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Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C > T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

BackgroundMinimally differentiated acute myeloid leukemia is heterogeneous in karyotype and is defined by immature morphological and molecular characteristics. This originally French-American-British classification is still used in the new World Health Organization classification when other criteria are not met. Apart from RUNX1 mutation, no characteristic molecular aberrations are recognized.Design and MethodsWe performed whole genome single nucleotide polymorphism analysis and extensive molecular analysis in a cohort of 52 patients with minimally differentiated acute myeloid leukemia.ResultsMany recurring and potentially relevant regions of loss of heterozygosity were revealed. These point towards a variety of candidate genes that could contribute to the pathogenesis of minimally differentiated acute myeloid leukemia, including the tumor suppressor genes TP53 and NF1, and reinforced the importance of RUNX1 in this leukemia . Furthermore, for the first time in this minimally differentiated form of leukemia we detected mutations in the transactivation domain of RUNX1. Mutations in other acute myeloid leukemia associated transcriptions factors were infrequent. In contrast, FLT3, RAS, PTPN11 and JAK2 were often mutated. Irrespective of the RUNX1 mutation status, our results show that RAS signaling is the most important pathway for proliferation in minimally differentiated acute myeloid leukemia. Importantly, we found that high terminal deoxynucleotidyl transferase expression is closely associated with RUNX1 mutation, which could allow an easier diagnosis of RUNX1 mutation in this hematologic malignancy.ConclusionsOur results suggest that in patients without RUNX1 mutation, several other molecular aberrations, separately or in combination, contribute to a common minimally differentiated phenotype.

Minimally differentiated acute myeloid leukemia (AML-M0) is defined by immature morphology and expression of early hematologic markers. By gene expression profiling (GEP) and subsequent unsupervised analysis of 35 AML-M0 samples and 253 previously reported AML cases, we demonstrate that AML-M0 cases express a unique signature that is largely separated from other molecular subtypes. Hematologic transcription regulators such as CEBPA, CEBPD, and ETV6, and the differentiation associated gene MPO appeared strongly down-regulated, in line with the primitive state of this leukemia. AML-M0 frequently carries loss-of-function RUNX1 mutation. Unsupervised analyses revealed a subdivision between AML-M0 cases with and without RUNX1 mutations. RUNX1 mutant AML-M0 samples showed a distinct up-regulation of B cell-related genes such as members of the B-cell receptor complex, transcription regulators RUNX3, ETS2, IRF8, or PRDM1, and major histocompatibility complex class II genes. Importantly, prediction with high accuracy of the AML-M0 subtype and prediction of patients carrying RUNX1 mutation within this subtype were possible based on the expression level of only a few transcripts. We propose that RUNX1 mutations in this AML subgroup cause lineage infidelity, leading to aberrant coexpression of myeloid and B-lymphoid genes. Furthermore, our results imply that AML-M0, although originally determined by morphology, constitutes a leukemia subgroup. (Blood. 2009; 114: 3001-3007)

Purpose and background: Variation in radiosensitivity of normal tissues is related to genetic susceptibility (Svensson et al. 2006). Persistence of gamma-H2AX foci after ionizing radiation is higher in DNA-DSB repair deficient cells than in normal cells. We investigated whether differences in number of ?-H2AX foci may be useful as a predictor of radiosensitivity. Materials and methods: Induction of gamma-H2AX foci was measured in various cell types: freshly isolated lymphocytes, fibroblast cells (AT+/+, AT+/-, AT-/-), DNA-DSB repair deficient and normal hamster cells and human tumour cells (lung carcinoma, SW-1573 and prostate cancer cell lines with different TP53 status: LNCaP: TP53 wt, PC3: TP53 null and DU145: TP53 mutant). A radiation dose of 2.4 Gy was given as a single dose or as pulsed doses of 100 mGy in 24 h (PDR: 240 pulses of 100mGy, 1 pulse every 6 min). The number of gamma-H2AX foci was determined 30 min and 24 h after single dose and directly after PDR. Results: Maximal number of γ-H2AX foci occurred after single dose at 30 min after irradiation without significant differences in numbers of foci between different cell lines. The number of gamma-H2AX foci was radiation dose dependent and linearly correlated with clonogenic survival. Both 24 h after a single dose and after PDR more remaining foci are observed in repair deficient cells than in normal cells. In addition the TP53 null cells have more remaining foci than the mutant and wt TP53 cells. Conclusions: The detection gamma-H2AX after in vitro irradiation of lymphocytes and cell lines is simple, reproducible and sensitive. The number of residual gamma-H2AX foci after single dose as well as after PDR enables us to distinguish cells with differences in DNA-DSB repair capacities. However, it remains to be established whether gamma-H2AX assay applied in lymphocytes can be used to predict for late normal tissue toxicity after radiotherapy. Sponsored by the Dutch Cancer Foundation UVA2008-4019

