Epithelial cells release proinflammatory cytokines and undergo c-myc-induced apoptosis on exposure to filarial parasitic sheath protein-Bcl2 mediates rescue by activating c-H-ras

Summary Circulating filarial proteins elicit strong immunologic reactions in humans leading to the chronic manifestations in human lymphatic filariasis such as lymphatic occlusion, fibrosis, edema, and in some cases, tropical pulmonary eosinophilia. Our earlier studies, in vitro, conclusively prove that filarial parasitic sheath proteins induce apoptosis in HEp2 cells, an epithelial cell line, by a pathwa inhibitable by bcl2. The present findings provide evidence that c-myc activation triggers apoptosis in HEp2 cells and that it is also responsible for the burst of abortive proliferation at 6 d of treatment of HEp2 bcl2 cells that overexpress bcl2, with filarial parasitic sheath protein, demonstrating the interplay between the two genes c-myc and bcl2, wherein bcl2 acts by restoring the prosurvival signal to c-myc and keeping its apoptotic tendency in check. This study also indicates that bcl2 upregulates c-H-ras, engaring ras to bring about the suppression of apoptosis through protein tyrosine kinase elevation, thus promoting the survival of the HEp2 bcl2 cells. In addition to the activation of these “signal switches,” we also observe that these cells release cytokines like IL-6 and IL-8 through the upregulation of c-fos, when exposed to filarial parasitic sheath protein, reflecting on the immunomodulatory capacity of the epithelium to elicit a host immune response by setting up a chemotactic gradient, attracting inflammatory cells to the site of infection.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of EJ-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Effects of oxygen free radicals on articular chondrocytes in culture: C-myc and c-Ha-ras messenger RNAs and proliferation kinetics

Since oxygen free radicals are believed to play an important role in cartilage degradation, we studied the effects of these radicals generated by the hypoxanthine-xanthine oxidase system on rabbit articular chondrocytes in culture. Among the damages induced by these radicals, cell proliferation inhibition and G2 arrest were observed. To elucidate the mechanisms involved in this phenomenon, the expression of c-myc and c-Ha-ras genes whose products are associated with cell growth control was studied. Results showed that in chondrocytes, c-myc and c-Ha-ras expression was particularly important during the G1 phase of the cell cycle and that oxygen reactive species, especially H₂O₂, induced an important decrease of c-myc and c-Ha-ras mRNA levels. Chondrocytes cell cycle analysis revealed an accumulation of cells in G2 phase. It led us to suggest that the chondrocyte cell cycle perturbations observed after oxygen free radicals treatment could be associated with the decrease of c-myc and c-Ha-ras expression.     

Purification and characterization of interferon-like antiviral protein derived from flatfish (Paralichthys olivaceus) lymphocytes immortalized by oncogenes

Flatfish leukocytes were transfected with the expression plasmids of the v-myc, c-myc, c-fos, v-myb and c-Ha-ras oncogenes. Only cotransfection of c-Ha-ras with c-myc or c-fos resulted in complete immortalization of the cells. Interferon-like anti-viral protein was found in the cultured medium of the immortalized lymphocytes. The protein was purified by DEAE-Toyopearl 650 M ion exchange chromatography and WGA agarose affinity chromatography. The protein was a glycoprotein of about 16 kDa. The antiviral activity of the protein was trypsin-sensitive and was fairly stable at pH values from 4 to 8. The protein retained about 60% of the activity even at 60°C and showed a broad antiviral activity for various fish cells and viruses.     

The CTCF Insulator Protein Is Posttranslationally Modified by SUMO

The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.     

A p53 mutation is required for stable transformation of REF52 cells by themyc andras oncogenes

