Significant role of macrophages in human cancers associated with Epstein-Barr virus (Review).

Epstein-Barr virus (EBV) is a ubiquitous pathogen that was first identified as a human cancer virus. Many human cancers are associated with EBV, and we demonstrated that EBV infects macrophages. Macrophages infected with EBV show a close correlation with many human cancers, and thus more attention must be given to the role of macrophages infiltrating into cancer tissues associated with EBV. In this review, I discuss the role of macrophages in the process of EBV-associated oncogenesis with regard to interleukin-10.

Involvement of NANOG upregulation in malignant progression of human cells.

Previously, we isolated cell lines that display various degrees of transformed phenotypes from a single-cell population of human diploid fibroblasts (RB) containing a large deletion (13q14-22) in one copy of chromosome 13. They included a cell line transfected with SV40 early genes (RBSV), an immortalized cell line (RBI), an anchorage-independent cell line (RBS), and a tumorigenic cell line (RBT). Here, we analyzed gene expression profiles in these cell lines and showed that expression of some fibroblast-specified or mesenchyme-specified genes were downregulated, and those of stem cell-specified genes, including NANOG, were upregulated during malignant progression. When NANOG expression was knocked down with a short hairpin NANOG expression vector (shNANOG vector) in the RBS and RBT cells, the anchorage independency and tumorigenicity were repressed. We next examined various cancer cell lines for NANOG expression and showed that some cancer cell lines expressed a high level of normal and/or variant NANOG proteins. Overexpression of NANOG mRNA in lung adenocarcinoma was also shown by in situ hybridization. All these data indicate the involvement of NANOG in tumorigenesis.

Macrophage involvement in Epstein-Barr virus-related tumors.

Epstein-Barr virus (EBV) is known as a causative agent of Burkitt's lymphoma, nasopharyngeal carcinoma and approximately 10% of stomach carcinoma cases. In other human cancers, EBV gene expression including lytic infection protein detected using in situ hybridization and immunofluorescence staining has been reported. Moreover, the expression and replication of EBV genes in cultured normal macrophages and in histiocytes of Langerhans' cell histiocytosis have been identified. The aim of this study was to examine EBV expression in macrophages in other EBV-associated human tumors. Forty-one cases of EBV-associated tumors, which had been confirmed to express EBV, were examined. Tissue sections after in situ hybridization were double-stained immunohistochemically with the monoclonal anti-CD68 antibody. EBV expression in macrophages in the lesions of nasopharyngeal carcinoma, oral cancer, thyroid carcinoma, renal cell carcinoma, testicular carcinoma, uterine carcinoma, cutaneous T-cell lymphoma and anaplastic large-cell lymphoma was identified, whereas macrophages in normal or non-cancerous lesions showed no EBV expression. Many tumor-associated macrophages in EBV-related tumors carry EBV, which appears to induce the EBV lytic infection of macrophages. Therefore, the possibility that the lytic infection of macrophages by EBV and the resulting inflammation play certain roles in the oncogenesis of EBV-associated human tumors was raised.

Downregulation of drs tumor suppressor gene in highly malignant human pulmonary neuroendocrine tumors.

Neuroendocrine tumors in the lung fall into four categories: typical carcinoid tumor (TC), atypical carcinoid tumor (AC), large-cell neuroendocrine carcinoma (LCNEC) and small-cell lung carcinoma (SCLC), in ascending order of malignancy. The drs gene was originally isolated as a suppressor against v-src transformation and was shown to induce apoptosis in human cancer cells. The expression of drs was markedly downregulated in various human cancer tissues and cell lines. Furthermore, drs knockout mice showed a tumor-prone phenotype, indicating that drs acts as a tumor suppressor gene in malignant tumor formation. To clarify the role of the drs gene in the development of human pulmonary neuroendocrine tumors, we examined the expression of drs mRNA in tissue specimens from 3 cases of TC, 4 cases of AC, 2 cases of LCNEC, and 11 cases of SCLC by in situ mRNA hybridization. Four cases of normal lung and bronchial epithelia, 8 samples of normal brain tissue, and 2 cases of tumorlets in the lung were also examined. The drs mRNA was definitely expressed in all normal tissues of the lung and brain, and 3 TC and 2 tumorlet tissues. The expression of drs mRNA was also detected in 2 of 2 LCNEC tissues and 3 of 4 AC tissues, although the signals were weak. On the other hand, drs mRNA was not detected in 10 of 11 SCLC tissues. Downregulation of drs mRNA was also observed in 3 of 4 SCLC cell lines that were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Neither gross deletion nor rearrangement of the drs genome was detected in these cell lines by Southern blot analysis. Our results indicate that the downregulation of drs is correlated with the development of SCLC, a highly malignant pulmonary neuroendocrine tumor.