Mutation induction by UV irradiation was studied in a retroviral vector integrated in one copy per cell at various chromosomal positions. As a mutational target, hamster hprt cDNA was present on the retroviral vector. To minimize the influence of repair we used repair-deficient hamster cells, V-H1 and UV5, as a recipient for the vector. There is no major influence of chromosomal position on UV-induced mutation frequency and spectrum because no statistically significant difference between mutation induction in retroviral cDNA copies integrated at different chromosomal sites was observed. However, a major difference was found in mutation induction between the endogenous hamster hprt gene and the retroviral cDNA copies. Most noticeable was the absence in the cDNA of the strong strand bias for mutation induction, which was reported for the endogenous hprt gene. Our results with the hprt cDNA exclude as a general phenomenon a difference in mutation induction for leading and lagging strand DNA replication, which was proposed as an explanation for this strand bias in the endogenous gene. The similarity of mutation induction in the different retroviral cDNA copies, all directly surrounded by the same DNA sequence elements, together with the marked difference between the mutation induction in the endogenous gene and the cDNA copies may point to an important role of chromatin structure in mutation induction.

The polypeptides encoded by the early region of the Mu genome (comprising approximately 8000 base-pairs of DNA extending from the immunity encoding end of the linear Mu genome) have been determined. Minicells were infected by wildtype, suppressor-sensitive-mutation-carrying Mu phages and by λpMu hybrid phages. Comparison of the polypeptides synthesized in these infected minicells with polypeptides synthesized in minicells containing recombinant plasmids, which carry the first 5100 base-pairs of the early region of Mu DNA, have permitted a definitive correlation of the region of DNA with the encoded gene product. The Mu represser protein, gpc (Mr, 26,000), is encoded by approximately 900 base-pairs of DNA extending from the c-end of the genome † . The region 1300 to 4300 base-pairs encodes gpA (Mr, 70,000) and gpB (Mr, 33,000) and the region 4300 to 5100 basepairs encodes gpcim (Mr, 7000) and gpkil (Mr, 8000). A 14,000 Mr polypeptide, which is probably gpgam, is located in the region 4800 to 5400 base-pairs, and three polypeptides of molecular weights 20,500, 19,000 to 22,000 and 11,500 are located in the region 5200 to 6600 base-pairs. One of these polypeptides is gpsot. The basis for the apparent range of molecular weights (19,000 to 22,000) exhibited by one of the polypeptides encoded in this region is unknown. The region 6200 to 8600 base-pairs encodes three polypeptides (Mr = 10,000, 10,000 and 8000) of unknown function. The polypeptides identified as early polypeptides from their location on the Mu genome are also those polypeptides detected earliest after infection of minicells and are the major translation products of Mu RNA synthesized in minicells infected in the presence of chloramphenicol. These early polypeptides, assuming no overlapping genes, require the expression of approximately 80% of the coding capacity of the early region of the Mu genome.

