We show for the first time that potential alteration in p53 function in childhood ALL is more common (P = .036) in cases of early treatment failure than in children who remain in long-term continuous remission.

Sézary syndrome is a leukaemic form of cutaneous T-cell lymphoma which presents with multiple cytogenetic abnormalities and responds poorly to chemotherapy. Because of the importance of the p53 tumour suppressor in maintaining genomic stability and in sensitizing transformed cells to DNA damaging agents, we looked for alternations which may affect p53 functions in 18 patients with Sézary syndrome. Cytogenetic analysis suggested frequent p53 gene inactivation since 6/18 patients had loss of one copy of 17p. However, single-strand conformational polymorphism (SSCP) revealed that p53 gene mutations are relatively rare, occurring in only two of 18 Sézary patients. Neither of these two patients was missing a copy of 17p. Possible abnormalities of p53 pathway function through mdm-2 over-expression were also investigated. Although all 18 patients had normal levels of mdm-2 RNA 4/18 over-expressed mdm-2 protein. One patient with advanced disease and the highest percentage of malignant cells overexpressed mdm-2 protein and possessed a nonsense p53 gene mutation. The five patients with abnormalities of p53 or mdm-2 were found to have significantly highest absolute lymphocyte counts and higher absolute numbers of Sézary cells (P=0-021 and 0.027 respectively). In summary, molecular alternations of 17p and potential p53 pathway abnormalities are a common event in Sézary syndrome and appear to be associated with more advanced disease.

No abstract

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.Genetic heterogeneity is one of the most remarkable and pervasive characteristics of established or immortal cell lines, especially those of high tumorigenicity (6,32,33,53). Whereas normal cells in vivo or in culture are predominantly or entirely diploid, tumors and immortal cell lines are invariably aneuploid. Such transformed cells exhibit frequent chromosomal alterations, in particular translocations (53) and gene amplifications (6, 37), some of which are associated with specific tumor types (6,24,53). They also undergo progressive dedifferentiative changes in gene expression, such as ectopic production of gene products (typically embryonal genes) which are not found in normal differentiated cells of that type (32), and clonal loss of gene products which would be expressed in normal cells of that type (33). These factors imply a fundamental shift toward genetic instability associated with transformation to immortality and tumorigenesis. Although homologous recombination is a likely common mechanism for DNA translocation and amplification (1, 10, 17) and a possible mechanism for alterations in gene expression (6, 24), recombination frequencies have not been directly compared between immortally transformed cell lines and normal diploid cells.Most carcinogens prove to be mutagenic when assayed in Salmonella His-reversion assays which are specific for point or frameshift mutations (6), but many are poorly detecte...

In this study, a new sequencing-based typing strategy for the HLA-A locus is presented which involves group-specific separate amplification of exon 2 and 3 of HLA-A alleles in a first step. Conserved HLA-A locus-specific primers of intron 1 or 3 were combined in 10 primer-mixes with group-specific primers hybridizing to the 5'- or 3'-end of exon 3 or 2 for pre-typing of the HLA-A alleles in 14 allelic groups. Maximally four overlapping short amplicons are produced under identical polymerase chain reaction (PCR) conditions with individual separate amplification of exon 2 and exon 3 of the haplotypic alleles in most heterozygous combinations. Time- and money-saving one-directional Big Dye Terminator cycle sequencing is shown to provide reliable high resolution typing of the HLA-A alleles, even in a few cases of two amplicons in one primer reaction mixture. In comparison, to other sequencing-based typing (SBT) techniques the applied typing strategy minimizes the risk of unequal amplification or of drop-outs of one of the haplotypic alleles and allows unequivocal definition of the cis/ trans linkage of polymorphic positions of the complete exon 2 and exon 3 in most heterozygous cells. This also includes detection of new alleles differing in the polymorphic template generating primer annealing sites as well as in unusual combinations of known exon 2 and 3 sequences. With 10 primer sets working under identical conditions for pre-grouping and separate amplification of the haplotypic alleles our SBT procedure also could be implemented in clinical settings of large-scale stem cell donor histocompatibility testing for fast molecular HLA-A matching.

The distribution of the different HLA-B*51 suballeles among patients with Behçet's disease (BD) of German (n=33) and Turkish (n=92) origin in comparison to their presence in the respective ethnically matched healthy control groups (German: n=325, Turkish: n=93) was studied. HLA-B*51x was significantly increased in both patient groups in comparison to the controls (Germans: 58% vs. 12%, OR 9.76, P<0.001; Turkish: 75% vs. 25%, OR 9.13, P<0.001). Molecular subtyping of B*51x revealed HLA-B*51011 and B*5108 as the predominant suballeles in both patient groups and controls although with a slightly increased frequency of HLA-B*5108 in the diseased individuals. HLA-B*5105 was the only further HLA-B*51x subtype detected in one Turkish patient heterozygous also for HLA-B*5101. HLA-B*5107 although present in a Turkish as well as German control was absent in the patient groups. There was also a tendency towards a higher degree of homozygosity for HLA-B*51x in both patient groups versus the matched controls (Germans: 10% in patients vs. 2,5% in controls; Turkish: 27% in patients vs. 13% in controls). Our study further supports previous hypothesis of an association of BD with B51 suballeles which share amino-acid residues at positions 63 and 67 as well as at positions 77-83 for specific peptide binding and natural killer (NK)-cell interactions. This applies to HLA-B*5101 and B*5108, but not to HLA-B*5107 different at position 67, which could be negatively associated with BD.

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.