It is known that neoplastic transformation of rodent primary embryonic fibroblasts culturedin vitro requires coexpression at least of two cooperating oncogenes. In the case of transduction into cells of oncogenesras andmyc, the cell transformation is poorly effective. To study some additional factors necessary for such transformation, c-myc and N-ras ^Asp12 were consecutively introduced into REF52 cells by retroviral infection, and the cell cultures obtained were analyzed. Expression ofmyc broke the regulation of the cell cycle, in particular, canceled the G1 phase arrest for cells with damaged DNA, despite the normal function of protein p53 and induction of the p53-responsive genep21 ^Waf1 in these cells. The subsequent transduction ofras led to morphological transformation of cells and an increase of p53 level. However, reversion of the transformed phenotype to normal morphology took place after less than five passages. On this background, rare clones generated the stable transformed cell lines characterized by accelerated proliferation and having a mutation in thep53 gene. Attempts to obtain stable transformed cell lines by transduction ofras into REF52 cells not expressing exogenousmyc were unsuccessful. Analysis of the stable transformed clones revealed a mutation at codon 271 of thep53 gene, a hot spot of mutations, which led to the replacement of arginine by cysteine. In these clones, p53 is accumulated owing to the increased life time, and has a flexible conformation, being able to interact with monoclonal PAb1620 and PAb240 antibodies recognizing alternative protein conformations. The results obtained suggest that p53 participates in negative regulation of the cell cycle under conditions of oncogenic stimulation, and its inactivation is necessary for full transformation of cells by cooperating oncogenesmyc andras.     

Influence of myc overexpression on the phenotypic properties of Chinese hamster lung cells resistant to antitumor agents

In the Chinese hamster lung fibroblast cell line DC-3F, the development of resistance to different drugs, through several mechanisms like MDR expression or alteration of the DNA topoisomerase II activity, has been shown to be associated with a decreased tumorigenicity. Multiple studies have shown that the myc oncogene, in cooperation with ras, plays a major role in the oncogenic transformation of fibroblasts. As an approach to a better understanding of the relationship between the different phenotypic traits, we analyzed the expression of myc and ras oncogenes in the drug-sensitive DC-3F cells and in variants resistant to 9-hydroxy-ellipticine (9-OH-E) (DNA topoisomerase II alteration) or to actinomycin D (AD) (multidrug (MDR) expression). Southern and Northern blot analyses revealed about a 10-fold amplification and a 20-fold overexpression of the c-myc gene in the DC-3F cells as compared to the normal lung fibroblasts. Both amplification and overexpression are markedly decreased in the two resistant variants. ras gene copy number and expression were found to be identical in all cell types. In order to analyze the contribution of the decreased myc expression on the different phenotypic traits, the DC-3F/9-OH-E cells were transfected with the plasmid pSV-c-myc, and six clones expressing high amounts of the transfected myc were isolated and characterized. Morphological and caryological alterations, as well as an increased cloning efficiency in soft agar, indicated that the myc gene product was made in these cells. However, the tumorigenicity of the sensitive parental cells was not restored, thus showing that the decreased myc expression alone does not account for the loss of tumorigenicity in the resistant cells. 9-OH-E resistance was not modified in the transfected cells, while the cross-resistance of these cells to MDR-sensitive drugs, such as vincristine, actinomycin D, and taxol, was reversed roughly in proportion of the expression of the transfected myc.     

Oncogene alterations in in vitro transformed rat tracheal epithelial cells

10 derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 nontumorigenic cell line transformed in vitro by treatment with 7,12-dimethyl-benz[a]anthracene (DMBA), benzo[a]pyrene (BP) and/or 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for oncogene alterations. No abnormalities of Ha-ras were seen that were suggestive of amplification, rearrangement or the presence of RFLPs. Analysis of specific-point mutations in Ha-ras using Pst I digestion (codon 12, GGA to GCA) or Ha-ras and Ki-ras using Xba I (codon 61, CAA to CTA) were negative. In one cell line derived by DMBA treatment, changes in the c-myc restriction digest pattern were seen after incubation with Bam HI and Hind III. Northern analysis revealed consistent differences between normal and transformed cells when probed with Ha-ras; c-myc expression was of low intensity, and the expression of Ki-ras could not be detected. Transfection of RTE cell DNAs into NIH/3T3 cells did not result in the appearance of morphologic transformants. The studies suggest that Ha-ras or Ki-ras codon 61 A to T transversions (CAA to CTA) are not associated with the immoral/tumorigenic phenotype in RTE cells transformed by DMBA or TPA, and are in contrast to results reported in some other biological systems.     

