Miki Ohira

High expression of Survivin, mapped to 17q25, is significantly associated with poor prognostic factors and promotes cell survival in human neuroblastoma

Survivin (SVV) is a family member of inhibitor of apoptosis proteins (IAPs) and its expression is cell cycle regulated. The gene is mapped to chromosome 17q25, the region of which is frequently gained in advanced stages of neuroblastoma (NBL). However, the role of SVV in NBL is poorly understood. Here we studied the clinical and biological role of SVV in NBL. A 1.9u2009kb SVV transcript was expressed in all of 9 NBL cell lines at higher levels than those in adult cancer cell lines. In 34 primary NBLs, high levels of SVV expression was significantly associated with age greater than 12 months (two sample t-test: P=0.0003), advanced stages (P=0.0136), sporadic tumors (P=0.0027) and low levels of TrkA expression (P=0.0030). In NBL cell lines, SVV mRNA expression was dramatically down-regulated in CHP134 and IMR32 cells undergoing apoptosis after treatment with all-trans retinoic acid (RA) or serum deprivation. It was only moderately decreased in cells (SH-SY5Y and CHP901) undergoing RA-induced differentiation. On the other hand, in proliferating NBL cells or RA-treated SK-N-AS line which is refractory to RA, the SVV mRNA remained at steady state levels or rather up-regulated. Furthermore, transfection of SVV into CHP134 cells induced remarkable inhibition of the RA-induced apoptosis. Collectively, our results suggest that high expression of SVV is a strong prognostic indicator for the advanced stage neuroblastomas, and that it could be one of the candidate genes for the 17q gain.

p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent

p73, a novel p53 family member, is a recently identified candidate neuroblastoma (NBL) suppressor gene mapped at chromosome 1p36.33 and was found to inhibit growth and induce apoptosis in cell lines. To test the hypothesis that p73 is a NBL suppressor gene, we analysed the p73 gene in primary human NBLs. Loss of heterozygosity (LOH) for p73 was observed in 19% (28/151) of informative cases which included 92 mass-screening (MS) tumors. The high frequency of p73 LOH was significantly associated with sporadic NBLs (9% vs 34%, P<0.001), N-myc amplification (10% vs 71%, P<0.001), and advanced stage (14% vs 28%, P<0.05). Both p73α and p73β transcripts were detectable in only 46 of 134 (34%) NBLs at low levels by RTu2009–u2009PCR methods, while they were easily detectable in most breast cancers and colorectal cancers under the same conditions. They found no correlation between p73 LOH and its expression levels (P>0.1). We found two mutations out of 140 NBLs, one somatic and one germline, which result in amino acid substitutions in the C-terminal region of p73 which may affect transactivation functions, though, in the same tumor samples, no mutation of the p53 gene was observed as reported previously. These results suggest that allelic loss of the p73 gene may be a later event in NBL tumorigenesis. However, p73 is infrequently mutated in primary NBLs and may hardly function as a tumor suppressor in a classic Knudsons 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 500u2009kb. 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 3½ years (HDNB1/UFD2, KIAA0591F/KIF1B-β, 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-β, 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.

Recent trends in microRNA research into breast cancer with particular focus on the associations between microRNAs and intrinsic subtypes.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate the function of target genes at the post-transcriptional phase. miRNAs are considered to have roles in the development, progression and metastasis of cancer. Recent studies have indicated that particular miRNA signatures are correlated with tumor aggressiveness, response to drug therapy and patient outcome in breast cancer. On the other hand, in routine clinical practice, the treatment regimens for breast cancer are determined based on the intrinsic subtype of the primary tumor. Previous studies have shown that miRNA expression profiles of each intrinsic subtypes of breast cancer differ. In hormone receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer, miRNA expressions are found to be correlated with endocrine therapy resistance, progesterone receptor expression and heat shock protein activity. Some miRNAs are associated with resistance to HER2-targeted therapy and HER3 expression in HER2-positive breast cancer. In triple-negative breast cancer, miRNA expressions are found to be associated with BRCA mutations, immune system, epithelial–mesenchymal transition, cancer stem cell properties and androgen receptor expression. As it has been clarified that the expression levels and functions of miRNA differ among the various subtypes of breast cancer, and it is necessary to take account of the characteristics of each breast cancer subtype during research into the roles of miRNA in breast cancer. In addition, the discovery of the roles played by miRNAs in breast cancer might provide new opportunities for the development of novel strategies for diagnosing and treating breast cancer.

Structure, expression profile and chromosomal location of an isolog of DNA-PKcs interacting protein (KIP) gene.

