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High expression of SQSTM1/p62 (p62) protein, which functions as a hub of oncogenic signaling pathways, has been detected in several human cancers. However, the clinicopathological and functional contribution of p62 expression is largely unknown in endometrial cancers (ECs). In this study, we assessed the expression status of p62 in primary ECs (n = 194) by immunohistochemistry and analyzed its clinical significance. Although p62 was expressed in the cytoplasm and/or nucleus in primary ECs, we observed that an expression subtype, high expression of cytoplasmic p62 but low expression of nuclear p62 (cytoplasm(High)/nucleus(Low)), significantly correlated with nonendometrioid types (P = 0.002), high grade (P < 0.001), deep myometrial invasion (P = 0.025), vascular invasion (P = 0.012), and poor prognosis (P < 0.001), and may be an independent prognostic marker of ECs (P = 0.011). Furthermore, RNA interference-mediated inhibition of p62 expression in the HEC-1A EC cell line led to the reduction of invasiveness and resistance to oxidative stress in vitro, as well as the suppression of in vivo tumor growth in an orthotopic mouse model of ECs. High expression of cytoplasmic p62 is a novel prognostic biomarker of ECs, and excess p62 expression may functionally contribute to the acquirement of malignant phenotypes in EC cells., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The nuclear factor-kappaB (NF-kappaB) transcription factors play important roles in cancer development by preventing apoptosis and facilitating the tumor cell growth. However, the precise mechanisms by which NF-kappaB is constitutively activated in specific cancer cells remain largely unknown. In our current study, we now report that NF-kappaB-inducing kinase (NIK) is overexpressed at the pretranslational level in adult T-cell leukemia (ATL) and Hodgkin Reed-Sternberg cells (H-RS) that do not express viral regulatory proteins. The overexpression of NIK causes cell transformation in rat fibroblasts, which is abolished by a super-repressor form of IkappaBalpha. Notably, depletion of NIK in ATL cells by RNA interference reduces the DNA-binding activity of NF-kappaB and NF-kappaB-dependent transcriptional activity, and efficiently suppresses tumor growth in NOD/SCID/gammac(null) mice. These results indicate that the deregulated expression of NIK plays a critical role in constitutive NF-kappaB activation in ATL and H-RS cells, and suggest also that NIK is an attractive molecular target for cancer therapy.

Background/aims: We conducted an analysis of chromosomal numerical aberrations and their clinical significance in hepatocellular carcinoma., Methods: We analyzed 87 hepatocellular carcinomas by array-based comparative genomic hybridization with an array containing 800 bacterial artificial chromosome clones., Results: Frequent (>30%) chromosomal losses on 1p36.1, 4q21-25, 4q34-35.1, 8p23.3b-11.1, 13q14.1-14.3, 16p13.3, 16q22.1-24.3b, 17p13.3-13.1 and 17p13.3-11, and gains on 1q21-44f, 2q21.2, 2q34, 3q11.2, 5p14.2, 5q13.2-14, 7p22, 7p14.2, 7q21.1, 7q22.3, 7q34, 8q12-24.3 and 17q23, were observed. Recurrent (>5%) amplifications were detected on 1q25, 8q11 and 11q11, and we discovered a novel homozygous deletion at 14q32.11. The extent of chromosomal aberrations correlated significantly with various clinicopathological characteristics of the tumors, and increased in a stepwise manner with the progression of hepatocellular carcinoma. We also identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Multivariate analysis revealed that both chromosomal loss on 17p13.3 and gain on 8q11 are independent prognostic indicators., Conclusions: Our results contribute to a complete description of genomic structural aberrations in relation to hepatocarcinogenesis and provide a valuable basis from which we can begin to understand the characteristics of tumors, predict patient outcomes and discover novel therapeutic targets for hepatocellular carcinoma.

We investigated DNA copy number aberrations in 37 cell lines derived from multiple myelomas (MMs) using comparative genomic hybridization, and 11 (29.7%) showed high-level gain indicative of gene amplification at 1q12-q22. A corresponding transcriptional mapping using oligonucleotide arrays extracted three up-regulated genes (IRTA2, PDZK1, and S100A6) within the smallest region of overlapping in amplifications. Among them PDZK1 showed amplification and consequent overexpression in the MM cell lines. Amplification of PDZK1 was observed in primary cases of MM as well. MM cell lines with amplification of PDZK1 exhibited the resistance to melphalan-, cis-platin-, and vincristin-induced cell death compared with MM cell lines without its amplifications. Furthermore, down-regulation of PDZK1 with an anti-sense oligonucleotide sensitized a cell line KMS-11 to melphalan, cis-platin, and vincristin. Taken together, our results indicate that PDZK1 is likely to be one of targets for 1q12-q22 amplification in MM and may be associated with the malignant phenotype, including drug resistance, in this type of tumor.

