Detection of MYCN gene amplification in neuroblastoma by fluorescence in situ hybridization: a pediatric oncology group study.

To assess the utility of fluorescence in situ hybridization (FISH) for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (< or =30% replacement) in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker.

The p53-inducible gene EI24/PIG8 localizes to human chromosome 11q23 and the proximal region of mouse chromosome 9.

Activation of the p53 tumor suppressor leads to either a cell cycle arrest or to apoptosis and the factors that influence these responses are poorly understood. It is clear, however, that p53 regulates these processes by inducing a series of downstream target genes. One recently identified p53-target gene, EI24 (alias PIG8), induces apoptosis when ectopically expressed. To better understand the biological properties of EI24 and its potential relevance to disease, in particular cancer, we determined the chromosomal location and pattern of gene expression of EI24. EI24 is widely expressed in adult tissues and throughout mouse embryogenesis. The genomic locus of EI24 was mapped to the proximal region of mouse chromosome 9 and human chromosome 11q23-->q24, a region frequently altered in human cancers. These results suggest that EI24 may play an important role in the p53 tumor suppressor pathway.

Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN.

Caspase 8 is a cysteine protease regulated in both a death-receptor-dependent and -independent manner during apoptosis. Here, we report that the gene for caspase 8 is frequently inactivated in neuroblastoma, a childhood tumor of the peripheral nervous system. The gene is silenced through DNA methylation as well as through gene deletion. Complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplification of the oncogene MYCN. Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme. Thus, caspase 8 acts as a tumor suppressor in neuroblastomas with amplification of MYCN.

Cloning and chromosomal localization of the gene encoding human cyclin D-binding Myb-like protein (hDMP1).

The murine transcription factor murine cyclin D-binding Myb-like protein (mDmp1) arrests the cell cycle in G1 phase, through an activity that can be overridden by direct interaction with the D-type cyclins. Here, we describe the identification, sequence, chromosomal localization, and expression of the human cognate, hDMP1. The hDMP1 cDNA contains a 2280bp open reading frame that shares a high degree of identity with the mDmp1 coding region. The 4.4kb hDMP1 messenger RNA is ubiquitously expressed in normal human tissues, with highest levels in testis and substructures within the brain. By use of fluorescence in situ hybridization with a human genomic P1 probe, we assigned hDMP1 to chromosome 7, band q21. This chromosomal region is frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of human acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We analyzed hDMP1 copy number by fluorescence in situ hybridization in leukemic blasts from nine patients with abnormalities of the long arm of chromosome 7, and in each case one allele of the hDMP1 gene was deleted. Functional analysis of the mDmp1 protein has shown that it negatively regulates cell proliferation, which suggests that this gene is a candidate suppressor of malignant transformation. Further study will be needed to determine whether gene-specific mutations implicate hDMP1 as a tumor suppressor in acute leukemias with deletions of the long arm of chromosome 7 or in other types of human malignancy.

Lack of homozygously inactivated p73 in single-copy MYCN primary neuroblastomas and neuroblastoma cell lines.

We examined 18 neuroblastoma cell lines and 32 primary single-copy MYCN tumor specimens to determine whether mutations of p73, a novel p53-related gene located in chromosome band 1p36.33, contribute to the genesis or progression of childhood neuroblastoma. By fluorescence in situ hybridization, 16 of the 18 cell lines, but only 3 of the 32 primary tumors, had evidence of a deleted p73 allele. Sequence analysis of the p73 coding region in the mRNAs expressed by these cell lines and tumors did not reveal inactivating mutations, suggesting that p73 is not homozygously inactivated in neuroblastoma. However, several novel splice forms of p73 mRNAs were identified, including one without exon 11 that predominated in multiple MYCN-amplified cell lines. Its encoded p73 protein differed from other splice forms in that the C-terminus was derived from an alternative reading frame. Further study of the functional properties of the protein encoded by this splice form of p73 will be needed to determine whether it contributes to the pathogenesis of childhood neuroblastoma with MYCN gene amplification.

