Misuzu Shimakage

Link of a new type of apoptosis-inducing gene ASY/Nogo-B to human cancer.

Although apoptosis plays an essential role in the embryogenesis and homeostasis of multicellular organisms, this mechanism has not yet been fully clarified. We isolated a novel human apoptosis-inducing gene, ASY, which encodes an endoplasmic reticulum-targeting protein without any known apoptosis-related motifs. This gene is identical to the Nogo-B, a splice variant of the Nogo-A which has recently been shown to be an inhibitor of neuronal regeneration in the central nervous system. Ectopic expression of the ASY gene led to extensive apoptosis, particularly in cancer cells. Furthermore, transcription of the ASY gene was suppressed in small cell lung cancer. These results suggest that a new type of apoptosis-inducing gene, namely, ASY, may be involved in the development of certain types of cancer.

Expression of Epstein-Barr virus in cutaneous T-cell lymphoma including mycosis fungoides.

Cutaneous T‐Cell lymphoma (CTCL) is a non‐Hodgkins lymphoma of unknown pathogenesis. Mycosis fungoides (MF) is a clinically determined subset of CTCL with intensive infiltration of lymphoma cells into the epidermis. To determine whether Epstein‐Barr virus (EBV) is associated with these lymphoma cells, we performed mRNA in situ hybridization in 5 cases of CTCL and 7 cases of MF using an RNA probe transcribed from BamHI W fragment of EBV genome. These transcripts were detected in the majority of lymphoma cells in all cases examined. We also detected intensive hybridization signals on epidermal squamous cells contiguous to strong infiltration with lymphoma cells into the subcutaneous connective tissue. Similarly, positive signals were detected using the probes transcribed from the sequences of EBV‐encoded small nonpolyadenylated RNAs‐1 (EBER1) and EBV‐determined nuclear antigen‐2 (EBNA2). The EBNA2 latent membrane protein‐1 (LMP1) and BZLF1 product (ZEBRA) were also detected by immunofluorescence staining using monoclonal antibodies. Further in the same experiment, we detected immunofluorescence of epidermal cells. EBV DNA was detected in all cases tested by DNA in situ hybridization. Moreover, we also identified the signals on epidermal cells via this technique. Polymerase chain reaction revealed amplified EBV DNA for most cases tested. Double staining with immunohistochemistry and RNA in situ hybridization showed that T‐cell marker‐positive cells, but not EBV‐carrying B‐cells, exhibited signals for the EB viral RNA. These findings suggest that EBV is involved in the neoplastic transformation of CTCL and MF.

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.

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.

TUMORIGENICITY OF EBNA2-TRANSFECTED CELLS

The Epstein‐Barr virus nuclear antigen 2 (EBNA2) gene is thought to be important for transformation by Epstein‐Barr virus (EBV), but the mechanism of this transformation is little understood. Here, to examine the transforming ability of EBNA2, we transfected a rat fibroblast cell line F2408 with a recombinant EBNA2 expression plasmid and examined cell morphology, colony formation in soft agar, and tumorigenicity in nude mice. The morphology of transfected clones was similar to those of untransfected cells, but two of seven clones grew in soft agar, and four clones of seven clones reproducibly formed tumors in nude mice. These four clones showed EBNA2 expression, but non‐tumorigenic clones did not. These results indicate that the expression of EBNA2 is correlated with tumorigenicity.

Down-regulation of drs mRNA in human prostate carcinomas

We have previously reported that the drs gene has the ability to suppress transformation by v-src and v-K-ras in the rat cell line F2808. We have also shown that the expression of drs mRNA is markedly reduced in a variety of human cancer cell lines, suggesting that down-regulation of drs mRNA is correlated with the development of human cancers. To clarify the role of the drs gene in prostate carcinogenesis, we examined the expression of the drs gene in 3 normal prostate, 13 prostate carcinoma, 5 benign prostate hyperplasia (BPH), and 2 prostatic intraepithelial neoplasia (PIN) tissue specimens by in situ hybridization and in 3 prostate carcinoma cell lines (PC3, LNCaP, and DU145) and 2 BPH tissues by Northern blot analysis. Furthermore, the deletion, and rearrangement of the drs gene were analyzed by Southern blot analysis. The drs mRNA was significantly expressed in normal prostate and BPH tissues, whereas it was markedly down-regulated in prostate carcinoma tissues and prostate carcinoma cell lines. In 2 tissues from PIN, drs mRNA was weakly expressed. There were no differences between prostate carcinoma cell lines and BPH tissues in terms of their banding patterns of Southern blot analysis. These results indicate that down-regulation of drs mRNA is closely correlated with development of prostate carcinoma, suggesting a tumor-suppressor function of the drs gene in this cancer.

