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.

Multi-functional gene ASY/Nogo/RTN-X/RTN4: apoptosis, tumor suppression, and inhibition of neuronal regeneration.

ASY, also designated Nogo, RTN-X, or RTN4, is a reticulon family protein containing two transmembrane domains and a C-terminal double lysine endoplasmic reticulum (ER) retrieval motif. Three protein variants are synthesized from the cognate mRNAs produced by alternative splicing, and are expressed in almost all tissues, localizing predominantly in the ER. The ASY protein induces apoptosis in various cancer cells when overexpressed, whereas normal cells are relatively resistant to ASY-dependent apoptosis. Furthermore, transcription of this gene is suppressed in certain types of cancers, suggesting that ASY may act to suppress tumor development. Although the physiological function of this protein has not been well defined, Nogo-A protein, the large variant of ASY, has been shown to inhibit neuronal regeneration in the central nervous system. Therefore, the products of this gene may be multi-functional, regulating apoptosis, tumor development, and neuronal regeneration.

Human peripheral blood polymorphonuclear leukocytes at the ovulatory period are in an activated state.

The ovulatory process has been compared with inflammation. We investigated the state of female peripheral polymorphonuclear leukocytes (PMN) during menstrual cycles, and found that PMN contained high levels of superoxide, hydrogen peroxide and nitric oxide (NO) during at the peri-ovulatory period. Assuming the cause of this elevation to be a luteinizing hormone (LH), the surge of which preceded the ovulation, we examined the responsiveness of PMN to pituitary LH. The results revealed that this hormone elevated dose-dependently the production of reactive oxygen intermediates (ROI). Furthermore, we demonstrated the mRNA expression of LH receptors and their presence on PMN. The data indicated that the LH surge before on the ovulatory day resulted in general activation of PMN, suggesting that this state of PMN may be a necessary step for initiation of ovulation, rather than a defensive role against infection.

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-Hodgkin's 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.

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.

Isolation of transformation suppressor genes by cDNA subtraction: lumican suppresses transformation induced by v-src and v-K-ras.

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.

Sp1 cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene (hTERT).

Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. The human telomerase catalytic subunit (hTERT) is a rate limiting determinant of the enzymatic activity of human telomerase. In the previous study, we identified the proximal 181 bp core promoter responsible for transcriptional activity of the hTERT gene. To identify the regulatory factors of transcription, transient expression assays were performed using hTERT promoter reporter plasmids. Serial deletion assays of the core promoter revealed that the 5'-region containing the E-box, which binds Myc/Max, as well as the 3'-region containing the GC-box, which binds Sp1, are essential for transactivation. The mutations introduced in the E-box or GC-box significantly decreased transcriptional activity of the promoter. Overexpression of Myc/Max or Sp1 led to significant activation of transcription in a cell type-specific manner, while Mad/Max introduction repressed it. However, the effects of Myc/Max on transactivation were marginal when Sp1 sites were mutated. Western blot analysis using various cell lines revealed a positive correlation between c-Myc and Sp1 expression and transcriptional activity of hTERT. Using fibroblast lineages in different stages of transformation, we found that c-Myc and Sp1 were induced to a dramatic extent when cells overcame replicative senescence and obtained immortal characteristics, in association with telomerase activation. These findings suggest that c-Myc and Sp1 cooperatively function as the major determinants of hTERT expression, and that the switching functions of Myc/Max and Mad/Max might also play roles in telomerase regulation.

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.

Malignant transformation of human diploid fibroblasts and suppression of their anchorage independence by introduction of chromosome 13.

Isolation of cell lines that display various degrees of transformed phenotypes may be very useful to clarify multistep mechanisms of oncogenesis, but malignant transformation of human diploid fibroblasts in culture is a very rare event. We attempted to isolate variously transformed cell lines from human diploid fibroblasts (RB) of a patient with hereditary retinoblastoma. The RB cells exhibited normal karyotypes with the exception of one copy of chromosome 13, which contained a large deletion at the q14-22 region, where the RB1 gene is located. By transfection with SV40 early genes and repeated passage, we succeeded in obtaining SV40-transfected mortal, immortalized, anchorage-independent, and tumorigenic RB cell lines. DNA fingerprinting showed that these cell lines were not contaminants, but derivatives of the original RB cells. The remaining RB1 allele may be wild-type even in the malignant cell lines, because the expression and the LT-binding ability were normal. Furthermore, we did not find any homozygous loss in 16 polymorphic markers located in the 13q14-22 region in the transformed cell lines. However, introduction of a copy of a normal chromosome 13 into the anchorage-independent cell line suppressed its anchorage-independent growth ability. All these data, together with the fact that the RB cells containing the deletion progressed to a tumorigenic state spontaneously, but normal fibroblasts did not, raise the possibility that a new tumor suppressor gene, located at 13q14-22, may play a critical role in neoplastic transformation. We conclude that these RB cell lines provide an excellent system for identification of genes involved in malignant transformation of human cells. Genes Chromosomes Cancer 26:47-53, 1999.