A method is described, which allows identification of early gene products of bacteriophage Mu. This method is based on ultraviolet irradiation of host cells prior to bacteriophage infection. It appears that only early genes of Mu are expressed in u. v. -irradiated cells. Two polypeptides with molecular weights of 23,000 and 35,000, as measured on sodium dodecylsulphate/polyacrylamide gels, were identified as Mu products, both after infection with Mu and after infection with a λdMu transducing phage. From the results obtained with various amber mutants we conclude that the polypeptide with a molecular weight of 35,000 is the product of gene B . The possibility that the Mu protein with a molecular weight of 23,000 is the product of a gene downstream of gene B will be discussed.

An Eco RI fragment, containing approx. 5100 base pairs (bp) of the immunity-end of bacteriophage Mu, was inserted into the multicopy plasmid pMB9 by in vitro recombination. The expression of early Mu genes, located on the cloned fragment, is thermo-inducible because of the presence of the ts mutation in gene c . The isolation of a transformant harbouring the recombinant plasmid, pGP1, was possible only when expression of Mu genes was prevented. pGP1 can be maintained at 28°C at high copy number, but at 42°C the pGP1 containing cells are killed due to the expression of the kil gene of Mu. The following Mu genes are present on pGP1: the ner gene, the integration and replication genes A and B , the cim gene, and the kil gene. pGP1 containing cells do not show Gam and Sot activity at 42°C, therefore the leftmost Eco RI site on the Mu DNA is located between genes kil and gam or sot , or within the gam or sot gene.

The G region of bacteriophage Mu is a 3,000-base pair (bp) invertible DNA segment that determines the host specificity of the phage1,2. Only phage with the G segment in the orientation designated (+) can adsorb to Escherichia coli K-12 (refs 3–5), whereas phage with the G segment in the (−) orientation infects other hosts such as Citrobacter freundii and E. coli C (ref. 1). It has been proposed that four genes are involved in host specificity—S, U, S′ and U′—of which S and U are expressed in the orientation (+) and S′ and U′ in the inverted orientation (−) of the G segment. We have presented two alternatives for the organization of these genes (Fig. 1). In model A the four genes involved are entirely contained within the G segment, whereas in the alternative model (B) it is assumed that the proximal part of the S gene is located in the a part of the Mu genome. We report here that the S and S′ genes are partially located outside the G segment and share a common part located in α (Sc). This common S region will be joined by G inversion to one of the two variable parts (Sv or Sv′) which results in the synthesis of either tail fibre protein S or S′.

The function of the invertible G region of bacteriophage Mu is apparently to confer different host specificities on Mu. Two products of genes S and U, situated in the G region are not needed for the infectivity of Mu G(-) particles. In the Mu G(-) phage the S gene product and the 21-K polypeptide, presumably the product of gene U, are missing. Instead, two other polypeptides with different molecular weights are observed.

The herpesvirus thymidine kinase ( tk ) gene integrated in the human cell line, 2.1-a, can be inactivated by limited de novo methylation. All these TK − clones show partial Eco RI digestion of the recognition site (cGAATTCg) in the tk promoter in contrast to complete digestion of this site in the original cell line. Studies on well-defined substrates prepared in vitro showed that methylation of one cytosine in the Eco RI recognition sequence resulted in partial and methylation of both cytosines in severe inhibition of digestion by Eco RI. This characteristic was used to determine whether no, one or both cytosines in the Eco RI site of the tk promoter were methylated in various TK − clones derived from 2.1-a and in TK + clones re-expressing the gene after 5-azacytidine treatment. A high correlation was found between inactivity of the tk gene and methylation of only one of the two cytosines in the Eco RI recognition site. The results also show that the tk promoter can be active despite the presence of a methylated cytosine.

The genome of bacteriophage Mu can integrate randomly in the chromosome of its host Escherichia coli, thereby causing mutations (Taylor, 1963). Random integration seems a more widespread phenomenon, since it was found that other DNA elements also show a more or less random integration: insertion sequences (IS) and transposons (Tn).