AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1.

The Wnt signaling pathway is essential for development and organogenesis. Wnt signaling stabilizes beta-catenin, which accumulates in the cytoplasm, binds to 1-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes. Mutations in CTNNB1 (encoding beta-catenin) or APC (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas (HCCs). Because HCC5 tend to show accumulation of beta-catenin more often than mutations in CTNNB1, we looked for mutations in AXIN1, encoding a key factor for Wnt signaling, in 6 HCC cell lines and 100 primary HCC5. Among the 4 cell lines and 87 HCC5 in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with beta-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXINI induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated beta-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers.

Mutations truncating the EP300 acetylase in human cancers.

The EP300 protein is a histone acetyltransferase that regulates transcription via chromatin remodelling and is important in the processes of cell proliferation and differentiation. EP300 acetylation of TP53 in response to DNA damage regulates its DNA-binding and transcription functions. A role for EP300 in cancer has been implied by the fact that it is targeted by viral oncoproteins, it is fused to MLL in Leukaemia and two missense sequence alterations in EP300 were identified in epithelial malignancies. Nevertheless, direct demonstration of the role of EP300 in tumorigenesis by inactivating mutations in human cancers has been lacking. Here we describe EP300 mutations, which predict a truncated protein, in 6(3%) of 193 epithelial cancers analysed. Of these six mutations, two were in primary tumours (a colorectal cancer and a breast cancer) and four were in cancer cell lines (colorectal, breast and pancreatic). In addition, we identified a somatic in-frame insertion in a primary breast cancer and missense alterations in a primary colorectal cancer and two cell lines (breast and pancreatic). Inactivation of the second allele was demonstrated in five of six cases with truncating mutations and in two other cases. Our data show that EP300 is mutated in epithelial cancers and provide the first evidence that it behaves as a classical tumour-suppressor gene.

UHRF1 is a novel diagnostic marker of lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer deaths worldwide. As the sensitivity and specificity of current diagnostic markers are not perfect, we examined whether ubiquitin-like with PHD and ring finger domains 1 (UHRF1), which is overexpressed in various cancers but not yet examined in lung cancer in large scale, can be a novel diagnostic marker of lung cancer. METHODS: Immunohistochemical analysis using surgical specimens obtained from 56 US and 322 Japanese patients with lung cancer was performed. RESULTS: The UHRF1 was stained specifically in the nuclei of cancer cells, but not in the other cells. The UHRF1 expression was observed in all histological types of lung cancer, especially in non-adenocarcinomas (non-ADCs), both in the US and Japanese cases. In 322 Japanese non-small cell lung cancer (NSCLC) cases, UHRF1 expression was associated with the histological type (higher in non-ADCs; P<0.00001), gender (higher in male; P=0.00082), smoking (higher in smokers; P=0.00004), pT factor (higher in advanced stage; P=0.00010), and pN factor (higher in cancers with metastasis in regional lymph nodes; P=0.00018). The UHRF1 expression was also associated with poor prognosis for NSCLC patients (P=0.0364). Although UHRF1 overexpression was associated with these malignant indicators, UHRF1 was detectable in half of lung cancer patients in an early pathological stage. CONCLUSION: The UHRF1 is overexpressed in various types of lung cancer from early pathological stage. Therefore, detection of UHRF1 expression in tissue specimens by immunohistochemistry can be useful for diagnosis of lung cancer in all pathological stages.

p300 is required for orderly G1/S transition in human cancer cells.

The role of the transcriptional coactivator p300 in cell cycle control has not been analysed in detail due to the lack of appropriate experimental systems. We have now examined cell cycle progression of p300-deficient cancer cell lines, where p300 was disrupted either by gene targeting (p300(-) cells) or knocked down using RNAi. Despite significant proliferation defects under normal growth conditions, p300-deficient cells progressed rapidly through G1 with premature S-phase entry. Accelerated G1/S transition was associated with early retinoblastoma (RB) hyperphosphorylation and activation of E2F targets. The p300-acetylase activity was dispensable since expression of a HAT-deficient p300 mutant reversed these changes. Co-immunoprecipitation showed p300/RB interaction occurs in vivo during G1, and this interaction has two peaks: in early G1 with unphosphorylated RB and in late G1 with phosphorylated RB. In vitro kinase assays showed that p300 directly inhibits cdk6-mediated RB phosphorylation, suggesting p300 acts in early G1 to prevent RB hyperphosphorylation and delay premature S-phase entry. Paradoxically, continued cycling of p300(-) cells despite prolonged serum depletion was observed, and this occurred in association with persistent RB hyperphosphorylation. Altogether, these results suggest that p300 has an important role in G1/S control, possibly by modulating RB phosphorylation.

