Molecular mapping of chromosome 2 deletions in murine radiation‐induced AML localizes a putative tumor suppressor gene to a 1.0 cM region homologous to human chromosome segment 11p11–12

Abstract

Radiation-induced acute myeloid leukemias (AMLs) in the mouse are characterized by chromosome 2 deletions. Previous studies showed that a minimal deleted region (mdr) of approximately 6.5 cM is lost from one homologue in chromosome 2-deleted AMLs. An AML tumor suppressor gene is proposed to map within this mdr. In this study, we refine the mdr to a I cM interval between markers D2Mit126 and D2Mit185 by microsatellite analysis of 21 primary radiation-induced F I AMLs. The construction of a partial yeast artificial chromosome (YAC) contig spanning the mdr and the location of six known genes indicated that the 1 cM mdr is homologous to human 11p11-12, a region implicated in some human AMLs. Screening of five cell lines derived from primary radiation-induced AMLs for homozygous loss of microsatellites and genes mapping within the mdr revealed loss of both copies of the hemopoietic tissue-specific transcription factor Sfpi1(PU.1/Spi1) in one cell line. Studies of primary and F1 AMLs failed to implicate Sfpi1 as the AML tumor suppressor gene. YAC contig construction, together with data suggesting that the critical gene flanks Sfpi1, represents significant progress toward identifying an AML tumor suppressor gene.

Mutations of the PU.1 Ets domain are specifically associated with murine radiation-induced, but not human therapy-related, acute myeloid leukaemia

“…One cell line (Mlp3) out of five lines derived from induced leukaemias had lost both copies abrogating PU.1 expression. PU.1 was found to be transcribed in all the F1 AMLs and cell lines tested with the exception of the Mlp3 cell line, which was null (Silver et al, 1999).…”

“…A mouse model of radiation-induced acute myeloid leukaemia (AML) has been used to localize a putative tumour suppressor gene to a 1.0 cM region homologous to the human chromosomal segment 11p11-p12 (Silver et al, 1999). PU.1 (Sfpi1) maps to this chromosomal region and the majority (>90%) of induced AMLs were found to have lost one copy of the gene.…”

“…In murine AMLs (n ¼ 45) induced by Xirradiation in F1 hybrids (CBA/H  C57BL/Lia and C3H/He  C57BL/6J) and the C3H/He inbred mouse (Seki et al, 1991;Silver et al, 1999), we identified a high frequency (67%, 30/45) of missense mutation leading to replacement of the arginine 235 (R235) residue within the Ets domain of PU.1 with cysteine (C; 49%, 22/45), serine (S; 16%, 7/45) or histidine (H; 2%, 1/45; …”

“…Normal DNA included matched normal brain DNA (n ¼ 23) and DNA from parents of F1's (n ¼ 8). F1 AMLs induced in CBA/H Â C57BL/Lia (n ¼ 27), including those referred to in Silver et al (1999), were checked for LOH for the PU.1 chromosomal segment using flanking microsatellites D2mit15, D2mit383, D2mit272 and D2mit44, and fluorescencebased genotyping on an ABI3100 sequencer Sussex, UK) made up to 25 ml using demineralized water. The PCR reaction conditions: 951C for 10 min, one cycle; 35 cycles of 951C for 35 s, annealing at optimum temperature for 35 s, and 721C for 35 s; and 721C for 10 min, one cycle.…”

Accommodating haploinsufficient tumour suppressor genes in Knudson's model

“…Haplosu ciency of tsg, and strong and early e ects of loss of their function during tumour development will promote homozygous loss, whereas haploinsu ciency, and weak and late e ects of loss will promote hemizygous loss chromosome 2 (Alexander et al, 1995;Silver et al, 1999). Somatic losses of the tsg at 5q and 7q tend to occur late in tumour development, often as a consequence of therapy for a pre-existing malignancy (Johansson et al, 1994;Pedersen-Bjergaard et al, 1995).…”