Masuo Yutsudo

Isolation of Transformation Suppressor Genes by cDNA Subtraction: Lumican Suppresses Transformation Induced by v-src and v-K-ras

ABSTRACT We have reported that suppressive factors for transformation by viral oncogenes are expressed in primary rat embryo fibroblasts (REFs). To identify such transformation suppressor genes, we prepared a subtracted cDNA library by using REFs and a rat normal fibroblast cell line, F2408, and isolated 30 different cDNA clones whose mRNA expression was markedly reduced in F2408 cells relative to that in REFs. We referred to these as TRIF (transcript reduced in F2408) clones. Among these genes, we initially tested the suppressor activity for transformation on three TRIF genes, TRIF1 (neuronatin), TRIF2 (heparin-binding growth-associated molecule), and TRIF3 (lumican) by focus formation assay and found that lumican inhibited focus formation induced by activated H-ras in F2408 cells. Colony formation in soft agar induced by v-K-ras or v-src was also suppressed in F2408 clones stably expressing exogenous lumican without disturbing cell proliferation. Tumorigenicity in nude mice induced by these oncogenes was also suppressed in these lumican-expressing clones. These results indicate that lumican has the ability to suppress transformation by v-src and v-K-ras.

Pro‐apoptotic ASY/Nogo‐B protein associates with ASYIP

We have previously shown that ectopic expression of the ASY/Nogo‐B gene induced apoptosis in various cancer cell lines. Nogo‐A, a splice variant of the ASY, has been reported to have an inhibitory effect on neuronal regeneration in the central nervous system. To investigate the mechanism of ASY‐induced apoptosis or inhibition of neuronal regeneration, we cloned a cDNA for the ASY‐interacting protein from the human cDNA library using the yeast two‐hybrid method, and obtained a cDNA we designated as ASYIP. The ASYIP protein contains two hydrophobic regions and a double lysine endoplasmic reticulum (ER) retrieval motif at its C‐terminus, which was shown to be identical to RTN3, a reticulon family protein of unknown function. We showed that ASY and ASYIP proteins formed a complex also in human cells. Mutational analysis indicated that both of the hydrophobic regions of the ASYIP protein were required for the association. By immunofluorescence analysis, the ASYIP protein was shown to be co‐localized with ASY in the ER. Characterization of the ASYIP gene may be very useful in clarifying the mechanism of ASY‐induced apoptosis or Nogo‐involved inhibition of neuronal regeneration in the central nervous system. J. Cell. Physiol. 196: 312–318, 2003.

Human papillomavirus type 17 DNA in skin carcinoma tissue of a patient with epidermodysplasia verruciformis.

Epidermodysplasia verruciformis (EV) is a serious skin disease caused by certain types of human papillomavirus (HPV), because the flat wart-like lesions of EV very frequently change to malignant squamous cell carcinoma. The relation between HPV and skin carcinoma was examined by studies on an EV patient who had a squamous cell carcinoma. HPV-17 was isolated from EV lesions of this patient. With HPV-17 DNA as a probe, cellular DNA prepared from the carcinoma tissue was analyzed by Southern blot hybridization. Results showed that cells contained about 100 copies of monomeric and oligomeric extrachromosomal HPV DNA. These results suggest that HPV-17 is involved in skin carcinogenesis in EV.

A novel apoptotic pathway induced by the drs tumor suppressor gene

The drs gene was originally isolated as a suppressor against v-src transformation. Expression of drs mRNA was markedly downregulated in a variety of human cancer cell lines and tissues, suggesting that the drs gene acts as a tumor suppressor. In this study, we found that ectopic expression of the Drs protein induced apoptosis in human cancer cell lines. Analyses using deletion mutants of drs revealed that both the C-terminal region and the three consensus repeats in the N-terminal region are essential for the induction of apoptosis. Caspase-12, -9, and -3 were sequentially activated by drs, and specific inhibitors of caspase-3 and -9 suppressed drs-induced apoptosis. The release of cytochrome c from the mitochondria into the cytoplasm was not observed in apoptosis by drs, suggesting that the mitochondrial pathway does not mediate drs-induced apoptosis. Furthermore, we found that the Drs protein can interact with ASY/Nogo-B/RTN-xS, an apoptosis-inducing protein localized in the endoplasmic reticulum, and that coexpression of these genes increased the efficiency of apoptosis. These results indicated that Drs induces apoptosis by a novel pathway mediated by ASY/Nogo-B/RTN-xS, caspase-12, -9, and -3.

Detection of Epstein-Barr virus transcripts in anaplastic large-cell lymphomas by mRNA in situ hybridization

Anaplastic large-cell lymphoma (ALCL) is a recently proposed subset of non-Hodgkins lymphoma. To determine whether Epstein-Barr virus (EBV) is associated with this lymphoma, we performed mRNA in situ hybridization on seven cases of ALCL using a probe consisting of an RNA sequence complementary to the transcripts of BamHIW fragment of the EBV genome. We detected BamHIW transcripts of EBV in the majority of atypical large cells of all cases of ALCL, but in none of three cases of lymphoblastic and small lymphocytic lymphomas. Furthermore, we detected latent membrane protein-1 (LMP1) in two cases of ALCL by means of immunofluorescence and immunoperoxidase stainings. These findings suggest that EBV is involved in the neoplastic transformation for ALCL as in the case of Hodgkins disease, which shares several clinicopathologic features with ALCL.

