A PCR-based clonal analysis of radiation-induced loss of heterozygosity in haemopoietic stem cells.

CBA mouse strains have been used for many years as a model of radiation-induced acute myeloid leukaemia and the leukaemias in CBA and their F1 hybrids are characterised by a specific loss of heterozygosity involving one homologue of chromosome 2. Previous cytogenetic studies of transplanted irradiated bone marrow, or of bone marrow obtained from irradiated mice significantly before the appearance of leukaemia, have been interpreted as the chromosome 2 deletion being a high frequency, possibly initiating event. However, these studies had not specifically addressed the question of whether the characteristic deletion was induced at a high frequency in stem cells. Using a PCR-based technique, we have studied the induction of chromosome 2 LOH in the progeny of (CBA/H x C57BL/6)F1 stem cells after a potentially leukaemogenic radiation exposure. Whilst chromosome 2 LOH can be induced directly by irradiation and there is a preferential loss of the CBA allele, the frequency is no greater than LOH induced in other chromosomal regions studied. The data do not support radiation-induced deletion involving one homologue of chromosome 2 in long-term repopulating stem cells (<1 in 200) being as high a frequency event as might be inferred by previous cytogenetic studies of total bone marrow.

DNA-PK activation by ionizing radiation-induced DNA single-strand breaks.

PURPOSE: To assess the ability of 60Co gamma-radiation-induced plasmid DNA single-strand breaks (gamma-ssb) to activate the DNA-dependent protein kinase (DNA-PK) in vitro. MATERIALS AND METHODS: Plasmid DNA was gamma-irradiated under aerobic conditions to yield 0-6 gamma-ssb and <0.1 double-strand breaks (dsb) per plasmid molecule. The irradiated DNA was used to stimulate DNA-PK in crude HF19 fibroblast nuclear extracts and/or purified HeLa cell DNA-PK protein, and the activation compared with that obtained with a single enzymatically generated plasmid DNA ssb (GpII endonuclease) or dsb (EcoRI endonuclease). RESULTS: Gamma-Irradiated plasmid DNA activates DNA-PK in both crude and purified preparations and the kinase activity increases linearly with dose. As significant DNA-PK activation was detectable using irradiated plasmids which contain <0.1 dsb/molecule, it was concluded that this activation is due to gamma-ssb. However, using purified DNA-PK, this activation is relatively weak as approximately 3 approximately-ssb is equivalent to one GpII-generated DNA ssb or one end of an EcoRI-generated dsb in DNA-PK assays. CONCLUSIONS: As gamma-ssb are in a approximately 20-fold excess of approximately-dsb in vivo for low LET radiation, gamma-ssb may contribute significantly to DNA-PK signalling of gamma-radiation-induced DNA damage in vivo.

p53-dependent X-ray-induced modulation of cytokine mRNA levels in vivo.

In vitro studies have shown that ionizing radiation can cause increases in some cytokine mRNA levels and activation of the nuclear NF-kappa B and/or AP1 transcription factors which have been implicated in the transcriptional regulation of many cytokine genes. Thus, radiation-induced upregulation of cytokine mRNAs appeared to be in part a direct consequence of transcription factor activation. To test this in vitro model in vivo, the effects of whole-body X-irradiation (0-10 Gy) on cytokine and other gene mRNA levels have been examined in mice. Increases and decreases in cytokine mRNA levels were detected in tissues which underwent an early wave of apoptosis (bone marrow and/or spleen), but not in more radioresistant tissues (kidney, liver, brain, and heart). Some mouse strain-specific differences were observed, but none of the changes in mRNA level was detected in p53-/- mice. As activation of the NF-kappa B and AP1 transcription factors was not detected in early-(spleen) or late-(liver) responding tissues in 10 Gy X-irradiated p53+/+ mice in vivo, it is concluded that the modulation of cytokine gene expression in vivo is p53-dependent and indirectly associated with apoptosis.