Molecular cloning of a candidate tumor suppressor gene, DLC1, from chromosome 3p21.3.

The short arm of chromosome 3 is thought to contain multiple tumor suppressor genes, because one copy of this chromosomal arm frequently is missing in carcinomas that have arisen in a variety of tissues. We have isolated a novel gene encoding a 1755-amino acid polypeptide, through large-scale sequencing of genomic DNA at 3p21.3. Mutational analysis of this gene by reverse transcription-PCR revealed the lack of functional transcripts and an increase of nonfunctional RNA transcripts in a significant proportion (33%) of cancer cell lines and primary cancers (4 of 14 esophageal cancer cell lines, 2 of 2 renal cancer cell lines, 11 of 30 primary non-small cell lung cancers, and 3 of 10 primary squamous cell carcinomas of the esophagus). However, no alterations of the gene itself were detected in any of the cancers examined. Introduction of the cDNA significantly suppressed the growth of four different cancer cell lines, two of which produced no normal transcript on their own. No such effect occurred when antisense cDNA, cDNA corresponding to an aberrant transcript, or the vector DNA alone were transfected. These data suggest that aberrant transcription of this gene, designated DLC1 (deleted in lung cancer 1), may be involved in carcinogenesis of the lung, esophagus, and kidney.

Isolation and characterization of a novel human gene, DRCTNNB1A, the expression of which is down-regulated by beta-catenin.

Beta-catenin plays significant roles in cell-to-cell adhesion and the Wnt/Wg signal transduction pathway. Accumulation of this protein in the cytoplasm and nucleus as a result of mutations of the adenomatous polyposis coli tumor suppressor gene or of the beta-catenin gene itself is often seen in a wide variety of tumors including carcinomas of the colon, liver, uterus, and brain. Interaction of accumulated beta-catenin with Tcf/Lef transcription factors is known to deregulate expression of some downstream genes, but the precise mechanisms whereby beta-catenin contributes to carcinogenesis remain to be disclosed. Here we report isolation of a novel murine gene, Drctnnb1a (down-regulated by Ctnnb1, a), the expression of which was experimentally down-regulated in response to the activated form of beta-catenin. To investigate a possible role of DRCTNNB1A in cancers, we also isolated the human homologue, DRCTNNB1A, the deduced product of which was 91% identical to the murine protein. The transcript was expressed in all human tissues examined, and we assigned the genomic location of DRCTNNB1A to chromosomal band 7p15.3 by in situ hybridization. Expression of DRCTNNB1A in SW480 colon cancer cells was significantly increased in response to reduction of intracellular beta-catenin by adenovirus-mediated transfer of the beta-catenin-binding domain of the adenomatous polyposis coli gene into the cells. Furthermore, we documented reduced expression of DRCTNNB1A in 12 of 15 primary colorectal cancers examined, compared with corresponding adjacent noncancerous mucosae. Our results implied that DRCTNNB1A is one of the genes involved in the beta-catenin-Tcf/Lef signaling pathway, and that reduced expression of DRCTNNB1A may have some role in colorectal carcinogenesis.

Absence of genetic alteration at codon 531 of the human c-src gene in 479 advanced colorectal cancers from Japanese and Caucasian patients.

Activation of c-src, a cellular human gene homologous in sequence to the v-src gene of Rous sarcoma virus, had been thought to play an important role in the progression of several types of human cancers, without having undergone any genetic changes. However, recently truncating mutations at codon 531 of the c-src gene were reported in 12% of the advanced colon cancers, and it was also demonstrated that this change was activating, transforming, tumorigenic, and metastasis promoting. To investigate whether the codon 531-specific mutation could be involved in the carcinogenesis of colorectal cancer in the Japanese and Caucasian populations, we examined a total of 479 advanced colorectal cancers from 421 Japanese patients (46 of them with liver or lung metastases) and from 58 Caucasian patients (11 of them with liver metastases). Using the PCR-RFLP assay and additional single-strand conformation polymorphism analysis, we detected no genetic alteration in any of the advanced colorectal cancers. Our results suggest that the codon 531-specific mutational activation of c-src is unlikely to play a significant role in the malignant progression of colorectal cancers among most Japanese and Caucasian patients.

