Oncogenic genes and human malignancy.

All vertebrates possess a series of genes which are homologs of the oncogenic genes of acute transforming retroviruses. Two lines of evidence suggest that these genes may play a role in the development of human malignancy: (1) DNA from a variety of human tumors transforms NIH 3T3 mouse fibroblasts and the transforming genes from a number of carcinomas, sarcomas, and hematological malignancies have been identified as members of a family of genes, the ras family, closely related to the oncogenic genes of the Harvey and Kirsten murine sarcoma viruses; and (2) correlations exist between the chromosomal localizations of certain oncogenes and the chromosomal breakpoints in specific translocations and deletions in certain human malignancies. In three separate hematological malignancies, alterations in more than one oncogenic gene may be involved in the neoplastic process.     

Oncogenic potential of EAG K(+) channels.

We have investigated the possible implication of the cell cycle-regulated K(+) channel ether à go-go (EAG) in cell proliferation and transformation. We show that transfection of EAG into mammalian cells confers a transformed phenotype. In addition, human EAG mRNA is detected in several somatic cancer cell lines, despite being preferentially expressed in brain among normal tissues. Inhibition of EAG expression in several of these cancer cell lines causes a significant reduction of cell proliferation. Moreover, the expression of EAG favours tumour progression when transfected cells are injected into immune-depressed mice. These data provide evidence for the oncogenic potential of EAG.     

Genetic transmission of retroviral genes and cellular oncogenes

Several different families of retrovirus genome have been found to exist, each in multiple copies, in the cellular DNA of rodents and primates. There are at least four distinct families of genome in rodents: two type C families, the MTV family and another related to mouse type A particles. In primates there are also at least two families of endogenous type C virogenes and a third type D virogene family. Both in rodents and in primates, the virus-related sequences constitute almost 0.1% of the cellular genome. We have been able to generate transforming viruses, starting with endogenous mouse 'helper' type C viruses by passing them through chemically transformed mouse cells and selecting for variant viruses that have acquired the ability to induce normal cells to display anchorage-independent growth. These viruses produce both sarcomas and carcinomas in the animal; clones that produce only pulmonary carcinomas have also been selected. These presumably have arisen by recombination between the helper and 'transforming' sequences derived from the cells. Moloney sarcoma virus-transformed cells produce a new peptide, called sarcoma growth factor (SGF), that makes normal cells take on some of the properties of transformed cells. Studies with temperature-sensitive viral mutants show that the production of SGF is under the control of the transforming genes of the sarcoma virus.     

Selection for retroviral insertions into regulated genes.

Gene traps can be used to monitor faithfully the changes in gene expression accompanying several cellular processes. Here, we present a strategy that combines retroviral gene trap vectors, efficient selection schemes based on fluorescence-activated cell sorting or dominant positive and negative drug selection, and appropriately responsive cell lines in order to enrich for retroviral insertions into regulated genes (i.e., genes participating in cellular differentiation processes and genes induced by growth factors, drugs, or neurotransmitters, etc.). As an example, we applied this approach to the identification of insertions into genes activated by a MyoD protein, using a MyoD-responsive fibroblast line. In a single experiment designed to demonstrate the feasibility of this approach, we have been able to screen thousands of gene trap integrations and to select those that represent direct or indirect targets of MyoD. Distinct patterns of regulation were observed during myogenic determination. Sequences flanking the integrations can be rescued with several approaches, and they can be used to isolate the host genes or can serve as entry points for genome-wide sequencing projects.     

Oncogenic potential in fibroblasts from individuals genetically predisposed to cancer