No association between p53 status and alpha-particle-induced chromosomal instability in human lymphoblastoid cells.

Previous work has demonstrated that alpha-particle irradiation of primary human bone marrow cells leads to the transmission of chromosomal instability in the descendants of the irradiated cell, although there is some interindividual variation. We have extended these studies to human EBV-transformed lymphoblastoid cell lines in order to establish an in vitro model system. The five cell lines analyzed, including one from a Fanconi anaemia patient, exhibited high levels of persistent chromatid aberrations up to approximately 40 cell generations after alpha-irradiation. The p53 status of the cell lines was defined according to whether cellular p53 levels were induced by irradiation, translocated to the nucleus and were able to bind a p53 DNA consensus recognition sequence in vitro. Together with the primary bone marrow cell studies, we conclude that alpha-particle induced chromosomal instability is independent of the p53 status of the cell as defined in these studies.

193 nm light induces single strand breakage of DNA predominantly at guanine.

Irradiation of DNA with 193 nm light results in monophotonic photoionization, with the formation of a base radical cation and a hydrated electron (phi PI = 0.048-0.065). Although > 50% of the photoionization events initially occur at guanine in DNA, migration of the "hole" from the other bases to guanine occurs to yield predominantly its radical cation or its deprotonated form. From sequence analysis, the data reveal that 193 nm light induces single strand breaks (ssb) in double-stranded DNA preferential 3' to a guanine residue. However, it has previously been reported that 193 nm light yields very low yields of ssb (< 2% of the yield of e-aq). The distribution of these ssb at guanine is nonrandom, showing a dependence on the neighboring base moiety. The efficiency of ssb formation at nonguanine sites is estimated to be at least one order of magnitude lower. The preferred cleavage at guanine is consistent with migration and localization of the electron loss center at guanine. It is argued that singlet oxygen and the photoionized phosphate group of the sugar moiety are not major precursors to ssb. At present, the mechanisms of strand breakage are not known although a guanine radical or one of its products remain potential precursors.

Enhancer dependent expression of the chicken beta-hatching globin gene during erythroid differentiation.

The activity of the chicken beta H globin gene promoter has been analysed in functional assays in both chicken and murine erythroleukaemia cells. Sequences between -251 and -146 bp, in the presence or absence of the chicken beta globin locus enhancer, strongly repress transcription in erythroid cells before and after the induction of terminal differentiation. A 50 bp sequence (-98 to -146 bp), which contains adjacent cGATA-1 and NF1 protein binding sites in vitro, and which is bound by non-histone protein in vivo, is essential for full promoter activity. Mutagenesis studies indicate that both protein binding sites are required. During terminal differentiation, both the absence of repressor and the presence of the erythroid enhancer are required for maximal promoter activity, suggesting that the beta A, beta epsilon and beta H globin gene promoters compete for the enhancer during development.

The purification of an erythroid protein which binds to enhancer and promoter elements of haemoglobin genes.

An erythroid nuclear protein (EF1), originally detected as a protein binding within the nuclease hypersensitive site upstream of the chicken beta H-globin gene, has been purified. This protein of 37,000-39,000 molecular weight binds to three sites within the hypersensitive region: one between the CCAAT and TATA boxes, the second (further upstream) next to a NF1 binding site, and the third adjacent to a regulatory element found in a number of beta-globin genes. The EF1 protein also binds to an erythroid-specific promoter element of the mouse alpha-globin gene and to two sites within the chicken beta A-globin enhancer. These six EF1-binding sites are related by the consensus sequence A/TGATAA/GG/C. A minor protein of molecular weight 72,000 which co-purifies with EF1 also binds to the same sequences.

TGGCA protein is present in erythroid nuclei and binds within the nuclease-hypersensitive sites 5' of the chicken beta H- and beta A-globin genes.