Significant differences in the frequency of transcriptional units, types and numbers of repetitive elements, GC content, and the number of CpG islands between a 1010-kb G-band genomic segment on chromosome 9q31.3 and a 1200-kb R-band genomic segment on chromosome 3p21.3.

We determined the nucleotide sequence of the entire 1,010,525-bp insert contained in CEPH YAC clone 867e8. This human genomic segment was derived from chromosome 9q31.3 and corresponds to a G-band region. We compared this segment, in terms of structure, with a previously characterized 1,201,033-bp sequence in CEPH YAC936c1 that had come from a portion of human chromosome 3p21.3 corresponding to an R-band region. The two segments were significantly different with respect to the frequency of transcriptional units, the types and numbers of repetitive elements present, their GC content, and the number of CpG islands. Alu elements, GC content, and CpG islands all showed positive correlations with the abundance of exons, but the distribution of LINE1s did not. These observations might reflect an influence of the first three of these features on the functions or expression of genes in the respective regions. In addition to a novel gene (F36) lying at the centromeric end of the 9q segment, we found a cluster of placenta-specific genes within a small section (about 400 kb) on the telomeric side of YAC867e8. This cluster consisted of four apparently unrelated ESTs and two genes, pregnancy-associated plasma protein-A (PAPP-A) and a novel gene (tentatively named EST-YD1). Our characterization of the two chromosomal regions provided evidence that genes are not evenly distributed throughout the human genome, and that gene richness is correlated with the GC content and with the frequency of either Alu elements or CpG islands.

Characterization of a 1200-kb genomic segment of chromosome 3p22-p21.3.

We previously determined the nucleotide sequence and characterized the 685-kb proximal half of CEPH YAC936c1, which corresponds to a portion of human chromosome 3p21.3. In the study reported here, we characterized the remaining 515-kb of this YAC clone corresponding to the telomeric half of its human insert. The newly sequenced region contained a total of ten genes including six reported previously: phospholipase C delta 1 (PLCD1), human activin receptor type IIB (hActR-IIB), organic cation transporter-like 1 (OCTL1), organic cation transporter-like 2 (OCTL2), oxidative stress response 1 (OSR1), and human xylulokinase-like protein (XYLB). The remaining four genes present in the telomeric region included two known genes, MyD88 and ACAA, and two novel genes. One (designated ENGL) of the novel sequences was found to encode an amino-acid sequence homologous to the family of DNA/RNA endonucleases, especially endonuclease G. The other gene F56 revealed no significant homology to any known genes. These results disclosed complete physical and transcriptional maps of the 1200-kb region of 3p present in YAC 936c1.

Differential gene expression in the small intestines of wildtype and W/W(V) mice.

UNLABELLED: Much of the evidence demonstrating the role of interstitial cells of Cajal (ICC) in pacemaking and neurotransmission in the gastrointestinal tract comes from studies of W/W(V) mice. These animals have few pacemaker ICC in the small bowel due to reduced functional Kit protein. We examined gene expression in the small intestines of wildtype and W/W(V) mice. RNA expression in the jejunums of wildtype and W/W(V) mutants was studied using a differential gene expression METHOD: Seven known genes were differentially expressed in wildtype and W/W(V) mice. COX7B (cytochrome c oxidase, subunit VIIb) and SORCIN (encoding multidrug-resistance complex, class 4) were suppressed in both fed and fasted W/W(V) mice. Expression of another five genes was increased in W/W(V) mice: ADA (adenosine deaminase), MDH1 (malate dehydrogenase), RPL-8 (ribosomal protein L8), SPTB2 (spectrin, nonerythroid, beta subunit), and p6-5 (encoding phosphorylcholine [PC] T-cell suppressor factor [TsF]). Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state. We conclude that several genes are differentially expressed in the small intestines of W/W(V) mice where the major lesion is loss of pacemaker ICC. Differential gene display may help develop a molecular profile of motility disorders in which ICC are lost.

A systematic approach to the development of novel therapeutics for lung cancer using genomic analyses.

