Using fluorescence in situ hybridization (FISH) to interphase nuclei, we examined the replication timing of 1 allele relative to its counterpart in PHA-stimulated peripheral blood lymphocytes of normal subjects and patients suffering from a solid tumor (renal cell carcinoma). In the FISH assay, an unreplicated DNA sequence is identified by a single dot-like hybridization signal, whereas a replicated region gives rise to a duplicated, bipartite signal. Accordingly, lymphocytes of normal individuals show 2 patterns of allelic replication: (i) synchronized replication of allelic counterparts, as exemplified by the biallelically expressed loci TP53 and D21S55; and (ii) non-synchronized replication of allelic partners, as exemplified by the early and late replicating alleles of GABRB3, an imprinted locus subjected to monoallelic expression. However, when present in lymphocytes of the cancer patients, all 3 loci change their replication mode: alleles of TP53 and D21S55 become asynchronous, whereas the early replicating allele of GABRB3 delays replication, leading to relaxation in the imprinted mode of replication. Based on the tight relationship between temporal order of allelic replication and allelic mode of expression, the modified order of allelic replication observed in nonmalignant cells of individuals diagnosed with cancer represents a novel genetic alteration associated with malignancy. This alteration detected by simple cytogenetic means, applied to peripheral blood lymphocytes, offers a potential test for cancer identification. Genes Chromosomes Cancer 27:270-277, 2000.
Copyright 2000 Wiley-Liss, Inc.