A hydrophobic transmembrane segment at the carboxyl terminus of thy-1.

The mode of integration of the glycoprotein thy-1 within the cell membrane has been controversial due to an apparent lack of a transmembrane hydrophobic segment. Rat and mouse complementary DNA and genomic clones encoding the thy-1 molecule have been isolated and sequenced. These studies have enabled us to determine the intron-exon organization of the thy-1 gene. Furthermore, they have revealed the existence of a sequence which would encode an extra segment (31 amino acids) at the carboxyl terminus of the thy-1 molecule. These extra amino acids include a 20-amino acid hydrophobic segment which may be responsible for integration of thy-1 within the plasma membrane.

Drosophila proliferating cell nuclear antigen (cyclin) gene: structure, expression during development, and specific binding of homeodomain proteins to its 5'-flanking region.

The genomic and cDNA clones for a Drosophila melanogaster proliferating cell nuclear antigen (PCNA) (cyclin) were isolated and sequenced. The coding sequence for a 260-amino-acid residue polypeptide was interrupted by a single short intron of 60 base pairs (bp), and about 70% of the deduced amino acid sequence of the Drosophila PCNA was identical to the rat and human PCNA polypeptides, with conserved unique repeats of leucine in the C-terminal region. Genomic Southern blot hybridization analysis indicates the presence of a single gene for PCNA per genome. The PCNA mRNA was detected at a high level in adult ovaries, unfertilized eggs, and early embryos and at low levels in the other developmental stages. The major transcription initiation site (cap site) was localized at 89 bp upstream from the ATG codon. Neither a TATA box nor a CAAT box was found within the 600-bp region upstream of the cap site. Clusters of 10 bp of sequence similar to the binding sites for Drosophila proteins containing homeodomains were found in the region from -127 to -413. DNase I footprint analysis revealed that the Drosophila homeodomain proteins coded by even-skipped and zerknüllt genes can specifically bind to these sites. These results suggest that the expression of the PCNA gene is under the control of genes coding for homeodomain proteins.

Correlation between concanavalin A agglutinability and cytotoxic sensitivity to antiserum against tumor-associated antigen in rat fibrosarcoma cells.

An ip transplantation of 3-methylcholanthrene-induced, transplanted fibrosarcoma KMT-17 cells (1 X 10(8)) grew rapidly and killed syngeneic WKA rats in 3-4 days. Agglutinability induced by concanavalin A (Con A) and antigenic expression of KMT-17 cells were investigated in relation to days after ip transplantation. Agglutinability was highest in 1-day-old cells and lowest in 3-day-old cells. The agglutinability of 3-day-old cells increased again when these cells were transplanted into normal rats. The cytotoxic sensitivity of tumor cells to antiserum against tumor-associated surface antigen (TASA) changed simultaneously with the degree of Con A agglutinability. This phenomenon disappeared after artificial infection of tumor cells with Friend murine leukemia virus. The result of the quantitative absorption test at 4 degrees C overnight was that 1- and 3-day-old cells did not differ in their absorbing capacities to anti-TASA sera. However, when the absorption test was done at 37 degrees C for 60 minutes, 1-day-old cells had approximately 16 times more absorbing capacity than 3-day-old cells. However, the cytotoxic sensitivity to antiserum against histocompatibility antigen did not change, regardless of the number of days after ip transplantation. Analysis based on the quantitative absorption test revealed no difference in antibody-absorbing capacities between 1- and 3-day-old cells at both 4 degrees C and 37 degrees C. The relationship between Con A agglutinability and cytotoxic sensitivity to anti-TASA serum is discussed from the viewpoint of "lateral receptor mobility" on the cell surface.

Increased oxidative DNA damage in mammary tumor cells by continuous epidermal growth factor stimulation.