Expression of latent and replicative-infection genes of Epstein-Barr virus in macrophage

SummaryUnlike other herpesviruses, Epstein-Barr virus (EBV) has not yet been shown to infect macrophages. Six macrophage cultures were isolated from normal and affected samples. Nested polymerase chain reaction revealed the existence of the EBV genome in all these macrophages. EBV latent genes expression in all cultures were detected by mRNA in situ hybridization and immunofluorescence staining. Some cultures also expressed EBV replicative-infection proteins, while in other cultures induction of these proteins was demonstrated. These findings are the first to show expression of several latent and replicative-infection genes of EBV in macrophages, indicating that EBV proliferates in macrophages.

Suppression of tumorigenicity, but not anchorage independence, of human cancer cells by new candidate tumor suppressor gene CapG.

Previously, we isolated a series of cell lines from a human diploid fibroblast lineage as a model for multistep tumorigenesis in humans. After passaging a single LT-transfected fibroblast clone, differently progressed cell lines were obtained, including immortalized, anchorage-independent and tumorigenic cell lines. In the present paper, we analysed the gene expression profiles of these model cell lines, and observed that expression of the CapG protein was lost in the tumorigenic cell line. To examine the possibility that loss of CapG protein expression was required for tumorigenic progression, we transfected CapG cDNA into the tumorigenic cell line and tested for tumor-forming ability in nude mice. Results showed that ectopic expression of CapG suppressed tumorigenicity, but not growth in soft agar or liquid medium. We also found that certain cancer cell lines including stomach cancer, lung cancer and melanoma had also lost CapG expression. One such cancer cell line AZ521 also became non-tumorigenic after the introduction of CapG cDNA. Moreover, we showed that CapG expression was repressed in small-cell lung cancer tissues. Together, our findings indicated that CapG is a new tumor suppressor gene involved in the tumorigenic progression of certain cancers.

Involvement of Epstein-Barr virus expression in testicular tumors

PURPOSE Because orchitis has been described as a symptom of infectious mononucleosis which is caused by Epstein-Barr virus (EBV), a human tumor virus, we tried to ascertain the relationship between EBV and testicular tumors. MATERIALS AND METHODS Sixteen seminomas, 11 embryonal carcinomas and 25 nonmalignant control testes were examined for persistence and expression of the EBV genome. To detect expression of EBV, mRNA in situ hybridization and immunofluorescence staining by monoclonal antibodies were performed. To confirm the EBV genome in testes, we used nested polymerase chain reaction (PCR). RESULTS Messenger RNA in situ hybridization showed that all 27 seminomas and embryonal carcinomas expressed EB viral RNA, but the 25 nonmalignant testes did not. Monoclonal antibody staining showed EBV-related nuclear antigen (EBNA) 2 and latent membrane protein (LMP) 1 expression in testicular tumors. Nested polymerase chain reaction detected the EBV genome in normal testes as well as in testicular tumors. CONCLUSIONS These results suggest that EBV is related to testicular tumors.

Expression of Epstein-Barr virus in mesopharyngeal and hypopharyngeal carcinomas

Epstein-Barr virus (EBV) causes various tumors, including nasopharyngeal carcinoma (NPC). There have been no reports as to whether the carcinogenicity of EBV is restricted to the nasopharynx or extends into the mesopharynx and hypopharynx. We attempted to ascertain the relation between EBV and mesopharyngeal (MPC) and hypopharyngeal carcinomas (HPC). Messenger RNA in situ hybridization showed that all 29 cases of MPC and 5 of 12 HPC expressed EBV mRNA. For further analysis, we established 7 cell lines from 5 MPC and 2 HPC. All cell lines and 5 tumors formed by these cultured cells in nude mice expressed EBV transcripts. Moreover, immunofluorescence staining showed expression of EBV-related nuclear antigen-2 and latent membrane protein-1 (LMP1) in the original tumors and the cell lines, as well as in nude mouse tumors. Study by reverse transcription polymerase chain reaction (RT-PCR) also showed EBER1 and LMP1 expression. Furthermore, lytic-cycle antigens of EBV were detectable in most cell lines. Nested PCR showed the EBV genome in 3 cases of MPC and 4 cases of HPC. These results suggest that EBV plays an important role in the development of MPC and HPC as well as in NPC.

Rat mutant cells showing temperature sensitivity for transformation by wild-type Moloney murine sarcoma virus.

To clarify the cellular target(s) of onc gene products of Moloney murine sarcoma virus (Mo-MSV), we isolated seven mutant cells that exhibit temperature sensitivity for transformation by wild-type Mo-MSV from Fischer rat cell line. Five strains of these mutant cells showed normal virus production at the nonpermissive temperature when infected with Mo-MSV, suggesting that viral replication is not affected by these cellular mutations. Four of these mutants were also temperature sensitive (ts) for transformation by Kirsten murine sarcoma virus (Ki-MSV), whereas the other three mutants were not ts, suggesting that our mutants isolated with Mo-MSV can be divided into two classes as regards temperature sensitivity to transformation by Ki-MSV.