Amplification of epidermal growth factor receptor (EGFR) gene and oncogenes in human gastric carcinomas

DNAs from 37 human gastric carcinomas and seven lymph node metastases were analyzed for alterations of the epidermal growth factor receptor (EGFR) gene and oncogenes by the Southern blot hybridization method. The probes used were EGFR gene, c-Ha-ras, v-Ki-ras, N-ras, c-myc, v-myb, v-fos, c-erbB-2, v-erbA, v-abl and v-fes. Amplification of the EGFR gene was detected in only one poorly differentiated adenocarcinoma. Amplifications of c-myc gene and c-erbB-2 gene were each observed in two well differentiated adenocarcinomas. One of these tumors had coamplification of c-erbB-2 and c-erbA genes but there were no amplifications nor rearrangements of other oncogenes. The poorly differentiated adenocarcinom with amplified EGFR gene also showed enhanced expression of EGFR gene by Northern blot analysis and additionally had strong synchronous immunoreactivity for EGFR and EGF. Supported in part by Grants-in Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan and for Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan     

Ras oncogene enhances the production of a recombinant protein regulated by the cytomegalovirus promoter in BHK-21 cells

In order to enhance recombinant protein productivity in animal cells, we developed the oncogene activated production (OAP) system. The OAP system is based on the premise that oncogenes are able to enhance promoter activity. To this end, we constructed reported plasmids by fusing various promoters to the human interleukin-6 (hIL-6) cDNA, and the effector plasmids by inserting individual oncogenes, for example c-myc, c-fos, v-jun, v-myb and c-Ha-ras, downstream from the human cytomegalovirus immediate early (CMV) promoter. Results of transient expression experiments with BHK-21 cells suggest that the CMV promoter is the most potent promoter examined and that theras product is able to transactivate the -actin, CMV and SR promoters. Recombinant BHK-21 cells producing hIL-6 under the control of the CMV promoter were contransfected with theras oncogene and dihydrofolate reductase gene, then selected with 50 nM methotrexate to coamplify theras oncogene. We were able to rapidly establish a stable and highly productive clone which exhibited a 35-times higher production rate as compared to the control value.     

Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.     

Dose effects of transfected c-Ha-ras oncogene in transformed cell clones

We have examined the expression of the transformed phenotype in a series of clonal lines of NIH/3T3 cells transfected with the human c-Ha-ras^{Val 12} oncogene and the neomycin phosphotransferase gene. Cells from individual transformed foci were cloned and subjected to detailed analyses of the ras sequences. Three clones were found that expressed approximately one, 2–4, or 4–8 copies of the human c-ras oncogene, respectively. A fourth clone had multiple copies of the transfected sequences, and expressed abundant c-Ha-ras RNA. Analysis of the tranformed phenotype of various clones indicated that cells expressing low levels of mutant c-Ha-ras had lost some of their extracellular fibronectin network, and were barely altered in their cytoskeleton. In contrast, cells expressing abundant c-Ha-ras had lost both their actin and fibronectin networks and showed an increase in plasminogen activator activity. Cells with amplified c-Ha-ras^{Val 12} grew better in low serum, formed large colonies in soft agar and showed enhanced activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis. These results show that the dosage level of the mutant oncogene makes a significant contribution to the transformed phenotype of c-Ha-ras oncogene-transformed cells.     

Selection of cells with different chromosomal localizations of the amplified c-myc gene during in vivo and in vitro growth of the breast carcinoma cell line SW 613-S

The c-myc gene is amplified in the human breast carcinoma cell line SW 613-S. At early in vitro passages, the extra copies of the gene were mainly localized in double minute chromosomes (DMs), as shown by in situ hybridization with a biotinylated c-myc probe. However, cells without DMs were also present in which the c-myc genes were found integrated into any of several distinct chromosomes (mainly 7q+, 4 and 4q+, and 1). When this cell line was propagated in vitro, the level of c-myc amplification decreased because cells with DMs and a high amplification level were lost and replaced by cells without DMs and having a low amplification level. On the contrary, when early passage SW 613-S cells were grown in vivo, as subcutaneous tumours in nude mice, cells with numerous DMs and a high level of c-myc amplification were selected for. In one cell line (SW 613-Tu1) established from such a tumour, the DM-containing cells were substituted at late passages for cells with a high number of c-myc copies integrated within an abnormally banded region, at band 17q24 of a 17q+ chromosome. When only cells with integrated genes were present, this cell line was still highly tumorigenic indicating that the localization of the c-myc genes in DMs was not required for these cells to be tumorigenic in nude mice. Furthermore, cells of the secondary tumours induced by SW 613-Tu1 did not contain any DMs showing that in vivo growth did not promote the release of integrated c-myc copies into DMs.     