The developmentally regulated 5'-flanking DNase-I-hypersensitive site of the chicken beta H-globin gene in nuclei contains a subregion which is resistant to DNase I and which disappears when nuclei are extracted with 0.3 M NaCl, suggesting that there are salt-extractable proteins bound to sequences within this region. The 0.3 M NaCl extract contains two proteins which bind in vitro to these sequences. One of the binding sequences has an inverted repeat very similar to that bound by TGGCA protein. Partially purified TGGCA protein from chicken liver binds to this sequence in vitro giving exactly the same footprint as that obtained with erythroid nuclear proteins. Similarly TGGCA protein binds to an inverted repeat with the beta A-globin 5'-hypersensitive site giving a footprint identical to that obtained with erythroid nuclear protein extracts. From competition footprinting experiments and the electrophoretic mobility of the protein-DNA complex, it is concluded that the erythroid proteins previously described as binding to the beta H- and beta A-globin inverted repeats within the 5'-flanking hypersensitive sites both belong to the TGGCA protein family.

Detection of Sequence-Specific Protein-DNA Interactions by the DNA-Footprinting Technique.

The initiation of transcription of eukaryotic genes by RNA polymerases is controlled by complex interactions between nonhistone proteins and specific regulatory DNA sequences (promoters and enhancers) (1). In order to characterize and purify such transacting protein factors, a sensitive and accurate assay for sequence-specific DNA-binding proteins is the DNA-footprinting technique, which can be used to analyze the interaction of a complex mixture of proteins with a gene regulatory sequence(s) that is known to be important for the expression of that gene.

Characterisation of chicken erythroid nuclear proteins which bind to the nuclease hypersensitive regions upstream of the beta A- and beta H-globin genes.

Chicken erythrocyte sequence-specific nuclear DNA-binding proteins, which bind to the 5'-flanking DNAseI hypersensitive sites of the erythrocyte chromosomal beta A- and beta H-globin genes, have been fractionated by HPLC gel filtration. Three beta A-globin gene DNA binding activities (to sites A, B and B' (10-12)) were separated. The erythroid precursor cell line HD3 has beta A-globin gene sites B and B' binding activities, but binding to site A is detected only after the HD3 cells are induced to differentiate. The fractionated protein binds to a redefined site B', which contains at its center the globin CACCC consensus sequence. The chromosomal beta H-globin gene has two 5'-flanking DNAseI hypersensitive sites which bracket two sequences (H and H') bound by erythrocyte and HD3 nuclear protein in vitro. The beta H- and beta A-globin gene binding sites (H and B) contain variants of the sequences bound by Nuclear Factor 1 and the TGGCA-binding protein, and their protein binding activity(ies) co-purify after HPLC gel filtration.

Sequence-specific DNA-binding proteins which interact with (G + C)-rich sequences flanking the chicken c-myc gene.

The interaction of nuclear sequence-specific DNA-binding proteins from definitive chicken erythrocytes, thymus and proliferating transformed erythroid precursor (HD3) cells with the 700-base-pair (700-bp) DNA 5'-flanking region of the chicken c-myc gene was investigated by in vitro footprint analysis. The major HD3 protein-binding activity binds to a site (site V) 200 bp upstream from the 'cap' site but, after further fractionation, a second distinct binding activity is detected to a site (site VIII) which contains both the 'CAAT' and 'SP1-binding' consensus sequences. Protein from thymus and erythrocyte cells which express c-myc at lower levels, bind to seven and eight sites respectively. In common with HD3 cell protein, they both bind to site VIII and, although binding to the sequence at site V is also detected, the footprint protection pattern is sufficiently different (site V') to suggest the involvement of different proteins in terminally differentiated and proliferating cells. The DNA-binding activities were partially fractionated by high-performance liquid chromatography gel filtration and include an erythrocyte-specific protein which binds to a c-myc gene poly(dG) homopolymer sequence similar to that found upstream of the chicken beta A-globin gene.

Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.

Effect of adenovirus infection on expression of human histone genes.

The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA synthesis, observed when DNA replication is inhibited by a variety of drugs, is not maintained after adenovirus infection.