SKP2, an F-box protein constituting the substrate recognition subunit of the SCF(SKP2) ubiquitin ligase complex, is implicated in ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27(KIP1). Our earlier studies revealed SKP2 as a target gene within the 5p13 amplicon that is often seen in small-cell lung cancers. In the present study we examined amplification status and expression levels of SKP2 in non-small-cell lung cancer (NSCLC) and investigated its clinicopathological significance in this type of tumor because amplification of DNA at 5p13 is observed frequently in NSCLCs as well as in small-cell lung cancers. SKP2 exhibited amplification in 5 (20%) of 25 cell lines derived from NSCLC, and the transcript was overexpressed in 11 (44%) of the 25 lines. Moreover, expression of SKP2 was up-regulated significantly in 60 primary NSCLC tumors as compared to nontumorous lung tissues (P < 0.0001). Elevated expression of SKP2 correlated significantly with positive lymph node metastasis (P = 0.007), with stage II or higher of the international TNM classification (P = 0.014), with poor or moderate differentiation (P < 0.001), and with the presence of squamous cell carcinoma (P = 0.037). Reduction of SKP2 expression by transfection of an anti-sense oligonucleotide inhibited invasion and migration of NSCLC cells in culture. Our results suggest that SKP2 may be involved in progression of NSCLC, and that targeting this molecule could represent a promising therapeutic option.

We investigated DNA copy-number aberrations in 22 cell lines derived from small cell lung cancers (SCLCs) using comparative genomic hybridization. A minimal common region at 5p13, within the 5p11-p13 amplicon that was most frequently involved, harbored the CDH6, PC4, and SKP2 genes. These three genes showed amplification and consequent overexpression in the SCLC cell lines. SKP2 positively regulates progression of cell cycle by targeting several regulators, such as the cell-cycle inhibitor p27(KIP1), for ubiquitin-mediated degradation. SKP2 was amplified in 7 (44%) of 16 primary SCLC tumors, and consequently overexpressed in 10 (83%) of the 12 of those tumors we examined. Expression levels of SKP2 protein were cell cycle-dependent in SCLC cells as well as in normal cells, and were correlated with the DNA copy-number of the gene. There was an inverse correlation between the expression of SKP2 and p27(KIP1) proteins. Down-regulation of SKP2 using an anti-sense oligonucleotide remarkably suppressed the growth of SCLC cells. Our results indicate that SKP2 is likely to be a target of the 5p13 amplification and to play an important role in the growth of SCLC cells.

Juvenile myoclonic epilepsy (JME) is the most frequent and, hence, most important form of hereditary grand mal epilepsy. Genetic linkage, haplotype, and recombination analyses have indicated that 6p11-12 (EJM1) is one of the candidate regions harboring a gene responsible for JME. In efforts to identify a gene responsible for JME, we identified several expressed sequences in the EJM1 critical region. Here we report the identification and characterization of a gene, named C6orf33, in the EJM1 region. Northern blot analysis showed that C6orf33 is predominantly expressed in brain but in mice, testis shows additional transcripts. C6orf33 is predicted to encode a novel approximately 40-kDa membrane protein, LMPB1, that defines a novel protein family by having highly conserved orthologs in eukaryotes and three putative paralogs in human. Biochemical and immunocytochemical studies revealed that LMPB1 is indeed an integral membrane protein that targets to lysosomal structures. LMPB1 may be involved in specialized lysosomal functions that are unique to brain and testis, and the C6orf33 gene is of interest as a candidate for EJM1., (Copyright 2001 Academic Press.)

The three-amino-acid loop extension (TALE) homeodomain proteins are highly conserved transcription regulators. Since cooperative function among members of this growing family is critical for regulating transcription, we have tried to explore novel members to understand their regulatory mechanisms in cellular proliferation and differentiation. Here we report identification of PKNOX2, a novel TALE homeodomain protein that shows distinct homology with PKNOX1, a stable partner of PBX proteins. PKNOX2 is composed of 460 amino acids and contains HR1, HR2, and homeodomain, which are highly similar to PKNOX1, suggesting that PKNOX2 may also interact with PBX proteins as well as the same DNA sequence as PKNOX1. Genomic organization of PKNOX2 also showed high similarity to PKNOX1, though PKNOX2 lies on a different chromosomal region, 11q24. Unlike PKNOX1, which was broadly expressed in many tissues, PKNOX2 showed a more restricted pattern of mRNA expression. Nuclear localization of PKNOX2 was confirmed by transfection of epitope-tagged cDNA. Taken together, these data indicate that PKNOX2 is a novel PKNOX-related protein and may interact with PBX proteins and play a tissue-specific regulation of transcription., (Copyright 2001 Academic Press.)

