David R. Turner

Integrative analysis of RUNX1 downstream pathways and target genes

BackgroundThe RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia.ResultsHere we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes.ConclusionThis work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

Use of DNA polymorphisms and the polymerase chain reaction to examine the survival of a human limbal stem cell allograft

PURPOSEnThe extent to which limbal epithelial stem cell allografts will repopulate the human corneal ocular surface, and the time frame over which such cells survive, are uncertain. We investigated the survival of donor-derived epithelial cells after limbal stem cell allotransplantation in a patient with bilateral limbal stem cell failure by using short tandem-repeat DNA polymorphisms to distinguish donor and recipient cells.nnnMETHODSnEpithelial cells were harvested by impression cytology from the grafted eye before and at various times after transplantation. DNA was extracted and amplified by the polymerase chain reaction at an informative locus, D8S264.nnnRESULTSnCells of donor genotype were present over the grafted areas at the time of surgery but were not detected in the central cornea until 12 weeks postoperatively, indicating that repopulation of the epithelial surface from transplanted limbal stem cells took considerable time. However, by the 20th postoperative week, only recipient-type cells were detected in the grafted eye, despite systemic immunosuppression of the recipient with azathioprine and cyclosporine.nnnCONCLUSIONSnDiscrimination between donor and recipient cells on the ocular surface after limbal allotransplantation was possible using genotypic variation at DNA polymorphic sites (microsatellites). Long-term survival of donor cells after limbal transplantation did not occur in this patient. Detection of DNA polymorphisms amplified by the polymerase chain reaction is a simple, rapid, and noninvasive method of following the course of transplanted cells at the ocular surface.

Human lymphocytes aged in vivo have reduced levels of methylation in transcriptionally active and inactive DNA

The amount of 5-methylated cytosine (5 mC) in the sequence CmCGG has been measured in DNA extracted from uncultured peripheral blood human lymphocytes obtained from 24 young (mean age 25) and 22 old (mean age 75) individuals. When compared with the young group the old group had significantly reduced levels of 5 mC in total genomic DNA, in transcriptionally active DNA, and in genomic DNA from which transcriptionally active sequences had been removed. In both the young and old groups transcriptionally active DNA contained 10% less 5 mC than the residual inactive DNA. These results indicate that loss of genomic DNA methylation may be involved in aging in vivo and underscore the association of gene regulation with the distribution of methylation in DNA.

Adaptive response for chromosomal inversions in pKZ1 mouse prostate induced by low doses of X radiation delivered after a high dose

Abstract Day, T. K., Zeng, G., Hooker, A. M., Bhat, M., Scott, B. R., Turner, D. R. and Sykes, P. J. Adaptive Response for Chromosomal Inversions in pKZ1 Mouse Prostate Induced by Low Doses of X Radiation Delivered after a High Dose. Radiat. Res. 167, 682–692 (2007). Adaptive responses are induced by stress such as X radiation and result in a lower than expected biological response. Two-dose adaptive response experiments typically involve a low priming dose followed by a subsequent high radiation dose. Here, we used a sensitive in vivo chromosomal inversion assay to demonstrate for the first time an adaptive response when a low dose (0.01–1 mGy) was given several hours after a high 1000-mGy radiation dose. The adaptive responses in this study were of similar magnitude to the two-dose adaptive responses previously observed in this test system when the low dose was given first. A chromosomal inversion adaptive response was also induced by two 1000-mGy doses and when a 1-mGy dose was preceded or followed by a dose of 0.01 mGy, but not by two 4000-mGy doses. This is also the first example of an adaptive response when both doses are low. Our data agree with previous reports of an on-off mechanism of adaptive response. The induction of an adaptive response by a low dose after a high damaging dose provides evidence that the mechanisms underlying radiation adaptive responses are not due to prevention of damage induced by the high dose but to modulation of the cellular response to this damage.

Extremely Low Priming Doses of X Radiation Induce an Adaptive Response for Chromosomal Inversions in pKZ1 Mouse Prostate

Abstract Day, T. K., Zeng, G., Hooker, A. M., Bhat, M., Scott, B. R., Turner, D. R. and Sykes, P. J. Extremely Low Priming Doses of X Radiation Induce an Adaptive Response for Chromosomal Inversions in pKZ1 Mouse Prostate. Radiat. Res. 166, 757– 766 (2006). An adaptive response is a response to a stress such as radiation exposure that results in a lower than expected biological response. We describe an adaptive response to X radiation in mouse prostate using the pKZ1 chromosomal inversion assay. pKZ1 mice were treated with a priming dose of 0.001, 0.01, 1 or 10 mGy followed 4 h later by a 1000-mGy challenge dose. All priming doses caused a similar reduction in inversions compared to the 1000-mGy group, supporting the hypothesis that the adaptive response is the result of an on/off mechanism. The adaptive response was induced by a priming dose of 0.001 mGy, which is three orders of magnitude lower than has been reported previously. The adaptive responses completely protected against the inversions that would have been induced by a single 1000-mGy dose as well as against a proportion of spontaneous background inversions. The distribution of inversions across prostate gland cross sections after priming plus challenge irradiation suggested that adaptive responses were predominantly due to reduced low-dose radiation-induced inversions rather than to reduced high-dose radiation-induced inversions. This study used radiation doses relevant to human exposure.

