Identification of the full-length KIAA0591 gene encoding a novel kinesin-related protein which is mapped to the neuroblastoma suppressor gene locus at 1p36.2

The distal region of a short arm of chromosome 1p is frequently deleted in many human cancers including neuroblastoma (NBL), in which it has been narrowed down to the smallest region of overlap between D1S244 and D1S214 (approximately 7 cM). During the search for the candidate tumor suppressor genes mapped within the region, we found the KIAA0591 gene which encoded a new human kinesin-related protein with a homology to human axonal transporter of synaptic vesicles (ATSV). The kinesin is an intracellular motor protein and often associated with neuronal differentiation and survival. Here we identified a complete open reading frame of the KIAA0591 gene by screening a cDNA library derived from human substantia nigra. The KIAA0591 protein contains a possible pleckstrin homology (PH) domain at its carboxy-terminus. However, it did not possess a force-generating motor domain which is well conserved among kinesin superfamily members (KIFs). Northern blot analysis demonstrated that KIAA0591 mRNA was preferentially expressed in both adult and fetal brains, kidney, skeletal muscle and pancreas. KIAA0591 was expressed in favorable NBLs at higher levels than in unfavorable NBLs, although RT-PCR SSCP analysis showed no mutation within the coding region of the KIAA0591 gene, when 8 neuroblastoma tissues and 15 neuroblastoma-derived cell lines were examined. Thus, the full-length KIAA0591 gene may be a novel member of human KIF superfamily which lacks motor domain and might function as a tumor suppressor in an epigenetic but not a classic Knudson's manner.     

Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Oncogene (2000) 19, 4302 - 4307     

Genomic organization of the canine p53 gene and its mutational status in canine mammary neoplasia

To determine whether canine malignancies share common genetic lesions with their human counterparts, and are thus potentially interesting model systems in which to pose questions regarding tumor etiology and progression, we have elucidated the entire exon/intron structure of the canine p53 gene. A search for p53 gene abnormalities in mammary tumor tissue was undertaken utilizing single strand conformation polymorphism analysis. Mutations were detected in exons 4, 5, 6, and 7 of the p53 gene and consisted of nonsense, splicing, and frameshift mutations. None of 11 benign tumors and 6 of 40 primary carcinomas (15%) were found to harbor subtle p53 mutations. In 14 carcinomas examined the results in primary tumors and metastases were the same. These findings implicate involvement of this gene in the genesis of some malignant canine tumors, in a fashion similar to their human counterparts.     

Analyses of apoptotic regulators CASP9 and DFFA at 1P36.2, reveal rare allele variants in human neuroblastoma tumours

The genes encoding Caspase-9 and DFF45 have both recently been mapped to chromosome region 1p36.2, that is a region alleged to involve one or several tumour suppressor genes in neuroblastoma tumours. This study presents an update contig of the 'Smallest Region of Overlap of deletions' in Scandinavian neuroblastoma tumours and suggests that DFF45 is localized in the region. The genomic organization of the human DFF45 gene, deduced by in-silico comparisons of DNA sequences, is described for the first time in this paper. In the present study 44 primary tumours were screened for mutation by analysis of the genomic sequences of the genes. In two out of the 44 tumours this detected in the DFFA gene one rare allele variant that caused a non-polar to a polar amino acid exchange in a preserved hydrophobic patch of DFF45. One case was hemizygous due to deletion of the more common allele of this polymorphism. Out of 194 normal control alleles only one was found to carry this variant allele, so in respect of it, no healthy control individual out of 97 was homozygous. Moreover, our RT-PCR expression studies showed that DFF45 is preferably expressed in low-stage neuroblastoma tumours and to a lesser degree in high-stage neuroblastomas. We conclude that although coding mutations of Caspase-9 and DFF45 are infrequent in neuroblastoma tumours, our discovery of a rare allele in two neuroblastoma cases should be taken to warrant further studies of the role of DFF45 in neuroblastoma genetics.     

Mutational study of the 1p located genes p18ink4c, Patched-2, RIZ1 and KIF1B in oligodendrogliomas

Loss of 1p heterozygosity is one of the most characteristic events in oligodendrogliomas. Several genes located in this region have been previously studied to find the target gene implicated in the development of this tumor without success. Patched-2, RIZ1 and KIF1B are novel oncosuppressor genes located at 1p and involved in different kinds of tumors. We have studied these genes and p18(ink4c) using PCR/SSCP methods to detect sequence variations in a series of 40 oligodendrogliomas in which the allelic status at 1p was analyzed. Polymorphisms or no sequence changes were detected in all four genes analyzed. None of the genes analyzed seem to be the target-gene mapped at 1p involved by mutation in oligodendroglioma development.     