Tumorigenic conversion resulting from inhibition of apoptosis in a nontumorigenic HeLa-derived hybrid cell line.

Although tumorigenicity in nude mice is one of the most important transformed phenotypes, its mechanism has been little analyzed. To understand the molecular basis of tumorigenicity, we characterized nontumorigenic CGL1 and tumorigenic CGL4 cell lines, both of which were originated from a common ancestral HeLa-human diploid fibroblast hybrid cell clone and retained a malignant state except tumorigenicity. When injected into nude mice, nontumorigenic CGL1 cells underwent apoptosis, but tumorigenic CGL4 cells did not. In vitro, CGL1 was also less resistant to various apoptotic stimuli than CGL4. These results suggested that inhibition of apoptosis may lead to tumorigenicity. To examine this hypothesis, we introduced antiapoptotic genes into the CGL1 cell line and injected the resulting clones into nude mice. The results showed that the ectopic expression of Bcl-2 or E1B19k, but not of crmA, converted CGL1 cells to tumorigenicity, suggesting strongly that this phenotype may be conferred by evasion of apoptosis.

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

Unlike 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.

Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells.

Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. Of the three major subunits comprising human telomerase, human telomerase catalytic subunit (hTERT) has been shown to be a rate-limiting determinant of the enzymatic activity of human telomerase. However, little is known concerning how expression of hTERT is regulated in human cells. To identify the regulatory elements controlling hTERT gene expression, approximately 3.5 kb of the 5'-flanking sequence of hTERT was cloned and characterized. The promoter of hTERT was GC rich and lacked both TATA and CAAT boxes. The CapSite Hunting method identified transcription start site 19 bp upstream of the first nucleotide of the published cDNA sequence. Transient expression assays revealed that transcription of hTERT was significantly activated in cancer cell lines but repressed in normal primary cells. Using the fibroblast lineage at various stages of transformation, we found that transcription occurred in strains that had overcome replicative senescence and expressed telomerase activity. Deletion analysis of hTERT promoter identified the 181-bp core promoter region upstream of the transcription start site. Gel shift analysis revealed two major factors binding to core promoter, an E box (CACGTG) binding factor and Sp1. Overexpression of c-Myc resulted in a significant increase in transcriptional activity of the core promoter. These findings suggest that hTERT expression is strictly regulated at the transcription machinery, and that the proximal core promoter containing an E box and Sp1 sites is required for transactivation of hTERT.

Dispensability of p53 degradation for tumorigenicity and decreased serum requirement of human papillomavirus type 16 E6.

Certain types of human papillomavirus (HPV), such as types 16 and 18, are etiological agents for carcinogenesis of the uterine cervix. These HPVs have two oncogenes, E6 and E7, that have transforming activities in established murine cells. Tumorigenicity and decreased serum requirement for cell growth are conferred by the E6 gene, whereas anchorage-independent growth is mainly governed by the E7 gene. To understand the mechanism of cellular transformation by the HPV16 E6 gene, we examined three mutant E6 proteins defective for p53 binding, p53 degradation, or transactivation of the adenovirus E2 promoter for the ability to induce tumorigenicity and decreased serum requirement. The results showed that tumorigenicity and decreased serum requirement were associated with the ability of E6 to bind to p53, although the subsequent degradation of p53 was not required for these functions.

Human papillomavirus-induced carcinogenesis with p53 deficiency in mouse: novel lymphomagenesis in HPV16E6E7 transgenic mice mimicking p53 defect.