Expression of Epstein-Barr virus in renal cell carcinoma.

There have been few studies regarding the etiology of renal cell carcinoma. To examine the possible involvement of Epstein-Barr virus (EBV) in this disease, 9 renal cell carcinoma (RCC), 2 nephroblastoma (Wilms' tumor) and 2 RCC cell lines were subjected to mRNA in situ hybridization and indirect immunofluorescence staining. Messenger RNA in situ hybridization using BamHIW, EBNA LP, EBNA 2 and EBER1 probes of EBV revealed signals in all the examined samples, although some samples showed weak signals using the EBNA LP probe. Indirect immunofluorescence staining using anti-EBNA LP, anti-EBNA2, anti-LMP1 and anti-BZLF1 antibodies showed definitive fluorescence. PCR also revealed EBV DNA in all 8 RCC specimens including 7 cases other than hybridization and fluorescence. EBV infected all the RCC and nephroblastoma irrespective of the histological or clinical stage. On the other hand, EBV expression was stronger in papillary and clear cell-type RCC than chromophobe cell-type, as well as being stronger in the higher grades of RCC. These results suggest that the expression of EBV may be involved in the pathogenesis of RCC and nephroblastoma.

Downregulation of drs mRNA expression is associated with the progression of adult T-cell leukemia/lymphoma.

Although adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-cell leukemia virus (HTLV-1), many other factors are thought to be required for the progression from indolent ATL to aggressive ATLL. The drs gene was originally isolated as a novel suppressor gene of v-src transformation and was shown to induce apoptosis in human cancer cells. To investigate the involvement of drs downregulation in the progression of ATLL, we examined the expression of drs in smoldering, chronic and aggressive ATLL, and found that drs expression was markedly reduced in clinically aggressive ATLL. In aggressive ATLL cell lines, expression of drs mRNA was not detected, although expression of drs mRNA was detected in T-cell lines infected with HTLV-1. A correlation between drs downregulation and expression of the Tax gene was not observed in these T-cell lines. Furthermore, introduction of drs into an ATL cell line, HUT102, by retrovirus vector suppressed the colony formation of the cells in soft agar and enhanced apoptotic cell death of the cells under low serum culture conditions. These results indicate that downregulation of drs is closely linked to the progression of ATLL, independently of Tax expression, suggesting that drs may suppress the progression of ATLL via enhancing apoptosis.

Expression of the Epstein-Barr virus in lymphoproliferative diseases of the lung.

There have been few studies regarding the etiology of lymphoproliferative disorders of the lung. To examine the possible involvement of the Epstein-Barr virus (EBV) in these diseases, EBV mRNAs, proteins and DNA, were detected. Two non-Hodgkin's lymphomas (NHL) originating in the lung, 5 mucosal-associated lymphoid tissue lymphomas (MALT lymphoma) of the lung, 1 lymphoid hyperplasia of the lung and 1 lymphoid interstitial pneumonia (LIP), were subjected to mRNA in situ hybridization, indirect immunofluorescence staining and PCR. mRNA in situ hybridization using BamHIW, BamHIY1Y2, the Epstein-Barr virus nuclear antigen (EBNA) and the EBV encoded small non-polyadenylated RNA (EBER1) probe revealed signals in all the examined samples, although some samples showed weak signals by using the EBER1 probe. Indirect immunofluorescence staining using the anti-leader protein, anti-EBNA2, the anti-latent member protein-1 and anti-BamHIZ coding leftward reading frame-1 antibodies showed definitive fluorescence. PCR also revealed EBV DNA in these specimens. EBV infected all the lymphoproliferative diseases of the lung irrespective of the histological or clinical stages. Furthermore, EBV infected not only the atypical lymphocytes but also the macrophages in the tissues of these diseases. These results suggest that the expression of EBV could be involved in the pathogenesis of many lymphoproliferative diseases of the lung.