Molecularly targeted drugs for cancer therapy represent a therapeutic advance, but the proportion of patients who receive clinical benefit is still very limited. We present here the rationale and initial results of our program to define molecules involved in lung carcinogenesis with the goal of identifying new therapeutic targets and/or predictive biomarkers for drug response. We have used gene expression analysis of 120 lung cancers followed by RNA interference, tumor-tissue microarray analysis, and functional analyses to systematically distinguish potential target molecules specifically expressed in cancer cells. Through this approach, we have identified oncoproteins that provide the starting point for the development of therapeutic antibodies, dominant negative peptides, small-molecule inhibitors, and therapeutic cancer vaccines. We believe that the approach we describe should result in new molecularly targeted therapies with minimal risk of adverse events.

Novel multilayer Ti foam with cortical bone strength and cytocompatibility.

The major functions required for load-bearing orthopaedic implants are load-bearing and mechanical or biological fixation with the surrounding bone. Porous materials with appropriate mechanical properties and adequate pore structure for fixation are promising candidates for load-bearing implant material. In previous work, the authors developed a novel titanium (Ti) foam sheet 1-2mm thick by an original slurry foaming method. In the present work, novel Ti foam is developed with mechanical properties compatible with cortical bone and biological fixation capabilities by layer-by-layer stacking of different foam sheets with volumetric porosities of 80% and 17%. The resulting multilayer Ti foam exhibited a Young's modulus of 11-12GPa and yield strength of 150-240MPa in compression tests. In vitro cell culture on the sample revealed good cell penetration in the higher-porosity foam (80% volumetric porosity), which reached 1.2mm for 21 days of incubation. Cell penetration into the high-porosity layers of a multilayer sample was good and not influenced by the lower-porosity layers. Calcification was also observed in the high-porosity foam, suggesting that this Ti foam does not inhibit bone formation. Contradictory requirements for high volumetric porosity and high strength were attained by role-sharing between the foam sheets of different porosities. The unique characteristics of the present multilayer Ti foam make them attractive for application in the field of orthopaedics.

Overexpressing PKIB in prostate cancer promotes its aggressiveness by linking between PKA and Akt pathways.

Prostate cancer (PC) is the most common malignancy in males. Despite high response rates and clinical benefits, androgen-ablation therapy is ineffective for advanced or relapsed PC because of the emergence of aggressive castration-resistant prostate cancer (CRPC). Through our genome-wide gene expression analysis of PC cells purified from clinical CRPC tissues, we here identified a novel molecular target, PKIB (cAMP-dependent protein kinase inhibitor-beta), which was overexpressed specifically in CRPCs and aggressive PCs. Immunohistochemical analysis confirmed its overexpression in CRPCs and its strong correlation with high Gleason scores of PCs. Knockdown of PKIB by siRNA resulted in drastic growth suppression of PC cells, and, concordantly, exogenous introduction of PKIB into PC cells enhanced their growth and mobility. We found the direct interaction between PKIB and cAMP-dependent protein kinase A catalytic subunit (PKA-C), and showed that knockdown of PKIB in PC cells diminished the nuclear translocation of PKA-C. Knockdown of PKIB also decreased the phosphorylation level of Akt at Ser473 in PC cells, and exogenous PKIB introduction enhanced Akt phosphorylation in PC cells by incorporating with endogenous PKA-C kinase. In vitro kinase assay validated the recombinant PKIB enhanced phosphorylation of Akt at Ser473 by PKA-C kinase. These findings show that PKIB and PKA-C kinase can have critical functions of aggressive phenotype of PCs through Akt phosphorylation and that they should be a promising molecular target for PC treatment.

Isolation and characterization of a novel serine threonine kinase gene on chromosome 3p22-21.3.

Through large-scale DNA sequencing of a genomic region on chromosome 3p22-p21.3, we isolated a novel gene encoding a 527-amino-acid protein. Its 18 exons spanned a genomic region of about 90 kilobases, and the 4536-nucleotide cDNA contained an open reading frame of 1581 base pairs. The gene was expressed in all 16 human tissues examined by Northern blotting. The amino acid sequence of the predicted protein was 39% identical to that of human SOK1 (Ste20/oxidant stress response kinase-1), a molecule that is activated by oxidative stress. In view of its significant similarity to SOK1, we suspect that the novel gene, which we named OSR1, is a member of the SOK family of kinases in terms of function.

Isolation and mutational analysis of a novel human cDNA, DEC1 (deleted in esophageal cancer 1), derived from the tumor suppressor locus in 9q32.