Amplification of the 3q26 region appears to occur frequently among esophageal squamous cell carcinomas (ESCs). We examined ESC cell lines for amplification and expression levels of four genes in this region: SNO and EVI1, which encode proteins antagonizing transforming growth factor-beta signaling, and two other putative target genes, TERC and PIK3CA. Amplification of SNO was accompanied by significant increases in its expression level, suggesting that this gene is activated in an amplification-dependent manner. SNO was also amplified in 5 of 44 primary ESCs (11.4%). However, expression levels of EVI1, TERC, and PIK3CA did not correlate with their copy-numbers, even though EVI1 and TERC showed the same amplification pattern as SNO. Taken together, the data suggest that of the four candidates, SNO is the most probable target in the 3q26 amplicon for involvement in the progression of ESC.

Homeodomain transcription factors play important roles in directing cellular proliferation and differentiation. A TALE-superclass homeodomain protein, multifunctional repressor of TGFbeta-induced transcription. Here we report identification of TGIF2, a novel TALE-superclass homeodomain protein that shows distinct homology with TGIF, especially in its DNA-binding domain. TGIF2 is expressed ubiquitously in human tissues, with the highest levels being found in heart, kidney, and testis. The TGIF2 product contains a putative nuclear localization signal; translocation of the protein to the nucleus was confirmed by transfection of epitope-tagged cDNA. TGIF2 lies on chromosome 20q11.2-12. Since amplification of 20q is often observed among ovarian cancers, we determined the status of DNA copy-number and expression of TGIF2 in 14 ovarian-cancer cell lines. This gene was over-expressed in all lines that showed amplification by FISH analysis. The results suggested that TGIF2 may play an important role in the development and/or progression of some ovarian tumors through a mechanism of gene amplification., (Copyright 2000 Academic Press.)

The human EBAG9 was previously identified as an estrogen responsive gene using CpG-genomic binding site cloning (Watanate et al., (1998) Mol. Cell. Biol. 18: 442-449). Recently it was revealed that the EBAG9 is identical with RCAS1 which is a cancer cell surface antigen implicated in immune escape. Here, we isolated and analyzed the 5'-flanking region of human EBAG9 gene. We determined transcription initiation site, which has a homology with an initiator element YYCAYYYY, and found that TATA motif was absent. Deletion analysis of the 5'-flanking region using MCF-7 breast cancer cells indicated that the sequences -86 to -36 containing the ERE had the basal level of promoter activity and the upstream GC-rich region positively regulated the activity. EBAG9 promoter luciferase reporters containing the ERE could respond to estrogen, and electrophoretic mobility shift assay showed that ERalpha bound to the ERE. Moreover, fluorescent in situ hybridization analysis has shown that the human EBAG9 gene is located at chromosome 8q23 which is frequently amplified in tumors. These findings suggest that the human EBAG9 might be involved in carcinogenesis as an estrogen responsive gene., (Copyright 2000 Academic Press.)

Malignant rhabdoid tumors (MRTs) are rare, pediatric soft-tissue tumors. Homozygous deletions at chromosome 22q11.2 are a recurrent cytogenetic characteristic of MRTs, an indication that this locus may harbor one or more genes conferring tumor-suppressor activity. We constructed a deletion map of the relevant part of 22q11.2 from a panel of seven MRT cell lines, and isolated a novel gene from the center of the region. As it showed a high degree of sequence homology to genes of the Rab family, we designated it Rab36. The protein encoded by Rab36 was localized at the Golgi body. Sequencing of Rab36 cDNAs from three cell lines that retained at least one allele of this gene revealed no nonsense or frameshift mutations. Experiments to induce over-expression of Rab36 by transfection to an MRT cell line similarly failed to justify designation of this gene as a tumor suppressor that would contribute to tumorigenesis by a loss-of-function mechanism., (Copyright 1999 Academic Press.)

Protein interaction cloning method was used to identify a novel molecule, Sab, which binds to the SH3 domain of Bruton's tyrosine kinase (Btk), the deficient cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia and murine X-linked immunodeficiency. Immunoprecipitation using the anti-Sab antibody identified the protein product of the gene as a 70 kDa molecule. While Sab does not have a proline-rich sequence, it was shown to bind to Btk through the commonly conserved structure among SH3 domains. Remarkably, Sab exhibited a high preference for binding to Btk rather than to other cytoplasmic tyrosine kinases, which suggests a unique role of Sab in the Btk signal transduction pathway.

