-ALL. The activation of the HOX11 gene by translocations to the TCR locus results in the inappropriate expression of a 2.3 kb transcript. In this paper we describe a t(10;14)(q24;q11) breakpoint from a T-ALL patient specimen. The breakpoint appears to be mediated by errors in the TCR/V(D)J recombination system, but is more complex than commonly described reciprocal translocations between the HOX11 and TCR genes, since it involves an inversion event of the TCRdelta genes. In addition, the breakpoint was characterised to a previously unsequenced area of the 10q24 locus, 3.4 kb upstream of the HOX11 gene. This breakpoint is more centromeric than the breakpoint cluster region previously shown to be involved in the majority of reported t(10;14)(q24;q11) translocations. Hence, our investigations of the translocation breakpoint in this patient identify another breakpoint region in the 10q24 locus and may define a novel recombination 'hot spot'. Surprisingly, our studies provide a mechanism for a previously unexplained complex translocation described by another group which involves the same region of the HOX11 promoter.

Multiple negative elements contribute to repression of the HOX11 proto-oncogene.

The HOX11 proto-oncogene is normally expressed in embryogenesis where it directs the synthesis of the spleen. In adult tissues, HOX11 expression is silenced by an unknown mechanism. Aberrant expression of HOX11 occurs in T-cell acute lymphoblastic leukaemia (T-ALL), where it is thought to be involved in T-cell immortalization. The deregulated expression of HOX11 is frequently associated with chromosomal translocations which juxtapose a T-cell receptor (TCR) gene upstream of the HOX11 gene. In these cases, it is presumed that the activation of HOX11 expression results from bringing the gene under the control of TCR enhancer elements. However, activation of HOX11 also occurs in the absence of an associated translocation in both T-ALL and erythroleukaemia cells, implying that an alternative activation mechanism may exist. We hypothesized that HOX11 may be repressed in normal T-cells and erythroid cells by the action of negative elements which may be deleted or mutated in leukaemia. We therefore conducted a search for negative elements in the human HOX11 promoter which may function to silence its expression in normal cells of the haematopoietic lineages. Since little sequence of the HOX11 promoter was available, we began our investigation by sequencing over 4.5 kilobases of untranslated DNA from upstream of HOX11. The human sequence that overlaps with the 2.1 kb of murine Hox11 is highly conserved, suggesting that a large region of DNA upstream of HOX11 may have a regulatory function. We then used transfection assays to test the ability of portions of the promoter to drive transcription of a reporter gene. These studies identified four negative elements. Two of them (NRE2 and NRE4) function in all cell lines tested, while the remaining two (NRE1 and NRE3) appear to be cell-type specific. The DNA sequences of three elements are conserved between the human and mouse HOX11/Hox11 promoters. We propose a model in which the combined action of these negative elements contributes to the overall repression of HOX11 expression in normal blood cells.

Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae.

Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions. To identify proteins that act in concert with topoisomerase II during chromosome segregation in S.cerevisiae, we have used a two-hybrid cloning approach. We report the isolation of the PAT1 gene (for protein associated with topoisomerase II), which encodes a novel 90 kDa proline- and glutamine-rich protein that interacts with a highly conserved, leucine-rich region of topoisomerase II in vivo. Strains lacking Pat1p exhibit a slow growth rate and a phenotype reminiscent of conditional top2 mutants grown at the semi-permissive temperature; most notably, a reduced fidelity of chromosome segregation during both mitosis and meiosis. These findings indicate that the PAT1 gene is necessary for accurate chromosome transmission during cell division in eukaryotic cells and suggest that the interaction of Pat1p and topoisomerase II is an important component of this function.

SGS1, a homologue of the Bloom's and Werner's syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability, sgs1 delta strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1 delta mutants, although meiosis I chromosome missegregation has been shown to be elevated sgs1 delta suppresses the slow growth of a top3 delta strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1 delta strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3 delta or a sgs1 delta strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae.

Failure to unwind causes cancer. Genome stability.

The recent cloning of the gene defective in individuals with Bloom's syndrome has revealed a link between DNA helicases, genetic instability and a predisposition to cancer.

Sgs1: a eukaryotic homolog of E. coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation.

Topoisomerase II (topo II) catalyzes the decatenation of interlinked DNA molecules and is essential for chromosome segregation. To test the hypothesis that the noncatalytic C-terminal domain of topo II is necessary for mediating interactions with other proteins required for chromosome segregation, we used a two-hybrid cloning strategy to identify proteins that interact with S. cerevisiae topo II in vivo. One protein identified (Sgs1p) is structurally related to E. coli RecQ protein and contains helicase signature motifs. Strains lacking Sgs1p exhibit elevated levels of chromosome misseggregation during both mitotic and meiotic division. We propose a model to account for the interaction of a topoisomerase and a helicase in the faithful segregation of newly replicated eukaryotic chromosomes.