To investigate the transforming activity of human papillomavirus (HPV) E6 and E7 genes in vivo, we previously established transgenic mouse lines containing HPV16E6E7, in which male mice develop a Leydig cell tumors with a very high incidence. Because HPV-induced carcinogenesis is highly related to p53, we changed the dose of p53 gene in the transgenic lines by the mice crossing with p53-disrupted mice. The transgenic mice with homozygous wild-type p53 alleles developed only the testicular tumor, whereas novel T cell lymphomagenesis occurred in the heterozygous p53-disrupted E6E7 (p53+/-E6E7) transgenic mice. In this tumor and even in the normal spleen, the absence of p53 protein was observed, whereas the p53 mRNA was expressed with a normal size, suggesting the degradation of p53 protein in these tissues. These results suggest that HPV16E6 could stimulate p53 protein degradation in mouse cells and induced the lymphomagenesis in a manner indistinguishable from p53 deficiency.

A mutant cell line resistant to Vibrio parahaemolyticus thermostable direct hemolysin (TDH): its potential in identification of putative receptor for TDH.

Thermostable direct hemolysin (TDH), a pore-forming toxin produced by Vibrio parahaemolyticus, is cytotoxic to Rat-1, a fibroblast cell line derived from rat embryo. Through mutagenesis of Rat-1 with nitrosoguanidine, we established a mutant cell line, MR-T1. MR-T1 was over 200 times more resistant to the cytotoxic activity of TDH than Rat-1. TDH increased membrane permeability of Rat-1 but not of MR-T1. Binding analysis showed that, while being able to bind to Rat-1. TDH failed to bind to MR-T1, indicating that MR-T1 is deficient in the putative receptor for TDH. Somatic hybrid cells between Rat-1 and MR-T1 were similarly sensitive to TDH as Rat-1. Moreover, TDH could bind to the hybrid cells as well as to Rat-1 cells. These results indicate that MR-T1 is promising for complementation cloning of a gene related to the putative receptor for TDH.

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-Hodgkin's 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 Hodgkin's disease, which shares several clinicopathologic features with ALCL.

Increased sensitivity of EBNA2-transformed rat fibroblasts to ionizing radiation.

To clarify whether expression of Epstein-Barr virus(EBV) nuclear antigen 2 (EBNA2) correlates with sensitivity to ionizing radiation, we tested EBNA2-transformed rat fibroblast clones for their radiosensitivity to X-rays. These transformed clones reproducibly generated tumors in mice. X-irradiation suppressed the growth of the tumors, and irradiated mice survived longer than non-irradiated ones. In contrast, tumors formed by activated H-ras or E6-E7 genes of human papillomavirus type 16 (HPV16) were strongly resistant to the same dose of X-irradiation. In in vitro culture, these EBNA2-expressing clones also showed higher radiosensitivity than cell lines transformed by activated H-ras and E6E7 genes. The averaged D(o) of EBNA2-expressing clones was 2.3 times lower than that of non-expressing and control clones. These results suggest that expression of EBNA2 is responsible for the radiosensitivity.

Immunophenotypic features and configuration of immunoglobulin genes in hairy cell leukemia-Japanese variant.

Immunophenotypes and Ig gene rearrangements were investigated in 12 patients with a variant form of hairy cell leukemia (HCL) termed HCL-Japanese variant (HCL-J), and in an HCL-J-derived cell line. The leukemic cells of HCL-J characteristically showed the phenotype of CD20+, CD5-, CD10-, CD11c+, CD22+, CD24- and CD25-. Ig light (L) chain was undetected in nine cases, and the remaining four cases expressed kappa chain. Expression of Ig heavy (H) chain was studied in nine cases. In addition to Igkappa+ cases showing expression of predominantly gamma H chain isotype, alpha chain was detected in one case without expression of L chain. Rearranged bands in Ig heavy chain (JH) genes were recognized in all 12 cases tested. Rearranged bands in kappa chain genes and germline configuration in chi chain genes were seen in all three Igkappa+ cases tested. Four of nine cases without expression of L chain had a rearranged chi chain gene. The other three cases had chi chain genes in the germline configuration and rearranged and/or deleted kappa chain genes. In the remaining two cases, no rearrangement in either kappa or chi chain genes was detected. The Ig gene configuration and expression in HCL-J, partially overlapping with those described for immature B cell leukemia, were dissociated from the cytological features and CD20+, membrane CD22+ phenotype characteristic of mature B cells.