A novel DNA-PKcs interacting protein, KIP (kinase interacting protein), was recently isolated using a two-hybrid analysis which showed a significant homology to calcineurin B. We found other ESTs showing significant similarity to KIP gene in the dbEST database and isolated a cDNA clone which encodes a 187 amino acid polypeptide from a human fetal brain cDNA library. This protein (termed KIP2 for kinase interacting protein 2) has sequence homology to KIP (46% identical and 64% similarity). RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-based analysis with a radiation hybrid cell panel and fluorescence in situ hybridization, the gene was localized to the q24 region of chromosome 15.

Isolation and chromosomal assignment of human genes encoding cofactor of LIM homeodomain proteins, CLIM1 and CLIM2

AbstractCofactors of LIM homeodomain proteins (CLIM) are transcriptional activators that associate with the LIM homeoproteins and coordinate transcription. LIM homeoproteins and CLIMs are involved in a variety of developmental processes. Two CLIMs, CLIM1 and CLIM2, have been identified in the mouse. Here we report the isolation of human CLIM1 and CLIM2 cDNAs and the determination of their chromosome locations by using a human-rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. The proteins deduced from human CLIM1 and CLIM2 cDNAs were composed of 373 and 375 amino acids, respectively, and had 97.3% and 98.7% amino acid identity, respectively, to their mouse counterparts. Human CLIM1 and CLIM2 proteins were 75.5% identical. Human CLIM1 and CLIM2 genes were mapped to the chromosome on 4p15.3 and 10q24–q25 regions, respectively. Mapping of a pair of developmentally important genes may provide new clues to the understanding of genetic disorders caused by these chromosome regions.

Analysis of loss of heterozygosity at 16p12-p13 (familial neuroblastoma locus) in 470 neuroblastomas including both sporadic and mass screening tumors

BACKGROUNDnNeuroblastoma (NBL) in children usually occurs in a sporadic form. However, it rarely occurs in families. Recently, the familial neuroblastoma (FNB) locus has been mapped to 16p12-p13 by linkage analysis.nnnPROCEDUREnHere we show the result of loss of heterozygosity in the region spanning 16p12-p13 (D16S406-D16S409, 46 cM) in 470 NBLs including both sporadic and mass screening cases.nnnRESULTSnAllelic loss was found in 61(13%) tumors. Deletion of 16p was associated with mass screening tumor (P = 0.035) and <1 year of age at diagnosis (P = 0.048). We found two commonly deleted regions: the sizes of the region were approximately 2 cM and approximately 6 cM.nnnCONCLUSIONSnOur data suggested that allelic loss of 16p was common in favorable NBLs, and there may be at least two candidate loci within the region of FNB.

Hunting the subset-specific genes of neuroblastoma: Expression profiling and differential screening of the full-length-enriched oligo-capping cDNA libraries

BACKGROUNDnNeuroblastoma (NBL) has a distinct nature in different prognostic subgroups.nnnPROCEDUREnTo understand the molecular mechanism of NBLs genesis and biology as well as that of the neural crest development, we constructed full-length-enriched cDNA libraries by an oligo-capping method from two different subsets of primary NBL, one with favorable biology and the other with MYCN amplification.nnnRESULTSnSequencing analysis of these libraries revealed that the expression profile was markedly different between both subsets. To identify the genes differentially expressed between the subsets, semi-quantitative RT-PCR analyses are proceeding.nnnCONCLUSIONnSo far, 54 transcripts have been found to be expressed at high levels in favorable NBLs, and significantly at low levels in unfavorable NBLs.

Assignment of the E4TF1-60 gene to human chromosome 21q21.2–q21.3 ☆

The gene encoding human transcription factor E4TF1-60 was previously mapped to chromosome 21q21. We analyzed the localization of the E4TF1-60 gene in more detail by genomic Southern hybridization and determined the sequence of the exons and the regions surrounding the intron boundaries. We report here that E4TF1-60 locates in the long arm of chromosome 21 at q21.2-q21.3 and contains a total of ten exons.

Identification of the homozygously deleted region at chromosome 1p36.2 in human neuroblastoma

BACKGROUNDnWe have identified for the first time a homozygously deleted region within the smallest region of overlap at 1p36.2-3 in two neuroblastoma cell lines.nnnPROCEDUREnThe 800-kb PAC contig covering the entire homozygously deleted region was made and sequenced. To date, approximately 70% of sequencing has been accomplished, and the estimated length of the deleted region was 500 kb.nnnRESULTSnCurrently, we have found six genes within the region, which include three known genes as well as three other genes that have been reported during processing of our present project for the last 3(1/2) years. We report here the results of expression and mutation analyses of those genes.nnnCONCLUSIONSnFull sequencing for the region of homozygous deletion as well as further analyses of the genes mapped within the region may reveal whether or not there is a neuroblastoma suppressor gene as proposed by the Knudsons two-hit hypothesis.