A Farnesyltransferase Inhibitor Induces Tumor Regression in Transgenic Mice Harboring Multiple Oncogenic Mutations by Mediating Alterations in Both Cell Cycle Control and Apoptosis

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)–v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV–v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV–v-Ha-ras mice were interbred with p53^−/− mice. Tumors in ras/p53^−/− mice treated with L-744,832 regressed as efficiently as MMTV–v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G₁ with a corresponding decrease in the S-phase fraction. MMTV–v-Ha-ras mice were also interbred with MMTV–c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV–v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV–c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.     

Localization in situ of c-myc mRNA and c-myc protein in adult mouse testis

Summary It has been suggested that c-myc, one of the proto-oncogenes, plays a role in normal somatic cell proliferation and differentiation. To define whether c-myc is only expressed during somatic cell division or is also expressed during meiotic cell division, the production of c-myc mRNA and protein were investigated in the mouse testis by usingin situ hybridization with non-radioactive DNA probes and enzyme immunohistochemistry respectively. Forin situ hybridization, T-T dimerized DNA probes were used and DNAs hybridizedin situ were detected immunohistochemically using specific antibody against T-T dimer. The results indicate that c-myc mRNA and protein are expressed in a cell-cycle-dependent manner only in spermatogonia and not in spermatocytes and spermatids.     

Docosahexaenoic acid-containing phosphatidylethanolamine enhances HL-60 cell differentiation by regulation of c-jun and c-myc expression

18:1/docosahexaenoic acid (DHA)-containing phosphatidylethanolamine (PE) enhanced cell differentiation and growth inhibition of HL-60 induced by dibutyryl cAMP (dbcAMP) in a dose-dependent manner. The combined treatment of 200 μM dbcAMP and 50 μM 18:1/DHA-PE increased the NBT reducing activity, which is as an indicator of cell differentiation, to more than 75% from 40% of cells treated with 200 μM dbcAMP alone. In HL-60 cells treated with 50 μM 18:1/DHA-PE and 200 μM dbcAMP for 24 h, the expression level of c-jun mRNA and c-Jun protein were remarkably elevated compared to cells treated with dbcAMP alone. In contrast, there was no difference in the expression levels of c-fos mRNA and c-Fos protein between the combination of 18:1/DHA-PE + dbcAMP or dbcAMP alone. On the other hand, the combine treatment of 18:1/DHA-PE and dbcAMP markedly reduced the expression level of c-myc oncogene during 48 h incubation. The decreases of c-myc mRNA by 18:1/DHA-PE and/or dbcAMP was correlated with growth inhibition effect. Thus, 18:1/DHA-PE might enhance dbcAMP-induced HL-60 cell differentiation and growth inhibition by regulation of c-jun and c-myc mRNA and their products.     

Tumors Induced in Mice by Direct Inoculation of Plasmid DNA Expressing Both Activated H-ras and c-myc

Vaccines contain residual DNA derived from the cells used to produce them. As part of our investigation to assess the risk of this cellular DNA, we are developing a quantitative in vivo assay to assess the oncogenicity of DNA. In an earlier study, we had generated expression plasmids for two oncogenes - human activated T24-H-ras and murine c-myc - and had shown that these two plasmids, pMSV-T24-H-ras and pMSV-c-myc, could act in concert to induce tumors in mice, although the efficiency was low. In this study, we took two approaches to increase the oncogenic efficiency: 1) both oncogene-expression cassettes were placed on the same plasmid; 2) transfection facilitators, which increase DNA uptake and expression in vitro, were tested. The dual-expression plasmid, pMSV-T24-H-ras/MSV-c-myc, is about 20-fold more efficient at tumor induction in newborn NIH Swiss mice than the separate expression plasmids, with tumors being induced with 1 µg of the dual-expression plasmid DNA. However, none of the transfection facilitators tested increased the efficiency of tumor induction. Based on these data, the dual-expression plasmid pMSV-T24-H-ras/MSV-c-myc will be used as the positive control to develop a sensitive and quantitative animal assay that can be used to assess the oncogenic activity of DNA.     

Transgenic animals as a tool for studying the effect of the c-myc proto-oncogene on cardiac development

Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.