The human erythroid-specific transcription factor EKLF localizes to chromosome 19p13.12-p13.13.

Erythroid Krüppel-like factor (EKLF) is a trans-acting factor that binds specifically to the beta-globin promoter CACCC box. EKLF is essential for adult erythroid development in mice. We have cloned and sequenced a cDNA encoding the human homologue of this gene, which shares 69% identity with the mEKLF protein. The expression of hEKLF is restricted to fetal liver and adult bone marrow. The genomic locus of hEKLF was mapped to chromosomal band 19p13.12-p13.13, using fluorescence in situ hybridization.

Molecular cloning, expression pattern, and chromosomal localization of human CDKN2D/INK4d, an inhibitor of cyclin D-dependent kinases.

Progression through the G1 phase of the cell cycle is dependent on the activity of holoenzymes formed between D-type cyclins and their catalytic partners, the cyclin-dependent kinases cdk4 and cdk6. p16INK4a, p15INK4b, and p18INK4c, a group of structurally related proteins, function as specific inhibitors of the cyclin D-dependent kinases and are likely to play physiologic roles as specific regulators of these kinases in vivo. A new member of the INK4 gene family, murine INK4d, has recently been identified. Here we report the isolation of human INK4d (gene symbol CDKN2D), which is 86% identical at the amino acid level to the murine clone and approximately 44% identical to each of the other human INK4 family members. The INK4d gene is ubiquitously expressed as a single 1.4-kb mRNA with the highest levels detected in thymus, spleen, peripheral blood leukocytes, fetal liver, brain, and testes. The abundance of INK4d mRNA oscillates in a cell-cycle-dependent manner with expression lowest at mid G1 and maximal during S phase. Using a P1-phage genomic clone of INK4d for fluorescence in situ hybridization analysis, the location of this gene was mapped to chromosome 19p13. No rearrangements or deletions of the INK4d gene were observed in Southern blot analysis of selected cases of pediatric acute lymphoblastic leukemia (ALL) containing a variant (1;19)(q23;p13) translocation that lacks rearrangement of either E2A or PBX1, or in ALL cases containing homozygous or hemizygous deletions of the related genes, INK4a and INK4b.

Cloning of genomic loci and chromosomal localization of the human PCTAIRE-1 and -3 protein kinase genes.

Recent studies on the molecular mechanisms controlling the mammalian cell cycle have disclosed a large family of cdc2-related serine/threonine kinases. Among this gene family, the PCTAIRE protein kinases comprise a distinct subfamily of unknown cellular function. To analyze the genomic structure and chromosomal location of the PCTAIRE-1 and -3 genes, we isolated human cosmid clones for each gene by screening a human genomic library with murine PCTAIRE cDNA probes. Overlapping clones encompassing approximately 60 kb of genomic DNA were obtained for both PCTAIRE-1 and -3. These clones were confirmed to encode authentic PCTAIRE genes by the detection of exon-intron structures and the coincidence of the nucleotide sequence of exons to that of the published human cDNAs. Using these cosmid clones as probes for FISH analyses, the chromosomal loci for PCTAIRE-1 and PCTAIRE-3 were assigned to bands Xp11 and 1q31-q32, respectively.

Localization of the gene encoding human BiP/GRP78, the endoplasmic reticulum cognate of the HSP70 family, to chromosome 9q34.

BiP/GRP78 is a member of the HSP70 family involved in the folding and assembly of proteins in the endoplasmic reticulum. Using PCR amplification of DNA from human x rodent somatic hybrids that segregate human chromosomes in conjunction with fluorescence in situ hybridization, we have assigned GRP78 to chromosome 9q34. This is in agreement with the localization of murine and bovine homologues based on the high degree of synteny in this region. Several interesting genes and disorders map to this region and are discussed.