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

Loss of heterozygosity (LOH) contributes significantly to the inactivation of tumor suppressor genes and may involve a variety of mechanisms. Studying loss of HLA-A2 alleles in human lymphoblastoid cell lines, we previously showed that mitotic recombination and chromosome loss with concomitant duplication of the non-selected chromosome were the most frequent mechanisms of LOH. In the present study we used the HLA system to determine the rate and spectrum of LOH mutations in the EBV transformed lymphoblastoid cell line R83-4915. Spontaneous loss of HLA-A2 in R83-4915 occurred with a rate of 7.9x10-7 which was 5 to 10-times lower compared to the previously observed rate of loss of HLA-A2 in other lymphoblastoid cell lines. Among the HLA-A2 mutants, 27% did not show LOH of additional chromosome 6 markers. Molecular analysis showed that neither large deletion nor gene conversion was the cause for their mutant phenotype. The remaining mutants showed LOH, which was caused by mitotic recombination (40%) and chromosome loss (33%). However, the chromosome loss observed in mutants of R83-4915 was not accompanied by the duplication of the remaining chromosome. Instead 3 out of 5 mutants became polyploid suggesting that different mechanisms exist to compensate for chromosome loss. In conclusion, the rate and types of LOH that can be observed in cell lines obtained from various donors may depend on the genetic make-up or the transformation status of these cells

Isolation and molecular characterization of spontaneous mutants of lymphoblastoid cells with extended loss of heterozygosity.

The human major histocompatibility complex comprising the HLA class I and II genes provides a versatile source of natural heterozygous loci. This polymorphic genetic system allows analysis of the mechanistic aspects of loss of heterozygosity (LOH), a major phenomenon observed at tumor suppressor genes in human cancer cells. Four lymphoblastoid cell lines, ORI, TK6, WI-L2-NS and VH, were used to adjust current HLA immunoselection protocols to quantify loss of HLA-A2 in human lymphoblastoid cell lines. The modified selection protocol was used to isolate independent spontaneous HLA-A2 mutants from the lymphoblastoid cell line ORI. The frequency of spontaneous loss of HLA-A2 in ORI was 1.7 x 10(-5). By HLA typing 35 spontaneous HLA-A2 mutants, we showed that 74% of the HLA-A2 mutants also lost expression of the HLA-B allele, which is located on the same haplotype as HLA-A2. Microsatellites on both arms of chromosome 6 were used for molecular characterization of the spontaneous HLA-A2 mutants. Loss of heterozygosity at various loci on the p-arm or loss of an entire chromosome 6 was found in 80% of the mutants. Surprisingly, it appeared that a presumed mitotic recombination event in the cell line ORI itself had resulted in homozygosity of all markers distal from the HLA locus up to the telomere. This greatly limited the detection of mitotic recombination, resulting in LOH up to the telomere, on the short arm of chromosome 6 in this cell line. However, gene dosage analysis detected two copies of the remaining D6S265 allele in mutants which showed LOH at various loci along the p-arm. This suggested that recombination resulted in LOH in these mutants. The lymphoblastoid cell line TK6 did contain informative microsatellites along the complete chromosome 6. Mutants of TK6 either retained heterozygosity of all p-arm markers, showed LOH of all p-arm markers or showed loss from a breakpoint up to the telomere. These data indicate that recombination and chromosome loss both are important mechanisms involved in loss of the HLA-A2 allele in vitro. Such mechanisms may be involved in LOH in vivo and contribute to loss of tumor suppressor alleles.

A search for novel tumour suppressor genes for adult acute leukaemia by allelotyping at sub-telomeric chromosomal regions.

A search was initiated towards the localization of novel mutated tumour suppressor genes that may be involved in adult leukaemia. For this purpose, we measured the occurrence of loss of heterozygosity (LOH) in nine patients with acute B-lineage leukaemia (ALL) and one with undifferentiated leukaemia (AUL). Eight leukaemias exhibited a diploid karyotype. For each patient, PCR products of 130 polymorphic microsatellite markers, located in subtelomeric areas of every autosomal chromosome arm were analysed to visualize LOH events resulting from reduplication of a single mutated chromosome or from mitotic recombination. These kinds of LOH events contribute most to LOH in model systems but cannot be detected by classical cytogenetic techniques. By comparing allelic PCR products in tumour cells with those in normal cells, LOH was found in tumour cells of one ALL patient at 9p which harbours the known p16INK4A tumour suppressor gene. In the AUL patient, however, LOH was detected at the telomeres of 4q and 21q, suggesting that these sites may contain novel tumour suppressor genes specifically involved in this form of leukaemia. In the DNA of tumour cells from eight out of 10 patients no LOH was detected. This is in contrast with the general assumption that LOH is a frequent phenomenon in ALL. However, some markers at telomeric regions of chromosomes were already homozygous in the control T-cells of several patients. For instance, we found in the DNA of control cells from one patient five consecutive microsatellites on 9p up to 9p43 which were homozygous and in three other patients homozygosity was observed in band 8q24, which includes the MYC gene. These observations indicate that LOH events already are present in non-cancerous putative stem cells and that mitotic recombination may be a very early event in leukaemogenesis.

Chromosome loss with concomitant duplication and recombination both contribute most to loss of heterozygosity in vitro.

Loss of heterozygosity (LOH) plays an important role in the expression of recessive mutations in mammalian cells. To gain insight into the rate and mechanisms of LOH the autosomal HLA-A gene was used as a model system. Spontaneous HLA-A2 mutants originated with a rate of respectively 4.1 x 10(-6) and 6.9 x 10(-6) per cell per generation in TK6 and WI-L2-NS, two isogenic lymphoblastoid cell lines which differ in TP53 status. The rate of loss of HLA-A2 is 10-50 times higher compared to the mutation rate of the X-linked HPRT gene. The homozygous TP53 mutation in WI-L2-NS had no effect on the rate of HLA-A2 loss or the spectrum of these mutations. Microsatellite analysis of most of the HLA-A2 mutants (84%) showed LOH for multiple markers on chromosome arm 6p telomeric of a recombination breakpoint, LOH for all 6p markers, or LOH for markers on both the 6p- and 6q-arms. Cytogenetic analysis showed that these mechanisms gave mutant cells which harbored two intact chromosomes 6 and which were indistinguishable from non-mutant cells. Therefore, loss of HLA-A2 is mainly caused by somatic recombination (33-50%) or chromosome loss with duplication of the remaining chromosome (34-40%). These findings correspond to the mechanisms behind loss of the wild-type RBI allele in retinoblastoma and suggest that both somatic recombination and chromosome loss followed by duplication contribute to tumorigenesis.

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

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.

Molecular methods for the detection of mutations.

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.