Molecular basis of X-ray-induced mutation at the HPRT locus in human lumphocytes

Human lymphocytes lacking functional HPRT enzyme after a dose of 300 rad X-radiation were cloned and the monoclonal populations expanded so that sufficient genomic DNA was obtained for Southern analysis. A total of 33 mutant clones were analysed. Wild-type clones showed no evidence of changes to the HPRT gene resolvable by Southern banding patterns whereas 17 of 33 mutant clones showed changes. The alterations observed included total gene deletions (3 clones) and partial gene deletions with or without the appearance of novel bands (12 clones). Two clones showed the appearance of novel bands only. There were no changes observed in 16 of the 33 mutant clones. Three clones showed changes inconsistent with deletion of portions of the gene. In these clones inversion seems to have been the most likely cause of the mutation. The spectrum of gene alterations following ionizing radiation appears different to that previously observed for spontaneous mutations. Consequently, ionizing radiation or radiomimetic agents would appear to be aetiologic, at the most, for only a minor proportion of in vivo somatic mutations.

Familial atopy in Australian pedigrees: adventitious linkage to chromosome 8 is not confirmed nor is there evidence of linkage to the high affinity IgE receptor

Atopy frequently displays autosomal dominant inheritance and recent studies have favoured genetic linkage between atopy and the human chromosome 11q13. We have studied 12 extended families with aggregation of atopy consistent with autosomal dominant inheritance. The families have been studied for linkage of asthma and atopy to loci on chromosome 8p following the observation that one family suggested preliminary evidence of linkage to an anonymous hypervariable locus cloned from a DNA fingerprint and mapped to 8pter‐p22. Subsequent analysis shows this putative linkage to be adventitious as the remaining 11 families do not support linkage between atopy and 8p, We have analysed the same families for evidence of linkage of atopy to loci on 11q13. In these families there is no evidence of association between atopy and the 11q loci stronger than that expected by chance alone; furthermore there is no suggestion subpopulation of these families display linkage between atopy and the loci. In addition neither the 8p loci nor the 11q loci exhibit evidence of linkage to atopy by affected sib‐pair analysis. This also conflicts with previously published data for 11q.

Interindividual Variation in Mitotic Recombination

Mitotic recombination (MR) between homologous chromosomes is a mutational event that results in loss of heterozygosity in half of the segregants at mitosis. Loss of heterozygosity may have important biological consequences. The purpose of this study was to describe human variation in the spontaneous frequency of MR. Using an immunoselection technique for isolating the somatic mutations that result in loss of expression of one of the codominant alleles at the HLA-A locus, we have measured the frequency and molecular basis of somatic mutations in lymphocytes from a population of young adults. Mutations were classified as being the result of intragenic changes, major deletions, or MR. Here we show that the MR mutation frequency in females was significantly greater than that in males but that intragenic mutation frequency showed no association with sex. Individual variation in MR frequency ranged over more than two orders of magnitude and was not normally distributed. Furthermore, the observed number of individuals from whom no mutants resulting from MR were obtained was significantly greater than was expected. The endogenous level of MR may be under genetic control. Given the association of loss of heterozygosity with cancer initiation and progression, low endogenous MR may confer a reduced lifetime risk of cancer, and the converse may apply.

Mitotic recombination is an important mutational event following oxidative damage

The mutagenic effects of hydrogen peroxide (H(2)O(2)), a source of reactive oxygen species (ROS) have been determined in human lymphocytes. T-lymphocytes mutated at the autosomal HLA-A locus on chromosome 6 have been clonally isolated (N = 2097 clones) and the molecular basis of each clonal mutation characterised as due to intragenic, deletion or mitotic recombination mutation. H(2)O(2) caused a dose dependent increase in mutation frequency. There was no significant increase in the frequency of intragenic mutations. Mitotic recombination (MR) was responsible for 87% of the increase in mutation frequency induced by H(2)O(2) and gene deletion was responsible for 13%. MR results in loss of heterozygosity (LOH) distal to the recombination site. It is known that LOH is important in the initiation and progression of cancer. These results suggest that the biologically important consequence of some ROS may be LOH as a by-product of recombination repair. They also suggest that if our observations apply to ROS generally, then many of the mutations which accumulate with ageing or which are observed in cancer may be due to factors other than ROS.

The contribution of exogenous and endogenous mutagens to in vivo mutations

There is abundant evidence of the potential for exogenous agents to cause cancer but the proportion of human cancers attributable to defined external agents is uncertain. With rare exceptions it is difficult to demonstrate a role for exogenous agents in increasing mutation above background rates. There are many sources of endogenous mutation including physico-chemical processes, free radicals and enzymatic processes controlling DNA damage and repair. Evidence for the role of diet and genetic factors as major determinants of endogenous mutagenesis is reviewed with reference to the spontaneous spectrum of mutations in human cells and the quantitative measurement of mutation frequency in dietary restriction and the senescence-accelerated mouse.