Genomic organization and mutation analysis of Hel-N1 in lung cancers with chromosome 9p21 deletions

Allelic loss of chromosome 9p21 is common in small cell lung cancer (SCLC), but inactivation of the tumor suppressor gene CDKN2a is rare, implying the existence of another target gene at 9p21. A recent deletion mapping study of chromosome 9p has also identified a site of deletion in non-small cell lung cancer (NSCLC) centered around D9S126. The Hel-N1 (human elav-like neuronal protein 1) gene encodes a neural-specific RNA binding protein that is expressed in SCLC. We have mapped this potentially important gene in lung tumorigenesis to within 100 kb of the D9S126 marker at chromosome band 9p21 by using homozygously deleted tumor cell lines and fluorescence in situ hybridization to normal metaphase spreads. Hel-N1 is, therefore, a candidate target suppressor gene in both SCLC and NSCLC. We have determined the genomic organization and intron/exon boundaries of Hel-N1 and have screened the entire coding region for mutations by sequencing 14 primary SCLCs and cell lines and 21 primary NSCLCs preselected for localized 9p21 deletion or monosomy of chromosome 9. A homozygous deletion including Hel-N1 and CDKN2a was found in a SCLC cell line, and a single-base polymorphism in exon 2 of Hel-N1 was observed in eight tumors. No somatic mutations of Hel-N1 were found in this panel of lung tumors. Hel-N1 does not appear to be a primary inactivation target of 9p21 deletion in lung cancer.     

Cloning, expression and chromosomal localization of Wnt-13, a novel member of the Wnt gene family

The Wnt genes, encoding structurally-related secreted glycoproteins, are implicated in mammary carcinogenesis induced by mouse mammary tumor virus. In search of the Wnt gene(s) expressed in human gastric cancer, a WTGC1 cDNA fragment sharing 66.9% amino-acid homology with human and mouse Wnt-2 was isolated by degenerate polymerase chain reaction. The human gene corresponding to WTGC1 was designated as Wnt-13 and overlapping Wnt-13 cDNAs were cloned. Nucleotide sequence analysis indicated that the Wnt-13 gene encodes the protein of 372 amino acids, including a signal peptide, two potential N-glycosylation sites and 24 cystein residues highly conserved among members of the Wnt gene family. The Wnt-13 mRNA of 2.5 kb in size was detected in heart, brain, placenta, lung, prostate, testis, ovary, small intestine and colon of adult human and also in brain, lung and kidney of fetal human. Among various cancer cell lines, the Wnt-13 mRNA was detected in HeLa (cervical cancer), MKN28 and MKN74 (gastric cancer). The Wnt-13 gene has been mapped to human chromosome 1p13. These results suggest that the Wnt-13 gene may be involved in normal human development or differentiation as well as in human carcinogenesis.     

Using a quantitative measure of communicative environment to compare two psychogeriatric day care settings

Class I HLA genes are expressed in almost all tissues, but expression is low or undetectable in many neuroblastomas. We analysed class I HLA methylation in normal tissues and in 28 neuroectodermal tumour cell lines. HLA-C is hypermethylated in normal adult tissues and 13 cell lines, while 15 cell lines show the hypomethylated phenotype. Hypomethylation of HLA-C strongly correlates with hemizygous deletion of a 9 cM interval on 1p35-36.1, suggesting that this region encodes a modifier of methylation for HLA-C. To test whether hypomethylation of class I HLA genes results from loss of a modifier gene, we fused a hypomethylating neuroblastoma cell line with a hypermethylating cell line. Methylation of class I HLA genes was induced in the hybrids. Furthermore, methylation of HLA-C, -E and -A genes, which are encoded in a 1.4 Mb region on 6p21, is correlated in most cell lines. Our results suggest that 1p35-36.1 encodes a modifier of methylation for class I HLA genes, that is deleted in many neuroblastomas.     