Intracystic papillary breast tumors, including intraductal papilloma and low-grade intracystic papillary carcinoma, constitute a group for which differential diagnosis is frequently difficult. We examined the status of chromosomes 16 and 1 by multicolor fluorescence in situ hybridization (FISH) analyses and the DNA ploidy patterns by flow cytometry in 26 intracystic papillary tumors. Alterations of chromosomes 16 and 1 were detected by FISH in 93 and 85%, respectively, of 14 low-grade papillary carcinomas, and the latter alterations always concurred with the former. Two-color FISH using probes for the D1Z1 (1q12) and D16Z2 (16cen) loci or the D1Z1 and D16Z3 (16q11) loci showed that fusion of chromosomes 16 and 1, mostly with breakpoints distal to 16q11.2 and proximal to 1q12, occurred in 77% of the papillary carcinomas. DNA aneuploidy was detected in 6% of these carcinomas. No papilloma showed these chromosome alterations or DNA aneuploidy. Chromosome 16 and 1 fusions appeared to occur frequently in diploid breast carcinomas and to be involved in the acquisition of a malignant phenotype by duct epithelial cells. We suggest that two-color FISH methods for detecting 1;16 fusions might be applicable as supportive methods for the differential diagnosis of intracystic papillary breast tumors.

Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions, and the cDNA encoding its mammalian homologue was identified very recently (Ando-Akatsuka, Y., M. Saitou, T. Hirase, M. Kishi, A. Sakakibara, M. Itoh, S. Yonemura, M. Furuse, Sh. Tsukita, J. Cell Biol. 133, 43-47 (1996)). Here we describe the basic properties of mammalian occludin in epithelial cells at the DNA, RNA, and protein levels. The human occludin gene was mapped to chromosome band 5q13.1 by fluorescent in situ hybridization. Northern blotting identified several occludin mRNA bands, suggesting the possible expression of several alternatively spliced products. Occludin mRNA was detected in cultured epithelial cells, but not in cultured fibroblasts. The mRNA level was high in the testis, kidney, liver, lung, and brain, which reportedly bear well-developed tight junctions. We then produced monoclonal and polyclonal antibodies using recombinant mouse occludin as the antigen, which reacted not only with mouse, but also human, dog and pig occludin. These antibodies recognized several bands around 60 kDa in epithelial cells but not in fibroblasts. Immunofluorescence microscopy of various tissues revealed that the staining intensity of occludin correlated well with the number of tight junction strands in epithelial cells. By contrast, the staining of ZO-1, a well-characterized tight junction-associated protein, was not specific for tight junctions. Furthermore, the exclusive concentration of occludin at tight junctions in epithelial cells was confirmed by immunoreplica electron microscopy.

We identified a novel gene, kf-1, highly expressed in the normal cerebellum but not in the cerebral cortex, the expression of which could have been augmented in the cerebral cortex of a sporadic Alzheimer's disease patient. We cloned human and mouse entire kf-1 cDNAs encoding conserved 79 kDa proteins containing a zinc-binding RING-H2 finger motif at the carboxy-terminus as found in acetylcholine receptor-associated protein (RAPsyn). The 3'-untranslated regions are highly conserved between human and mouse as to constitute a common mRNA secondary structure. In situ hybridization analysis of mouse brain sections revealed strong kf-1 expression in the cerebellum and hippocampus. We propose that KF-1 is involved in membranous protein-sorting apparatus similarly to RAPsyn. We mapped the human kf-1 gene to 2p11.2.

In our efforts to explore possible roles of proteins with a LIM domain, which is a cysteine-rich Zinc-binding motif, in differentiation and oncogenesis in the lung, we have cloned a human LIMK2 gene and identified two alternative transcripts, LIMK2a and LIMK2b, which are probably due to variation in transcriptional initiation. The former encodes a protein containing two LIM domains, a PDZ domain, and a kinase domain, while the latter has only one and half LIM domains. The predominance of the two transcripts appears to be regulated in a tissue-specific manner. Alteration of the regulation is also observed in some cancer cell lines. Transfection studies have shown an association of 63-kDa and 58-kDa proteins with the LIMK2a and LIMK2b protein; the former is distributed in the cytoplasm and nucleus and the latter occurs mainly in the cytoplasm and is scarcely translocated to the nucleus. In contrast, a truncated LIMK2-Kinase has a nuclear location, not showing the protein association.