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.

Expression of Epstein-Barr virus in Langerhans' cell histiocytosis.

Langerhans' cell histiocytosis (LCH) is a proliferative histiocytic disorder of unknown etiology. We previously reported that Epstein-Barr virus (EBV) infects and proliferates in macrophages, and investigated the possibility that EBV exhibits etiologic effects in LCH. To detect EBV expression, paraffin sections from 17 LCH cases were examined by mRNA in situ hybridization for EBV BamHIW, Epstein-Barr virus nuclear antigen-2 (EBNA2), and Epstein-Barr virus-encoded small nonpolyadenylated RNA (EBER1) sequences, and by indirect immunofluorescence staining for EBNA2, latent membrane protein 1 (LMP1), and BamHIZ-coding leftward-reading frame 1 (BZLF1). To detect EBV DNA, polymerase chain reaction (PCR)-Southern blotting was used. All cases showed positive hybridization signals by BamHIW mRNA in situ hybridization. Also, 13 and 14 cases showed positive signals for EBNA2 and EBER1 RNA in situ hybridization, respectively. Furthermore, almost all cases exhibited fluorescence after immunofluorescence staining with monoclonal anti-EBNA2 and anti-BZLF1 antibodies, and 15 cases were positive after treatment with monoclonal anti-LMP1 antibody. PCR-Southern blotting detected an amplified EBER1 sequence in all 9 cases examined. EBV expression was confirmed in LCH using in situ hybridization and immunofluorescence. Furthermore, EBV DNA was also detected by PCR-Southern blotting. These positive results of BZLF1 suggest that EBV replicates in LCH tissues.

Expression of Epstein-Barr virus in thyroid carcinoma correlates with tumor progression.

There have been few studies regarding cancer progression from differentiated thyroid carcinoma to the undifferentiated one. To examine the possible involvement of Epstein-Barr virus (EBV) in this progression, 10 papillary carcinomas and 11 undifferentiated carcinomas were subjected to mRNA in situ hybridization, indirect immunofluorescence staining, polymerase chain reaction (PCR), and reverse-transcriptase PCR. mRNA in situ hybridization using a BamHIW probe revealed signals in all of the examined samples, although the signal strength was weaker in the papillary carcinomas than in the undifferentiated carcinomas. EBV nuclear antigen-2 (EBNA2) in situ hybridization produced almost the same results; however, the signals were detected less frequently in the papillary carcinomas. Indirect immunofluorescence using anti-EBNA2, anti-latent membrane protein-1 (LMP1), and anti-BZLF1 antibodies also showed positive results with high frequency and with more prominent fluorescence in undifferentiated carcinomas than in papillary carcinomas. An examination of thyroid carcinoma cell lines also confirmed these findings. EBV infected all of the thyroid carcinomas irrespective of the degree of pathological differentiation. The expression of EBV, especially of EBNA2 and LMP1 (both of which are oncogene products of EBV), was stronger in the undifferentiated carcinomas than in the papillary carcinomas. These results suggest that increased expression of EBV may be involved in the progression of thyroid papillary carcinoma to undifferentiated carcinoma.

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.

Suppression of anchorage-independent growth of human cancer cell lines by the TRIF52/periostin/OSF-2 gene.

In searching for genes that suppress the viral transformation of primary cells, we have isolated a number of TRIF (transcript reduced in F2408) genes that are expressed well in primary rat embryo fibroblasts (REFs) but poorly in spontaneously immortalized rat fibroblast cell lines derived from REFs. One of these genes, TRIF52, is a rat homologue of the mouse protein periostin, which is suspected of being involved in oncogenesis. We found here that periostin mRNA expression is markedly downregulated in a variety of human cancer cell lines and human lung cancer tissues. Human cancer cell lines with reduced endogenous periostin gene expression that were infected with a recombinant retrovirus containing the periostin gene had reduced anchorage-independent growth. Mutational analysis revealed that the C-terminal region of periostin is sufficient to convey the anchorage-independent growth-suppressive activity of the protein. These observations together suggest that periostin may serve to inhibit the development of human cancers by acting as a tumor suppressor.

Association of Epstein-Barr virus with oral cancers.