BACKGROUND: Growth factors can enhance the malignant potential of tumor cells. To examine the relationship between growth factors and tumor progression, we previously established a weakly malignant cell line, ER-1. We found that a 24-hour exposure of ER-1 cells to epidermal growth factor (EGF) induced malignant properties (tumor progression) that were reversible but that, after a 1-month exposure, these changes were irreversible. In this study, we investigated the irreversible changes induced in ER-1 cells by a 1-month exposure to EGF and the possible involvement of oxidative stress. METHODS: ER-1 cells were treated with EGF (100 ng/mL) for 1 month in the presence or absence of an antioxidant, N-acetylcysteine or selenium, and compared with untreated control ER-1 cells. We assessed tumor progression by measuring intracellular peroxide levels, 8-hydroxydeoxyguanosine (a marker for oxidative DNA damage) levels, in vitro invasiveness, and in vivo tumorigenicity and metastatic ability. All statistical tests are two-sided. RESULTS: After ER-1 cells were treated for 1 month with EGF, levels of intracellular peroxide and 8-hydroxyguanosine in the DNA of treated cells were higher than those in the DNA of control cells, and treated ER-1 cells were more tumorigenic and metastatic in vivo and more invasive in vitro than untreated control cells (all P<.001). Levels of 8-hydroxyguanosine in DNA increased as the length of the EGF treatment increased (P<.001). However, when N-acetylcysteine or selenium was added with EGF for 1 month, levels of intracellular peroxide and 8-hydroxyguanosine in DNA were comparable to those in control cells (r =.795). Both tumorigenicity (P =.008) and metastatic ability (P<.001) decreased after addition of N-acetylcysteine or selenium. CONCLUSION: The irreversible changes caused by continuous EGF stimulation of ER-1 cells result from increased oxidative damage in the DNA, which generates tumor cells with more malignant characteristics.

Preliminary study on the effects of bar placement on the thorax after the nuss procedure for pectus excavatum using bone scintigraphy.

AIM: Bone scintigraphy was performed to elucidate the effects of the Nuss procedure for pectus excavatum on the bony thorax. METHODS: Eight boys and 6 girls (5 - 24 years of age) underwent bone scintigraphy, using (99m)Tc-HMDP. Eleven patients were studied 5 to 21 days after the Nuss procedure; 6 were studied 20 to 24 months after the operation before bar removal. Three of 14 were studied twice after the Nuss procedure and before bar removal. RESULTS: In the early postoperative phase, RI accumulation was found at the sternum and ribs in only 1 of 6 patients under 9 years of age, whereas in all 5 older patients, RI had accumulated at the sternum. Scintigrams before bar removal revealed, regardless of age, hot spots at the lateral ribs in contact with the bar and at the costochondral junctions where the bar passed through the intercostal spaces. Furthermore, chest roentgenograms showed the deformed lateral ribs in contact with the bar. CONCLUSIONS: The Nuss procedure creates minute fractures at the sternum and the ribs, especially in older patients. The bar deforms the ribs and restrains the growth of the thorax. Furthermore, it constantly rubs against the ribs and can therefore cause late complications. Bone scintigraphy may determine the appropriate timing for bar removal.

Heterologous antiserum to chemically induced rat non-T, non-B leukemia and its application to characterization of rat leukemias.

Antisera to the 1-butyl-1-nitrosourea-induced "non-T, non-B" rat leukemia line DBLA-6 were raised in rabbits. Following absorption with syngeneic hepatoma cells, the antisera were very similar in specificity to antisera raised to rat Thy-1 antigen. Anti-DBLA-6 serum was cytotoxic in the presence of complement against 70 to 90% of thymocytes and 40 to 50% of neonatal spleen cells. In contrast, no significant cytotoxicity was observed against cells from bone marrow, lymph node, spleen, and peritoneum. An absorption test revealed that an antigen recognized by anti-DBLA-6 serum was present in brain tissue but absent in liver and kidney tissues. Nineteen rat leukemias and lymphomas were divided into six groups based on antigenic and morphological characteristics and the presence of receptor for guinea pig red blood cells. These tumors were investigated for the presence of the antigen recognized by anti-DBLA-6 serum. Of the leukemias and lymphomas studied, anti-DBLA-6 serum reacted with all thymic (Group 1) and extrathymic (Group 2) lymphomas and unclassified leukemias (Groups 3 and 4), while all myelogenous leukemias (Group 5) and erythroleukemias (Group 6) were negative. The position of leukemias and lymphomas reactive with anti-DBLA-6 serum in the lymphocyte maturational pathway is discussed.

Characterization and classification of rat leukemias and lymphomas by membrane markers.