The long arm of chromosome 9 is thought to contain one or more putative tumor suppressor genes that are mutated in squamous cell carcinomas. This region shows frequent loss of heterozygosity (LOH) in carcinomas arising in several developmentally related tissues, including the esophagus, head and neck, lung, and urinary bladder. We previously delineated the commonly deleted region in a panel of esophageal squamous cell carcinomas to a approximately 200 kb genomic segment at 9q32. Here we report the isolation of a novel gene, DEC1 (deleted in esophageal cancer 1), from the target region. Mutational analysis of this gene by reverse-transcriptase polymerase chain reaction disclosed significantly reduced expression of DEC1 in 8 of 13 (62%) esophageal cancer cell lines and in 16 of 30 (53%) primary squamous cell carcinomas of the esophagus. However, no genetic alteration was detected in any of the cancers examined. Introduction of DEC1 cDNA into 3 cancer cell lines that lacked expression of DEC1 significantly suppressed cell growth, whereas antisense cDNA or the vector DNA alone did not. Given the reduced expression of the DEC1 gene in esophageal cancer, the high frequency of LOH at 9q32 in esophageal carcinomas, and the fact that the DEC1 cDNA can suppress growth of some cancer cells in vitro, we suggest that the DEC1 gene is a candidate tumor suppressor in 9q32. Genes Chromosomes Cancer 27:169-176, 2000.

Genomic structure of a novel human gene (XYLB) on chromosome 3p22-->p21.3 encoding a xylulokinase-like protein.

We isolated a novel human cDNA of 1963 nucleotides that included an open reading frame encoding a protein of 528 amino acids. Homology analysis indicated that the predicted gene product, XYLB, bore 22% identity to the xylulokinase of Haemophilus influenzae that plays an important role in energy metabolism. The gene consists of 18 exons and spans about 28 kb of genomic DNA on chromosome 3p21.3.

Molecular cloning, mapping, and characterization of two novel human genes, ORCTL3 and ORCTL4, bearing homology to organic-cation transporters.

Through a large-scale sequencing of genomic DNA at 3p22-->p21.3, we have isolated two human genes (ORCTL3 alias OCTL1 and ORCTL4 alias OCTL2) encoding novel members of the family of organic-cation transporter molecules. The predicted proteins revealed the highest similarities to recently- isolated organic-cation transporter proteins, rat OCT-1r, rat NLT and mouse NKT. The transcripts of both genes were expressed ubiquitously in various human tissues, but some tissue-specific transcripts were also observed in kidney, testis, or skeletal muscle. The two genes are clustered within a 52-kb region of genomic DNA and ORCTL4 lies about 27 kb telomeric to ORCTL3 in the genomic DNA sequence in a tail-to-head orientation.

Changes of in vivo gastrointestinal motor pattern in pacemaker-deficient (WsRC-Ws/Ws) rats.

In vitro studies on pacemaker-deficient W-mutants have revealed a disappearance of rhythmic contraction in their gastrointestinal tracts. Their contractile force has not been diminished, however. In contrast, W-mutants often present dysmoility-like symptoms with distension of the gastrointestinal tract in vivo. Gastrointestinal motility of W-mutant rats was examined in vivo by an extraluminal strain-gauge force transducer method. We examined a normal gastrointestinal motor pattern in the rats with two distinct motor phases, digestive and interdigestive. Moreover, we detected a failure to form an interdigestive contractile complex in pacemaker-deficient rats. The interdigestive motor activity of the gastrointestinal tract is important for cleaning gastrointestinal tract in preparation for the next meal. The impairment of the interdigestive contractile complex may be related to the dysmoility-like symptoms of W-mutant rats in vivo.

Isolation and characterization of a human cDNA homologous to the Xenopus laevis XCAP-C gene belonging to the structural maintenance of chromosomes (SMC) family.

We have isolated a human cDNA encoding a protein of 1288 amino acids that shows 77% identity in amino acid sequence to XCAP-C, Xenopus chromosome-associated polypeptide-C, belonging to the family of structural maintenance of chromosomes (SMC), which is known to play a crucial role in the proper condensation and segregation of mitotic chromosomes. In particular, an almost 200-amino-acid domain in the N-terminal, including an NTP-binding motif and that in the C-terminal domain, including a DA-box, were well conserved and showed 95% identity between human and frog, indicating that these two domains are functionally very important. The transcript of this gene was expressed ubiquitously in various human tissues, but thymus, testis, and colon seemed to express this gene more abundantly. We also determined its chromosomal location at 3q26.1 by fluorescence in situ hybridization.