C/EBP alpha and C/EBPbeta play similar roles in the transcription of the human Cu/Zn SOD gene

Since alpha B-crystallin is known to be expressed in glial tissues of human brain and neuroectodermal tumors, the alpha B-crystallin content of neuroblastomas, may be related to the degree of glial or neuronal differentiation. The alpha B-crystallin content of 73 neuroblastomas, was determined by enzyme immunoassay. The concentration of alpha B-crystallin was examined in light of neuroblastoma prognostic factors. Neuroblastomas from patients who received chemotherapy (n = 23) contained higher concentrations of alpha B-crystallin than those from patients who did not receive chemotherapy (n = 50) (P > 0.05). There was a statistically significant difference in alpha B-crystallin concentrations in advanced stage patients who received preoperative chemotherapy (P < 0.01). Immunohistochemistry demonstrated alpha B-crystallin expression in the nerve-like fibers and a few ganglion-like cells. Staining was not apparent in the less differentiated cells in the tumor cell nest. alpha B-crystallin may play a role in the response to cellular stress in neuroblastoma.     

Co-amplification of a novel gene, NAG, with the N-myc gene in neuroblastoma

Substantial evidence implicates amplification of the N-myc gene with aggressive tumor growth and poor outcome in neuroblastoma. However some evidence suggests that this gene alone is not the sole determinant of outcome in N-myc amplified tumors. We have searched for genes that co-amplify with N-myc in neuroblastoma by means of two-dimensional analysis of genomic restriction digests. Using this approach, we have identified and cloned a novel genomic fragment which is co-amplified with N-myc in neuroblastomas. This fragment was mapped in close vicinity to N-myc on chromosome arm 2p24. It was amplified in 5/8 N-myc amplified neuroblastoma cell lines and in 9/13 N-myc amplified tumors. Using a PCR-based approach we isolated a 4.5 kb c-DNA sequence that is partly contained in the genomic fragment. The open reading frame of the cDNA encodes a predicted protein of 1353 amino acids (aa). The homology of the predicted protein, which we designated NAG (neuroblastoma amplified gene), to a C. elegans protein of as yet unknown function, and its ubiquitous expression suggest that NAG may serve an essential function. By Northern blot analysis we showed that amplification of the cloned gene correlates with over-expression in neuroblastoma cell lines. Amplification and consequent over-expression of NAG may, therefore, contribute to the phenotype of a subset of neuroblastomas.     

The p16 (CDKN2A) gene is involved in the growth of neuroblastoma cells and its expression is associated with prognosis of neuroblastoma patients

We previously reported that loss of heterozygosity (LOH) on chromosome 9p21 correlates with poor prognosis of neuroblastoma and the p16 gene is not expressed in approximately two thirds of neuroblastoma cell lines. Here we demonstrated that p16 expression was induced by 5-aza-2-deoxycytidine treatment in cell lines with 5' CpG island methylation but not in cell lines without methylation. Furthermore, the cell cycle of neuroblastoma cell lines significantly delayed with accumulation of cells in G1 phase by transfection of a wild-type p16 expression vector. These results indicate that p16 is inactivated in part by DNA methylation and its expression is involved in the growth of neuroblastoma cells in vitro. To assess the biological and clinical significance of p16 expression in primary tumors, we undertook immunohistochemical analysis in 74 paraffin sections of neuroblastomas. p16 protein was undetectable in 45 of 74 cases (61%) and lack of p16 expression significantly correlated with poor prognosis of patients and advanced stage of the disease. There was no correlation between loss of p16 expression and N-myc amplification in these tumors. These results indicate that inactivation of the p16 gene is involved in the progression of neuroblastoma independently of N-myc amplification.     

NRP/B, a novel nuclear matrix protein, associates with p110(RB) and is involved in neuronal differentiation

The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in the regulation of gene expression, the cell cycle, and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain-like structure in the predicted NH2 terminus, and a "kelch motif" in the predicted COOH-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes. NRP/B expression was upregulated during the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells. Overexpression of NRP/B in these cells augmented neuronal process formation. Treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as the neuronal process formation during neuronal differentiation of PC-12 cells. Since the hypophosphorylated form of retinoblastoma protein (p110(RB)) is found to be associated with the nuclear matrix and overexpression of p110(RB) induces neuronal differentiation, we investigated whether NRP/B is associated with p110(RB). Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and can bind to the functionally active hypophosphorylated form of the p110(RB) during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. Our studies indicate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110(RB) and participates in the regulation of neuronal process formation.