An immunological characterization of leukemias and lymphomas was made in the rat by using a panel of membrane markers in combination with morphological analysis. In the present study, five antigen markers and three surface markers were used for the characterization of 20 rat leukemias and lymphomas, and it was indicated that they could be divided into at least six groups. Of the lymphomas studied, six thymic lymphomas (Group 1) had the Thy-1.1 antigen, T-cell antigen, and receptors for guinea pig red blood cells; five extrathymic lymphomas (Group 2) lacked T- and B-cell antigens, receptors for guinea pig red blood cells, and surface immunoglobulin, but three of them had complement receptors. An absorption test revealed that Group 2 lymphomas possess a very low amount of the Thy-1.1 antigen compared to Group 1 lymphomas. None of the leukemias studied had detectable T- and B-cell antigen. Four leukemias had undifferentiated blast cell morphology and bore the Thy-1.1 antigen; three leukemias (Group 3) reacted with anti-lymphocyte serum, but one leukemia (Group 4) did not. Two leukemias (Group 5) had only the complement receptor and morphologically showed granulocytic appearance. Three leukemias (Group 6) had none of the membrane markers used and morphologically resembled erythroblasts. Based on these results, an attempt was made to classify these leukemias and lymphomas into T-cell lineage, B-cell lineage, stem cell, myeloid, and erythroid groups, respectively. Furthermore, the stage of differentiation in the lymphocyte maturational pathway of the leukemias and lymphomas belonging to Groups 1 to 4 is discussed.

Establishment and characterization of a differentiating myeloid cell line obtained from a rat myelomonocytic leukemia.

A long-term suspension culture line (c-WRT-7) was successfully established from a transplantable myelomonocytic leukemia induced by a neonatal injection of Rauscher leukemia virus in a WKA/Hok rat. A c-WRT-7 cell line was capable of being transplanted into syngeneic rats, and when transplanted, increased numbers of macrophage-like cells were observed in the peripheral blood of rats after i.v. injection. In in vitro culture, about 10% of the c-WRT-7 cells naturally differentiated into macrophage-like cells, which adhered to the bottom of a culture flask, and also possessed phagocytic activity. By means of cytological examination, about 30% of the c-WRT-7 cells were observed to be monoblastic with alpha-naphthyl butyrate esterase activity. The nature of these c-WRT-7 cells as a myelomonocytic leukemia line was constant during in vitro passages of more than 30 generations. In vitro treatment of c-WRT-7 cells with lipopolysaccharide, 12-O-tetradecanoylphorbol-13-acetate, or retinoic acid increased the numbers of differentiated cells with phagocytic activity to 80%. Treatment of the c-WRT-7 cells with the inducers also induced 15 to 20% of the cells to differentiate into metamyelocytes and segmented neutrophils. The Fc receptor and the complement receptor both became detectable on the surface of c-WRT-7 cells after treatment with lipopolysaccharide, 12-O-tetradecanoylphorbol-13-acetate, or retinoic acid. However, rosette-forming activity of sheep erythrocytes pretreated with neuraminidase which has been known as a marker of normal rat macrophages was not induced in c-WRT-7 cells. This shows that differentiated leukemic cells are not exactly identical with normal macrophages.

Establishment and characterization of a transplantable rat myelomonocytic leukemia.

A transplantable myelomonocytic leukemia was established from a leukemia of a WKA/Hok rat which had been inoculated with Rauscher virus at birth. The tumor grew in ascites form in normal syngeneic rats and, after the middle stage of i.p. transplantation, leukemia cells consisting of a mixed population of monocytic and granulocytic cells were observed in the peripheral blood. A complement-dependent cytotoxicity test failed to demonstrate Rauscher virus-related antigen on the tumor cell surface. Membrane marker analysis revealed that most of the tumor cells possessed receptors for both complement and neuraminidase-treated sheep RBC. More than 90% of ascitic tumor cells displayed phagocytic activity and a positive nonspecific esterase reaction. Serum from rats bearing this tumor contained high levels of muramidase. Ultrastructurally, the tumor cells resembled both immature and mature cells of the monocyte-macrophage series. On serial transplantation into the peritoneal cavity, the tumor displayed consistent differentiation from undifferentiated blast cells to monocytes and cells indistinguishable from granulocytes. The karyotype analysis revealed that the modal number of chromosomes of the tumor cells was 81, and no structural abnormalities of chromosomes were observed after quinacrine mustard staining. This transplantable leukemia will provide a useful experimental model for the study of granulocyte-monocyte differentiation and for human myelomonocytic leukemia.