Epstein-Barr virus (EBV) persists in the epithelial cells of oral mucosa and often replicates on them. EBV is known to be a causative agent of nasopharyngeal carcinoma. We suspect that EBV may be associated with oral cancers, and thus examined EBV expression on 28 tongues and 9 other oral cancers. We also examined 6 metastatic lesions in the lymph nodes. All cancers were squamous cell carcinoma (SCC). We used mRNA in situ hybridization, immunofluorescence staining, reverse transcriptase-polymerase chain reaction (RT-PCR), and polymerase chain reaction (PCR). The mRNA in situ hybridization using a probe comprising the transcripts of the BamHIW fragment of the EBV genome demonstrated EBV mRNA in the majority of tumor cells in all cases of oral cancer, but in none of the normal tissues. RNA in situ hybridization using an EBER1 probe detected RNAs in 16 out of 24 cancers. Also, mRNA in situ hybridization using a probe of the EBV-determined nuclear antigen-2 (EBNA2) region detected positive signals in 9 out of 12 cancers. Furthermore, EBNA2, latent membrane protein-1 (LMP1) and BZLF1 were detected in these cancers by immunofluorescence staining, but were not detected in any of the epithelial cells of the normal tissues. Four out of 6 metastatic tissues showed stronger fluorescence than that in the primary tissues. RT-PCR analysis also showed EBER1 expression in 1 of the 3 tongue cancers. PCR detected the BamHIW sequence of EBV DNA in all cases, including the normal tissues tested. These findings indicate that EBV may be involved in neoplastic transformation in oral cancers, such as nasopharyngeal carcinoma.

Expression of drs mRNA in human lung adenocarcinomas.

The drs gene was originally isolated from a rat primary embryo fibroblast cDNA library as a suppressor gene against v-src transformation. We have previously shown that expression of drs mRNA was markedly reduced in a variety of human cancer cell lines, including those of the colon, bladder, and ovary. Furthermore, introduction of drs cDNA by retrovirus vector into these cancer cell lines caused suppression of anchorage-independent growth without affecting cell proliferation. These findings suggest that down-regulation of drs mRNA is closely correlated with expression of malignant phenotypes in development of human cancers. To clarify the correlation between down-regulation of drs mRNA and malignant tumor formation in human tumor tissues, we examined the expression of drs mRNA in well-differentiated, moderately differentiated, and poorly differentiated lung adenocarcinoma tissues by in situ mRNA hybridization. The results clearly indicated that expression of drs mRNA was markedly reduced in 5 of 5 poorly differentiated lung adenocarcinomas examined but significantly expressed in normal lung tissues and 5 of 7 moderately-differentiated and 3 of 5 well-differentiated lung adenocarcinoma tissues. Neither gross deletion nor rearrangement of the drs genome was detected in these tissues. Down-regulation of drs mRNA was also observed in human lung adenocarcinoma cell lines derived from poorly differentiated adenocarcinomas. Our results suggest that down-regulation of drs mRNA is correlated with a poor degree of differentiation and progression of lung adenocarcinoma.

Down-regulation of drs mRNA in colorectal neoplasms.

The drs gene was originally isolated as a transformation suppressor gene against the v-src oncogene. Expression of drs mRNA is down-regulated by retroviral oncogenes such as v-src and v-K-ras in the rat cell line F2408. Expression of drs mRNA is also 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 colorectal neoplasms, we examined expression of drs mRNA in a variety of colon cancer tissues by in situ hybridization. A total of 53 morphologically distinct neoplastic specimens were divided into the following five groups according to morphology: low and high grade adenoma in 7 and 12 cases, respectively (groups A, B), protruded-type carcinoma in 16 (group C), superficial-type carcinoma with an adenomatous component in 10 (group D) or superficial-type carcinomas without any adenomatous component in 8 (group E). Expression of drs mRNA was detected in normal mucosa, low-grade adenoma and most superficial-type carcinomas without any adenomatous component. On the other hand, the rate of drs mRNA expression was significantly lower in protruded-type adenocarcinoma and superficial-type carcinoma with an adenomatous component. Our results indicate that down-regulation of drs mRNA is closely correlated with carcinomas which arise from adenomatous polyps in the course of the adenoma-carcinoma sequence, but that most carcinomas arising de novo are independent of the tumor suppressor function of the drs gene.