The INK4A locus encodes two independent but overlapping genes, p16INK4A and p19ARF, and is frequently inactivated in human cancers. The unusual structure of this locus has lead to ambiguity regarding the biological role of each gene. Here we express, in primary mouse embryonic fibroblasts (MEFs), antisense RNA constructs directed specifically towards either p16INK4A or p19 ARF. Such constructs induce extended lifespan in primary MEFs; this lifespan extension is reversed upon subsequent elimination of the p16INK4A or p19ARF antisense constructs. In immortal derivatives of cell lines expressing antisense p16INK4A or p19ARF RNA, growth arrest induced by recovery of p16INK4A expression is bypassed by compromising the function of the retinoblastoma protein (Rb), whereas growth arrest induced by re-expression of p19ARF is overcome only by simultaneous inactivation of both the Rb and the p53 pathways. Thus, the physically overlapping p16INK4A and p19ARF genes act in partly overlapping pathways.

The TEL (ETV6)؊AML1 (CBFA2) gene fusion is the most common reciprocal chromosomal rearrangement in childhood cancer occurring in Ϸ25% of the most predominant subtype of leukemia-common acute lymphoblastic leukemia. The TEL-AML1 genomic sequence has been characterized in a pair of monozygotic twins diagnosed at ages 3 years, 6 months and 4 years, 10 months with common acute lymphoblastic leukemia. The twin leukemic DNA shared the same unique (or clonotypic) but nonconstitutive TEL-AML1 fusion sequence. The most plausible explanation for this finding is a single cell origin of the TEL-AML fusion in one fetus in utero, probably as a leukemia-initiating mutation, followed by intraplacental metastasis of clonal progeny to the other twin. Clonal identity is further supported by the finding that the leukemic cells in the two twins shared an identical rearranged IGH allele. These data have implications for the etiology and natural history of childhood leukemia.An extraordinary diversity of chromosomal molecular abnormalities has been identified in hematopoietic malignancies (1, 2). Among the most prominent are reciprocal chromosomal translocations that produce, via genetic recombination, inframe fusion genes and hybrid proteins (3, 4). Although details of the mechanisms involved remain to be elucidated, many of these genes encode transcription factors; their novel products are thought to endow clonal advantage via the imposition of an altered pattern of gene regulation (3, 4). One of the most frequent gene fusions so far described is that between TEL (or ETV6) and AML1 (or CBFA2). This rearrangement, although cryptic at the level of chromosome karyotype, occurs in approximately 25% of the predominant subtype of pediatric cancer and leukemia-common acute lymphoblastic leukemia (cALL)-in children diagnosed between the ages of 2 and 10 years (5, 6). The translocation t(12;21)(p13;q22) in ALL consistently involves the fusion of the protein dimerization encoding 5Ј region of the ETS-like gene TEL with almost the entire AML1 gene including its DNA binding region (with homology to Drosophila runt) and transactivation domain (reviewed in ref. 5). The chromosome 12 breakpoints cluster within a single intron of the TEL gene whereas AML1 breaks occur within the large and currently unsized first two introns of the AML1 gene on chromosome 21 (5-7). As with other fusion genes in leukemia, each patient's intronic breakpoints and subsequent fusion sequence are unique, providing a stable genomic marker of the derivative clone of cells. In the context of the etiology and natural history of childhood ALL, a key issue is when and how the TEL-AML1 fusion gene is generated and whether this is an early or initiating event. We report here a molecular analysis of the genomic fusion region of TEL-AML1 in the unusual situation of concordant leukemia in monozygotic twins. This analysis provides unequivocal evidence that this genetic lesion can be acquired during fetal hematopoiesis in utero.

Fetal nucleated cells in the maternal circulation constitute a potential source of cells for the non-invasive prenatal diagnosis of fetal genetic abnormalities. We have investigated the use of the Magnetic Activated Cell Sorter (MACS) for enriching fetal nucleated erythrocytes. Mouse monoclonal antibodies specific for CD45 and CD32 were used to deplete leucocytes from maternal blood using MACS sorting, thus enriching for fetal nucleated erythrocytes which do not express either of these antigens. However, significant maternal contamination was present even after MACS enrichment preventing the accurate analysis of fetal cells by interphase fluorescence in situ hybridisation (FISH). To overcome this problem, we used simultaneous immunophenotyping of cells with the mouse antifetal haemoglobin antibody, UCHy, combined with FISH analysis using chromosome X and Y specific DNA probes. This approach enables selective FISH analysis of fetal cells within an excess of maternal cells. Furthermore, we have confirmed the potential of the method for clinical practice by a pilot prospective study of fetal sex in women referred for amniocentesis between 13 and 17 weeks of gestation.

The multi tumor suppressor genes MTS1 (CDKN2 p16 INK4A) and MTS2 (CDKN1, p15 INK4B ) located at 9p21-22 are inactivated in some human cancers via several mechanisms including deletion and hypermethylation. We have investigated the deletion and methylation status of MTS1 and MTS2 in childhood acute lymphoblastic leukemia (ALL) of both T-cell (17 cases) and B-cell phenotypes (29 cases), and p16INK4A and p15 INK4B mRNA expression in 36 of these cases. Biallelic or monoallelic loss of both MTS1 and MTS2 was observed in 12 cases of B-ALL and nine cases of T-ALL. Two cases of T-ALL showed deletion of MTS1 but not MTS2. The 5' CpG region of MTS2 was hypermethylated in 12 cases of precursor B-ALL and eight cases of T-ALL but no hypermethylation was found in the 5' CpG region of MTS1. All cases with homozygous deletion of MTS1 or MTS2 had no or low levels of mRNA expression and similar low levels of expression were found in cases in which MTS2 was present but fully methylated. Thus hypermethylation of MTS2, in contrast to MTS1, is frequent in childhood ALL. Furthermore our data show that although inactivation of MTS1 by deletion is common, inactivation of MTS2 by a combination of deletion and hypermethylation is more frequent in both B-ALL (20/29, 69%) and T-ALL (17/ 17, 100%). This suggests that both MTS1 and MTS2 are important targets of the 9p21-22 deletion.

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Through differential screening of established human leukaemia cell lines, we have identified and molecularly cloned lymphopain, a novel cysteine proteinase of the papain family. Lymphopain exhibits a remarkably restricted cellular pattern of expression, being predominantly expressed in cytotoxic T-lymphocytes and natural killer cells. The human lymphopain locus maps to chromosome 11q13, encodes a polypeptide of 376 amino acids and is conserved in the mouse. Both human and murine forms appear more closely related to protozoan papainlike enzymes than to other mammalian members of the papain family. The cellular distribution of lymphopain expression, together with the functional demonstration of lymphopainassociated proteinase activity in vitro, is suggestive of a role for lymphopain in immune cell-mediated, cell killing.

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