Dominant-negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme.

Previous experiments have suggested that some mutant forms of p53 are able to inactivate the endogenous wild-type p53 protein in a dominant-negative fashion. However, it remains unknown whether tumors with such dominant-negative (transdominant) p53 mutants have a biological significance that is different from that of recessive p53 mutants. In this study, we examined the dominant-negative potential of various p53 mutants using a yeast-based assay in which both wild-type and mutant p53 were efficiently expressed. We tested a total of 106 p53 mutants, which were identified in brain tumors, glioblastoma multiforme-derived cell lines, breast cancers, or premalignant lesions and squamous cell carcinomas of oral epithelium or were otherwise created by mutagenesis. In agreement with the previous studies, our results demonstrated that transdominant mutations affected amino acid residues that are essential for the stabilization of the DNA-binding surface in the p53 core domain and for the direct interaction of p53 with its DNA-binding sequence. Among 40 patients with sporadic glioblastomas, the average age at diagnosis was significantly younger in the patients with tumors harboring dominant-negative mutations (30.4 +/- 14.7 years, n = 7) than it was in those with recessive mutations (55.2 +/- 18.6 years, n = 9, P < 0.012) and in those without mutations (54.7 +/- 17.1 years, n = 24, P < 0.003). Our data suggest that dominant-negative p53 mutants accelerate development and/or growth of glioblastoma anlagen.

Detection of PTEN nonsense mutation and psiPTEN expression in central nervous system high-grade astrocytic tumors by a yeast-based stop codon assay.

We have developed a new yeast-based assay for the detection of PTEN nonsense mutation, and applied it to a total of 42 astrocytic tumors. The assay utilizes homologous recombination of PCR-amplified PTEN cDNA samples to a yeast vector which expresses an in-frame PTEN::ADE2 chimera protein. An allele of nonsense mutation in the sample PTEN mRNA gives a truncated chimera protein in a yeast cell, resulting in the formation of a red colony. The assay and subsequent sequence analysis demonstrated nonsense mutations as red colonies of more than 10% in one of 10 anaplastic astrocytomas and six of 18 glioblastomas, but none in six pilocytic astrocytomas or in eight astrocytomas. Sequence analysis of white colonies showed one missense mutation in a glioblastoma. Interestingly, four of seven nonsense mutations were frame-shifts due to exon skipping. In addition, pink colonies were found in one of six pilocytic astrocytomas, three of eight astrocytomas, two of 10 anaplastic astrocytomas, and 10 of 18 glioblastomas. Sequence analysis of the pink colonies revealed a sequence similar to those reported as psiPTEN/PTH2. By testing mRNA and genomic DNA, it was found to be a processed pseudogene which was transcribed. The psiPTEN expression was complementary to PTEN mutation, for 14 of 18 glioblastomas showed either PTEN mutation or psiPTEN expression and only one case showed both PTEN mutation and psiPTEN expression (P<0.046), suggesting a pathological role of psiPTEN expression as an alternative to PTEN mutation in glioblastomas.

High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay.

To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT-PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR-SSCP) of exon 5-8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR-SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.

Increased E1AF expression in mouse fibrosarcoma promotes metastasis through induction of MT1-MMP expression.