Frequent loss of heterozygosity in the region of D1S450 at 1p36.2 in myelodysplastic syndromes

To understand the underlying mechanisms in myelodysplastic syndromes (MDS) by identifying target tumor suppressor genes, we performed a detailed deletional mapping of the short arm of chromosome 1 in 38 paired samples of bone marrow and peripheral blood obtained from individuals with MDS by PCR amplification of a total of 23 highly informative microsatellite sequences. We identified the commonly deleted region between D1S508 and D1S244. LOH of this region was found in five patients (13%). In addition, LOH at 1p was associated with a poor clinical outcome, suggesting that the deletion of a gene in this region may be involved in the course of this disease. By analyzing the chromosomal map of this region, we found TNFRSF12 as a candidate tumor suppressor gene. However, our search for mutations in this gene did not identify somatic mutations in MDS. Our findings are consistent with the possible existence of an as-yet unknown tumor suppressor gene in this region that is altered in MDS.     

Functional evidence for a nasopharyngeal carcinoma-related gene BCAT1 located at 12p12

Nasopharyngeal carcinoma (NPC) is a malignancy that is prevalent among populations from Southeast Asia. The carcinogenesis of NPC is thought to be a multistep process involving several genetic changes. Our previous study based on distance and branching-tree models for NPC carcinogenesis indicated +12p11-p12 was an early event and should play an important role in NPC development. To understand the role of +12p11-p12 as the tree model predicted and evaluate which gene located at 12p11-p12 might be involved in NPC development, semiquantitative RT-PCR was applied to examine the expression status of 18 genes selected from 12p11-p12 in 36 NPC and 8 normal nasopharynx (NP) biopsies. The results revealed that BCAT1, KCNJ8, PTX1, and KRAS2 genes were overexpressed in NPC tissues and BCAT1 was of particular interest based on its function reported in other tumors. To further elucidate the function of BCAT1 gene in NPC, BCAT1 expression was specifically suppressed in 5-8F NPC cell line by RNA interference (RNAi), confirmed by RT-PCR and Western blotting. As expected, the depletion of BCAT1 could effectively block the proliferation of NPC cells. The BCAT1 identified in the amplified 12p11-p12 region may play a certain role in NPC development.     

Genomic organization of the human WT1 gene.

We have analyzed the genomic structure of the human WT1 gene, one of the recessive oncogenes for Wilms' tumor at chromosome 11p13. By analyses of three cosmids covering the WT1 gene as well as products generated by polymerase chain reaction, cleavage sites for 10 restriction enzymes were mapped in a region of about 80 kb, and the positions of 10 exons were defined. We also mapped two polymorphic sites for TaqI. Our genomic map will be useful to analyze DNA abnormalities sometimes found in the tumors, as well as loss of heterozygosity.     

Genomic structure of the human beta-PIX gene and its alteration in gastric cancer

beta-PIX, a newly identified p21-activated kinase (PAK)-interacting exchange factors (PIX), encodes a guanine nucleotide exchange factor for Rho guanosine triphosphatases. Characterization of beta-PIX gene was performed using the BAC Library method. The beta-PIX gene has 17 exons and an A/T polymorphism at the 32nd base upstream of the intron/exon junction of exon 7. The frequencies of genotypes A/T, A/A and T/T were 23.6% (13/55), 72.7% (40/55) and 3.6% (2/55), respectively; these frequencies are in Hardy-Weinberg equilibrium. Two out of 14 informative tumors (14.3%) were shown to have lost their heterozygosity at this locus, but no mutations in the remaining alleles were detected. In addition, we examined the gene-expression profile in another set of 30 gastric samples, but no significant over-expression of either the beta-PIX gene or the alpha-PIX gene was found. Though the beta-PIX gene has been speculated to potentially have tumor-related biological characteristics, the findings of the present study suggest that the involvement of beta-PIX gene in human gastric carcinogenesis is minimal.     

