Both the rate and spectrum of loss of heterozygosity differ between human lymphoblastoid cells derived from various donors

Dalen, Arnolda G. de Nooij-van; Giphart, Marius J.; Lohman, P.H.M.; Giphart-Gassler, Micheline

doi:10.1016/s0027-5107(98)00204-8

Cited by 5 publications

(5 citation statements)

References 31 publications

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“…Displacement and loss of any chromosome is commonly observed in karyotypes prepared from short‐term lymphoblastoid cultures (Abruzzo et al, 1986). LOH at the HLA‐A locus associated with whole chromosome 6 loss and duplication of the remaining chromosome has been observed at high frequencies in EBV transformed lymphoblastoid clones grown in vitro (de Nooij‐van Dalen et al, 1998, 1999). However, LOH at the HLA‐A locus associated with whole chromosomal loss is very rare in lymphocytes in vivo when the assay for LOH involves cloning in vitro (Turner et al, 1988; Morley et al, 1990; de Nooij‐van Dalen et al, 2001).…”

Section: Illustrationmentioning

confidence: 99%

Evidence for whole chromosome 6 loss and duplication of the remaining chromosome in acute lymphoblastic leukemia

McEvoy

Morley

Firgaira

2003

Genes Chromosomes & Cancer

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HLA class I molecules serve the essential immunological function of presenting antigen to CD8+ T lymphocytes. Tumor cells may present tumor-specific antigen to T cells via these molecules, but many tumors show a loss or down-regulation of HLA class I expression and this may serve as an immune escape mechanism. Using a microsatellite marker-based method, we have searched for loss of heterozygosity (LOH) mutations at 3 genomic regions implicated in HLA class I expression in a cohort of 56 acute lymphoblastic leukemia (ALL) samples. The regions analyzed consisted of the HLA class I heavy chain genes located within the MHC genomic region on chromosome arm 6p, the HLA class I light chain (beta-2-microglobulin, B2M) gene on chromosome arm 15q, and the putative HLA modifier of methylation gene (MEMO1) located on chromosome arm 1q. Results revealed low frequencies of B2M (2/55) and MEMO1 (5/42) LOH but a high frequency of MHC LOH (19/56) that was usually associated with whole chromosome 6 loss (13/19). Cytogenetic data were available for 30 samples, including nine of those that exhibited apparent whole chromosome 6 loss. No cases of chromosome 6 monosomy were observed. We propose that whole chromosome 6 loss with reduplication of the remaining chromosome is common in ALL and that it is driven by the presence of tumor-inhibiting factors on chromosome arm 6p (the HLA loci) along with previously localized tumor-suppressor genes on chromosome arm 6q.

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Section: Illustrationmentioning

confidence: 99%

Evidence for whole chromosome 6 loss and duplication of the remaining chromosome in acute lymphoblastic leukemia

McEvoy

Morley

Firgaira

2003

Genes Chromosomes & Cancer

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“…To exclude the possibility that HLA class I expression is impaired in the HLA-A2 mutants, clones were tested for complement-mediated killing following incubation with a monoclonal antibody directed against a common HLA class I epitope (W6.32). The CMC method has been used to determine the spontaneous spectrum of HLA-A2 loss in the four cell lines using fluctuation analysis [26][27][28].…”

Section: Specific Mutation Assaysmentioning

confidence: 99%

“…To determine the extent of LOH informative CA repeat markers located along chromosome 6 were amplified by PCR and analyzed to scan HLA-A2 mutant DNA for loss of CA repeat alleles. To distinguish between homozygous and hemizygous alleles or chromosomal regions, the copy number of a microsatellite allele close to HLA-A was determined by quantitative PCR and the number of chromosomes 6 or chromosomal rearrangements involving chromosome 6 were determined by fluorescent in situ hybridization [26][27][28].…”

Section: Specific Mutation Assaysmentioning

confidence: 99%

“…By using CMC, the spontaneous mutation rate of loss of HLA-A2 was determined by fluctuation analysis in TK6 and WI-L2-NS, two cell lines which were both derived from the cell strain WI-L2 but differ in their p53 status: TK6 harbors a p53 wild-type gene, while WI-L2-NS harbors a homozygous mutation in the p53 gene [43].The spontaneous mutation rate of loss of HLA-A2 was 4.1 × 10 -6 in TK6 and 6.9 × 10 -6 in WI-L2-NS per cell per generation [27], while the rate was significantly lower (7.9 × 10 -7 ) in the EBV transformed cell line R83-4915 (VH) [28]. The mean frequency of loss of HLA-A2 from 11 subclones of ORI was 1.7 ± 2.4 × 10 -5 [26].…”

Section: Loss Of Heterozygosity Mutations At Hla-amentioning

confidence: 99%

“…The mean frequency of loss of HLA-A2 from 11 subclones of ORI was 1.7 ± 2.4 × 10 -5 [26]. Independent HLA-A2 -mutants of TK6 [32],, WI-L2-NS [30], and VH [15], and 35 ORI mutants were isolated for analysis of LOH within the MHC region by HLA typing and of LOH on chromosome 6 by using CA repeat analysis [26][27][28]. Figure 4 depicts the theoretical mechanisms which may exist for the loss of HLA-A2 and summarizes the contribution of the various mechanisms to the spontaneous loss of HLA-A2 observed in the cell lines ORI, VH, TK6, and WI-L2-NS.…”

Section: Loss Of Heterozygosity Mutations At Hla-amentioning

confidence: 99%

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Molecular methods for the detection of mutations

Monteiro

Marcelino

Conde

et al. 2000

Teratog. Carcinog. Mutagen.

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We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

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Intrinsic genetic instability of normal human lymphocytes and its implication for loss of heterozygosity

Dalen¹,

Morolli²,

Keur³

et al. 2001

Genes Chromosom. Cancer

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A combination of flow cytometry and microsatellite analysis was used to investigate loss of expression of HLA-A and/or HLA-B alleles and concurrent LOH at polymorphic chromosome 6 loci both in freshly isolated lymphocytes (in vivo mutations) and in lymphocytes cultured ex vivo. The fraction of in vivo mutants that showed LOH at 6p appeared to vary from 0%-49% for various donors. During culturing ex vivo, HLA-A(-) cells arose at a high rate and showed simultaneous loss of expression at the linked HLA-B locus. Up to 90% of the ex vivo arisen HLA-A2(-) cell population showed LOH of multiple 6p markers, and 50% had lost heterozygosity at 6q. This ex vivo spectrum resembles that found in HLA-A2 mutants obtained from lymphoblastoid cells. The HLA-A2 mutants present in vivo may reflect only a small fraction of the mutants that can be detected ex vivo. In normal lymphocytes, in vivo only mitotic recombination appears to be sustained, indicating the importance of this mechanism for tumor initiation in normal cells. Although mutations resulting in LOH at both chromosome 6 arms were shown to result in nonviable cells in normal lymphocytes, they have been shown to result in viable mutants in lymphoblastoid cells. We hypothesize that these types of mutations also occur in vivo but only survive in cells that already harbor a mutated genetic background. In light of the high rate at which these types of mutations occur, they may contribute to cancer progression.

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Both the rate and spectrum of loss of heterozygosity differ between human lymphoblastoid cells derived from various donors

Cited by 5 publications

References 31 publications

Evidence for whole chromosome 6 loss and duplication of the remaining chromosome in acute lymphoblastic leukemia

Evidence for whole chromosome 6 loss and duplication of the remaining chromosome in acute lymphoblastic leukemia

Molecular methods for the detection of mutations

Intrinsic genetic instability of normal human lymphocytes and its implication for loss of heterozygosity

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