In this study, we investigated the role of E1AF, a member of ets family transcription factor, in the acquisition of metastatic capacity by non-metastatic mouse fibrosarcoma cell clone, QR-32. The QR-32 cell clone grows progressively after co-implantation with gelatin sponge in syngeneic C57BL/6 mice. The cell lines (QRsP) established from arising tumors after the co-implantation exhibited enhanced tumorigenicity and pulmonary metastasis in vivo as compared with parent QR-32 cells. The enhanced pulmonary metastasis of QRsP cells was correlated well with augmented production of matrix metalloproteinase-2 (MMP-2) and increased expression of membrane-type 1-MMP (MT1-MMP). The QRsP cells also acquired higher chemokinetic activities to fibronectin and higher invasive activities through a reconstituted basement membrane. Furthermore we observed the elevated mRNA expression of E1AF in QRsP cells compared to parent QR-32 cells. Therefore, we transfected QR-32 cells with E1AF cDNA. Overexpression of E1AF in the QR-32 cells resulted in the induction of MT1-MMP expression and converting an exogenously added precursor MMP-2 into active form. E1AF transfectants exhibited more motile and invasive activities, and moderately increased pulmonary metastatic activities than parental QR-32 cells in vivo, although their metastatic activities were lower than those of QRsP cells. These findings suggest that the increased expression of E1AF in fibrosarcoma contributes to invasive phenotypes including MT1-MMP expression and enhanced cell migration, but not sufficient for exhibiting highly metastatic activity in vivo.

Cloning and sequencing of the human homeobox gene HOX4A.

The HOX4A gene, one of the homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with a HOX4B cDNA probe. The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. According to conceptual translation, the HOX4A protein is predicted to be composed of 416 amino acid residues. Interestingly, the HOX4A protein has a sequence, Pro-Ala-Ser-Gln-Ser-Pro-Glu-Arg-Ser, eight amino acids downstream from the homeodomain, which is similar to that containing a phosphorylation site in pp60c-src, Pro-Ala-Ser-Gln-Thr-Pro-Asn-Lys-Thr. However, the HOX2G protein, which exhibits a paralogous relationship with the HOX4A protein, does not possess the sequence which is similar to that in pp60c-src. A comparison of the predicted HOX4A protein with the HOX2G protein revealed four regions of amino acid sequence similarities: an N-terminal tetrapeptide, a pentapeptide (pre-box) upstream of the homeodomain, the homeodomain and a C-terminal octapeptide.

Analysis of the 5' flanking region of the rat proliferating cell nuclear antigen (PCNA) gene.

The proliferating cell nuclear antigen (PCNA), highly conserved among eukaryotes, is an auxiliary factor for DNA polymerase delta. In this report we sequenced 1560 nucleotides (nt) of the 5' flanking region of the rat PCNA gene and located the transcription initiation site. The sequence contains 1435 nt upstream of the cap site and promotes transcription of a linked heterologous reporter gene in rat, mouse and human cells. Transient expression assays using a series of 5' deletion mutants revealed that 240 nt of the upstream sequence are sufficient for full promoter activity. Three GC boxes and several other binding sites of transcription factors were observed, but neither a TATA nor a CCAAT sequence was found in this region. The results also suggested the existence of a negative regulatory element(s) between -968 and -691. Cotransfection with early region 1 (E1) genes of human adenoviruses activated the expression of the reporter gene, suggesting that an E1-responsive element is located at the proximal promoter region within 81 nt upstream of the transcription initiation site.

[Experience with thoracoscopy for rifle gunshot penetrating trauma of the chest; report of a case].

A 57-year-old man came to our hospital by ambulance for a chest injury by a rifle gunshot. He had a penetrating injury of the chest wall, hemopneumothorax and pulmonary laceration. He was managed with chest drainage, oxygen inhalation. His respiratory and cardiac status was stable. However, for the purpose to prevent the development of empyema or pneumonia, and to check the existence of damage of intrathoracic structures by the gunshot injury, thoracoscopy was performed next day. He discharged without postoperative complications 17 days after the injury. Open thoracotomy is reported to be required in only about 10-15% of patients with chest injuries. However, operative indication of the chest injuries may spread in the future with the spread of thoracoscopy and its low invasiveness.

Induction of apoptosis in melanoma cell lines by p53 and its related proteins.