Down-regulation of drs mRNA in human colon adenocarcinomas

We have previously reported that the drs gene, whose mRNA expression is down‐regulated by retroviral oncogenes such as v‐src and v‐K‐ras, has the ability to suppress transformation by v‐src and v‐K‐ras in the rat cell line F2408. We have also isolated a human homolog of this gene (h‐drs) and shown that expression of h‐drs mRNA is markedly reduced in a variety of human cancer cell lines, including those of carcinomas of the colon, bladder, and ovary, suggesting that down‐regulation of drs mRNA is correlated with the development of human cancers. To clarify the correlation between down‐regulation of the drs gene and malignant tumor formation in human cancer tissues, we examined expression of drs mRNA in human normal tissues, colon adenoma, and adenocarcinoma tissues by in situ hybridization. Expression of drs mRNA was detected in most normal tissues tested, including those of the colon, bladder, and ovary. However, drs mRNA was hardly expressed in any of the colon adenocarcinoma tissues examined. Northern blot analyses confirmed these results. Neither gross deletion nor re‐arrangement of the drs genome was detected by Southern blot hybridization in these adenocarcinoma tissues. drs mRNA was significantly expressed in colon adenoma with mild atypia but down‐regulated in adenomas with moderate atypia and focal carcinoma. Our results indicate that down‐regulation of drs mRNA is closely correlated with development of colon adenocarcinoma, suggesting a tumor‐suppressor function of the drs gene in human cancers. Int. J. Cancer 87:5–11, 2000.

Down-regulation of ASY/Nogo transcription associated with progression of adult T-cell leukemia/lymphoma

Adult T‐cell leukemia/lymphoma (ATLL) is an aggressive form of human leukemia/lymphoma. Although this disease is initiated by infection with human T‐lymphotropic virus type 1 (HTLV‐1), many HTLV‐1 carriers survive for a long period without aggressive illness, suggesting that other factors may play roles in the progression of ATLL to an aggressive state. However, the mechanism involved in this progression still remains unclear. Previously, we have reported that ASY/Nogo mRNA was markedly down‐regulated in human small‐cell lung carcinomas, whereas it was expressed in normal tissues and other lung carcinomas, such as adenocarcinoma and squamous cell carcinoma. To understand whether or not ASY/Nogo gene is involved in the progression of ATLL, we examined the expression of ASY/Nogo mRNA in smoldering, chronic and aggressive ATLL, and found that the expression level of ASY/Nogo mRNA was markedly reduced in clinically aggressive ATLL. HTLV‐1 Tax expression was not affected by the down‐regulation of ASY/Nogo mRNA. These results indicate that the ASY/Nogo gene may act as a suppressor against ATLL progression, independent of Tax expression.

Detection of Epstein-Barr virus-determined nuclear antigen-2 mRNA by in situ hybridization

A mRNA in situ hybridization method was developed to detect mRNA of Epstein--Barr virus-determined nuclear antigen-2 (EBNA2). Strong in situ hybridization signals were detected in EBNA2-transfected cells with the antisense probe but not with the sense probe of this mRNA. Hybridization signals were also found in formalin-fixed paraffin-embedded tissue samples from patients with invasive uterine cervical cancer and with cervical intraepithelial neoplasia. The sensitivity of mRNA in situ hybridization was higher than that of Northern blotting, and almost the same as that of immunofluorescence staining using monoclonal anti-EBNA2 antibody. It is concluded that this method is useful for sensitive and convenient detection of EBNA2.