Genomic structure of the human ING1 gene and tumor-specific mutations detected in head and neck squamous cell carcinomas

We characterized the genomic structure of the human ING1 gene, a candidate tumor suppressor gene, and found that the gene has three exons. We also demonstrated that four mRNA variants were transcribed from three different promoter regions. Of 34 informative cases of head and neck squamous cell carcinoma, 68% of tumors showed loss of heterozygosity at chromosome 13q33-34, where the ING1 gene is located. Here we present the first report that three missense mutations and three silent changes were detected in the ING1 gene in 6 of 23 tumors with allelic loss at the 13q33-34 region. These missense mutations were found within the PHD finger domain and nuclear localization motif in ING1 protein, probably abrogating the normal function.     

Methylation-independent silencing of the p73 gene in neuroblastoma

p73 is a p53 homolog that, in vitro, inhibits cell growth and induce apoptosis. In some tumors p73 is monoallelically expressed and this raised the possibility that this gene is subjected to imprinting. Silencing of p73 in acute leukemia and in Burkitt's lymphoma occurs in association with the aberrant methylation of the first exon of the gene. We have analysed the methylation pattern of the p73 promoter and of upstream and downstream sequences in neuroblastoma. Our results demonstrate that p73 expression in this tumor is not regulated by methylation. We concluded that it is unlikely that p73 is imprinted in neuroblastoma and that the methylation-dependent silencing of this gene, thus far, is a characteristic of hematologic malignancies. Oncogene (2000) 19, 4553 - 4556.     

Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens

In lung and liver cancers, p53 mutations are mostly G:C to T:A transversions. This type of mutation is known to be induced by benzo(a)pyrene and aflatoxin B1 which are associated with the etiology of lung and liver cancers, respectively. Using a novel assay based on DNA polymerase fingerprint analysis, we identified p53 nucleotides targeted by these carcinogens. Thirteen of 14 nucleotide residues of the p53 gene which underwent G:C to T:A mutations in lung cancers were targeted by benzo(a)pyrene. Similarly, aflatoxin B1 formed adducts at a mutational hotspot specific for liver cancer. The same nucleotide (third base of codon 249), which mutates rarely in lung cancers, was not a target for benzo(a)pyrene. These in vitro observations indicate that p53 mutational hotspots identified in different tumors are selected targets specifically for the etiologically defined environmental carcinogens.     

Deletion mapping of chromosomes 14q and 1p in human neuroblastoma.

It has been suggested that loss of heterozygosity (LOH) on the short arm of chromosome 1 is a critical event for the development of neuroblastoma, and we have previously shown frequent LOH on chromosome 14 in neuroblastoma. To pursue these observations, especially to define further the regions which are commonly deleted in the tumor, we examined for allelic losses in 27 cases of neuroblastomas by using a number of polymorphic DNA markers for chromosomes 14q and 1p. LOH was observed in 10 out of the 25 informative cases (40%) on chromosome 14q and in eight out of the 21 informative cases (38%) on 1p. The commonly deleted regions were distal to the D14S13 locus (14q32-qter) on chromosome 14 and distal to the D1S112 locus (1p36.1-pter) on chromosome 1. These results strongly suggest that tumor-suppressor genes important in the pathogenesis of human neuroblastoma are located on the distal part of both chromosomes 14q and 1p.     

The gene for a new brain specific RhoA exchange factor maps to the highly unstable chromosomal region 1p36.2-1p36.3.

Guanine nucleotide exchange factors from the Dbl family are proto-oncogenic proteins that activate small GTPases of the Rho family. Here we report the characterization of GEF720, a novel Dbl-like protein related to p115Rho-GEF. GEF720 activated RhoA both in our recently developed Yeast Exchange Assay and in biochemical in vitro exchange assays. GEF720 induced RhoA dependent assembly of actin stress fibers in REF52 fibroblastic cells. In NIH3T3 cells this Dbl-like protein elicited formation of transformation foci with a morphology similar to RhoA-V14 induced foci. In the PC12 neuron-like cell line, expression of GEF720, whose mRNA is brain specific, inhibited NGF-induced neurite outgrowth. Finally, GEF720 gene is located on human chromosome 1 on band 1p36, between Tumor Protein 73 and Tumor Necrosis Factor Receptor 12, two genes rearranged in many neuroblastoma cell lines. Together, these results show that this new Dbl related protein, GEF720, is an exchange factor that can directly activate RhoA in vivo and is potentially involved in the control of neuronal cell differentiation. GEF720 is also a new candidate gene involved in the progression of neuroblastoma and developmental abnormalities associated with rearrangements in the 1p36 chromosomal region.