The t(11;19)(q23;p13.1) translocation is thought to play an important role in pathogenesis of myeloid leukemias in older patients. The MLL gene involved in other 11q23 abnormalities was also rearranged by this translocation. Screening of cDNA libraries of the t(11;19)(q23;p13.1)-carrying leukemic cells resulted in the isolation of several species of fusion cDNAs between the MLL gene and an unknown gene on 19p13.1, named MEN (myeloid eleven-nineteen translocation), which is ubiquitously expressed. Although the MLL gene was alternatively spliced, the fusion protein should contain an N-terminal half of the MLL, including AT hook motifs, that is fused to the MEN protein with a lysine-rich sequence, suggesting that the MLL/MEN fusion protein could be a chimeric transcription factor. The MLL/MEN fusion transcripts of 8.0 kb were detected in leukemic cells of two cases with the translocation. The MLL/MEN fusion was consistent in all three cases of the t(11;19)(q23;p13.1)-carrying leukemia examined by RNA-based polymerase chain reaction. These findings strongly suggest that the t(11;19)(q23;p13.1) results in the fusion formation encoding a new class of potential chimeric transcription factor that contributes to leukemogenesis of myeloid lineage.

A family suggesting cosegregation of beta-thalassemia minor and bipolar affective disorder was reported. The monogenic hereditary anemia was surveyed over three generations in this family, and three patients with the beta-thalassemia and the bipolar affective disorder were observed over two generations. The localization of a gene responsible for bipolar affective disorder was discussed on the basis of previous reports.

Protein-tyrosine phosphatases (PTPases) are considered to play an important role in signal transduction. We previously identified partial sequences of three novel PTPases in a human leukemic cell line. F-36P. We describe here cloning, characterization, and chromosomal localization of one of the newly identified PTPases, termed as HPTP eta (human protein-tyrosine phosphatase eta). The deduced amino acid sequence was composed of an extracellular region homologous to fibronectin type III repeats, a transmembrane region, and a cytoplasmic region containing a single PTPase-like domain. Based on its primary structure, this clone belongs to type-III receptor-type PTPases. The PTPase-like domain showed PTPase activity when expressed in Escherichia coli. Antibody against the extracellular region detected a protein of 220 to 250 kD in human hematopoietic cell lines expressing HPTP eta mRNA. The antibody also recognized a protein of approximately the same molecular weight in COS cells transfected with HPTP eta cDNA, indicating that the antibody specifically recognized HPTP eta gene product and that the cloned cDNA contained full-length coding region. The chromosomal localization determined by fluorescence in situ hybridization showed that the HPTP eta gene was located at chromosome 11p11.2 on the short arm of chromosome 11, which is frequently lost or deleted in human carcinomas.

Abnormality of the dihydrolipoamide succinyltransferase gene may be a cause of familial Alzheimer's disease linked to chromosome 14q24.3. However, the locus of the dihydrolipoamide succinyltransferase gene on this chromosome was uncertain. An unspliced cDNA of about 2.3 kb for human dihydrolipoamide succinyltransferase was isolated. This cDNA contained three exons and four introns and the nucleotide sequences at the 5' donor and 3' acceptor sites of all introns conformed to the gt-ag rule. The amino acid sequences of the three exons support our previous observation that human dihydrolipoamide succinyltransferase lacks a sequence motif for an E1 and/or E3 binding site. The unspliced cDNA was mapped only on human chromosome 14q24.2-q24.3 by fluorescent in situ hybridization. Thus the dihydrolipoamide succinyltransferase gene is concluded to be located on human chromosome 14q24.2-q24.3.

We have isolated a series of overlapping cDNA clones encoding a 2,628 bp transcript, which potentially codes for a 198 amino acid protein with predicted molecular mass of 21,641 daltons, for the human ryudocan core protein. The deduced core proteins of the human and the rat ryudocan have high structural conservation, particularly in the NH2 and COOH terminus regions of the putative mature core protein, including the combined transmembrane/cytoplasmic domains with conserved positions of all 4 tyrosine groups and 3 conserved glycosaminoglycan chain attachment regions, which might serve important roles for biological function of ryudocan. A major 2.7 kb transcript was detected in all tissues tested, with relatively high levels of expression observed in mRNA from lung, liver, skeletal muscle and kidney. A minor 1.9 kb transcript was also observed in some of tissues, which would be caused by alternative polyadenylation. Human ryudocan gene has been localized on the chromosome 20q12 by fluorescence in situ hybridization.