Melanoma cells rarely contain mutant p53 and hardly undergo apoptosis by wild-type p53. By using recombinant adenoviruses that express p53 or p53-related p51A or p73beta, we tested their apoptotic activities in melanoma cells. Yeast functional assay revealed a mutation of p53 at the 258th codon (AAA [K] instead of GAA [E]) in one cell line, 70W, out of six human melanoma cell lines analyzed (SK-mel-23, SK-mel-24, SK-mel-118, TXM18, 70W, and G361). Adenovirus-mediated transfer of p53, p51A, and/or p73beta suppressed growth and induced apoptotic DNA fragmentation of SK-mel-23, SK-mel-118, and 70W cells. Interestingly, p51A induced DNA fragmentation in them more significantly than p53 and p73beta. By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family members. Apoptotic Bax and antiapoptotic Bcl-2 were not significantly upregulated or downregulated by expression of p53, p51A, or p73beta, except for p53-expressing 70W cells, which contained a larger amount of Bax protein than LacZ-expressing cells. Activation of caspase-3 was demonstrated only in p51A-expressing SK-mel-118 cells. We show here that p51A can mediate apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and p73beta. It is also suggested that in melanoma cells (i) cellular target protein(s) other than Bcl-2 and Bax might be responsible for induction of p51A-mediated apoptosis and (ii) caspase-3 is not always involved in the apoptosis by p53 family members.

A novel control system for polymerase chain reaction using a RIKEN GS384 thermalcycler.

We have developed a novel high-throughput thermalcycler, the RIKEN GS384, which has a maximum of 1536 wells and whose temperature can be controlled accurately and simultaneously for a very small volume of a reaction mixture. In practice, the reaction is carried out using four 384-well (3.5 mm in diameter) plate formats which can be automatically moved using a robotic arm. To achieve accurate temperature control with high thermo-conductivity, we adopted Teflon-coated aluminum well plates closely sandwiched between silicon sheet-covered lids on top and a graphite sheet below. The lids were kept at a higher temperature (2 to 5 degrees C) than the reaction wells. The temperature of the 1536 sample wells was controlled accurately without temperature variability among the wells or evaporation, even for samples of very small volume (minimum 2 microliters). We also developed a new type of plate format which is similar to the 384-well place in terms of plate size, shape, and material, but which differs in the number (1536) and size (1.6 mm in diameter) of the wells. Since the amplification reactions could be done precisely as well, a total of 6144 reactions can potentially be carried out simultaneously using the GS384 thermalcycler. This is very promising for DNA microfabrication technology. This thermalcycler offers the advantage of high-throughput DNA analysis which should be useful for DNA diagnoses or for the human genome project.

The human plasma glutathione peroxidase-encoding gene: organization, sequence and localization to chromosome 5q32.

A genomic clone encoding the human plasma glutathione peroxidase (PGPx), a major enzyme in reducing lipid hydroperoxide and hydrogen peroxide in plasma, was isolated and 5618 nucleotides (nt) were determined. The nt sequence data revealed that the PGPx gene is composed of five exons spanning approx. 10 kb. Primer extension experiments mapped the transcription start point at 298 nt upstream from the predicted start codon. Twenty nt upstream from the polyadenylation site of the gene, an uncanonical polyadenylation signal, AGTAAA, was found. Human PGPx was localized on chromosome 5 band q32 by fluorescence in situ hybridization.

Interaction of MCC2, a novel homologue of MCC tumor suppressor, with PDZ-domain Protein AIE-75.

AIE-75 is a protein identified as an autoantigen in patients with autoimmune enteropathy and as a colon cancer-related antigen. It has recently been assigned to be a causative gene for Usher type 1C congenital syndromic hearing loss. The novel protein has three PSD-95/Dlg/ZO-1 (PDZ) protein-protein interaction domains and is therefore implicated to function as a molecular anchor or sorter. We have identified a novel protein that binds to AIE-75 by yeast two-hybrid screening. The protein has a high homology to the tumor suppressor MCC (mutated in colon cancer; or MCC1 hereafter) and was named MCC2. MCC2 protein binds the first PDZ domain of AIE-75 with its C-terminal amino acids -DTFL. Since the MCC1 does not bind to AIE-75 and the MCC2 displays different expression patterns in various organs compared to MCC1, they appear to play distinct roles in cells. The MCC2 gene is located on chromosome 19p13 in the vicinity of APCL gene, while MCC1 maps near to APC tumor suppressor gene. Because of negative expression of MCC2 in a panel of cancer cell-lines compared to the corresponding normal tissues, we suggest that further study is necessary to investigate a possible role of MCC2 as a tumor suppressor.