There is now considerable evidence to suggest that genetic factors can influence the incidence of cancer. Although expression of this susceptibility to cancer appears to be tissue-specific, the normal skin fibroblasts from individuals predisposed to cancer (predisposed fibroblasts) have also been shown to express the risk of the target cell in the development of cancer. In the context of the 2-stage theory of chemical carcinogenesis predisposed fibroblasts may, therefore, exist in a pre-neoplastic or initiated state. The purpose of the present study was to determine whether predisposed fibroblasts would be oncogenically transformed in vitro by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone. TPA treatment induced similar changes in cellular morphology, cytoskeleton, and epidermal growth-factor binding, in predisposed and normal cells. None of these cell lines acquired anchorage-independent growth or an unlimited growth potential in culture after chronic application of TPA. Fluorescent microscopy with an F-actin probe, in the absence of TPA, showed a disorganization of the microfilament and intermediate filament network in skin fibroblasts from individuals with familial polyposis coli, hereditary and sporadic retinoblastoma, basal cell nevus syndrome, and Gardner's syndrome, as compared to normal skin fibroblasts. Single and 2-dimensional electrophoresis also indicated that the incorporation of 35S-methionine into actin in predisposed fibroblasts was 2-fold greater than in normal fibroblasts, and the turnover rate of actin in predisposed fibroblasts was less than 5 h, compared to 48 h in normal fibroblasts. These observations clearly suggest that predisposed fibroblasts may not exist in a pre-neoplastic or initiated state, and that the mechanism of genetic susceptibility to cancer may be different from that of chemical carcinogenesis. In contrast, the results of this study indicate that genetic susceptibility to a variety of cancers may be associated with a rapid turnover of actin and a disorganization of the microfilament and intermediate filament networks.     

Functional cloning of drug resistance genes from retroviral cDNA libraries

To improve the curative success of chemotherapy, it will be essential to understand the molecular basis of drug resistance (DR) and sensitivity. We have developed a cell culture system that enables the functional cloning of mammalian DR genes based on phenotypic selection after overexpression of mammalian retroviral cDNA libraries and validated our system using the anticancer drug cisplatin. ERCC1-deficient and therefore cisplatin-hypersensitive mouse embryonic fibroblast target cells were transduced with a human placenta retroviral cDNA library. Subsequent cisplatin selection yielded 20 DR clones, each containing a recurring human ERCC1 gene. Surprisingly, nine of these clones contained 5'-truncated ERCC1 sequences that required alternative splicing of the vector sequence to encode a functional ERCC1 protein. The usage of cryptic splice sites in the vector sequence should be taken into consideration when interpreting results from retroviral gene expression applications, and might have consequences for the safe application of retroviral constructs in gene therapy.     

Oncogenic potential of a dominant negative mutant of interferon regulatory factor 3

Interferon regulatory factor 3 (IRF3) is activated in response to various environmental stresses including viral infection and DNA-damaging agents. However, the biological function of IRF3 in cell growth is not well understood. We demonstrated that IRF3 markedly inhibited growth and colony formation of cells. IRF3 blocked DNA synthesis and induced apoptosis. Based on this negative control of cell growth by IRF3, we examined whether functional loss of IRF3 may contribute to oncogenic transformation. IRF3 activity was specifically inhibited by expression of its dominant negative mutant. This mutant lacks a portion of the DNA binding domain like IRF3a, an alternative splice form of IRF3 in the cells. This dominant negative inhibition blocked expression of specific IRF3 target genes. Mutant IRF3 efficiently transformed NIH3T3 cells, as demonstrated by anchorage-independent growth in soft agar and tumorigenicity in nude mice. These results imply that IRF3 may function as a tumor suppressor and suggest a possible role for the relative levels of IRF3 and its dominant negative mutant in tumorigenesis.     

Syncytins - retroviral envelope genes captured for the benefit of placental development

In the past, germline infections by retroviruses have led to vertical transmission of "endogenized" retroviruses. Escaping genetic drift, some of the viral genes have been conserved until now because of beneficial effects on their host. Here we present the syncytin genes that encode envelope proteins from endogenous retroviruses. Syncytins have inherited fusogenic properties from their infectious ancestor and are specifically expressed in the placenta. Both properties have suggested their involvement in the formation of the syncytiotrophoblast, a multinucleated layer that mediates feto-maternal exchanges in the placenta. The capture of syncytin genes occurred on several independent occasions during evolution of mammals. Knock-out experiments of syncytins in the mouse definitively confirmed the role of these genes in placentation. Finally, a second function for syncytins, i.e. an immunosuppressive activity, could contribute to materno-fetal immune tolerance. This constitutes a remarkable example of convergent evolution where the properties of retroviral envelope genes are subverted to play a major physiological role.     

Oncogenic potential of Nrf2 and its principal target protein heme oxygenase-1

Nuclear factor erythroid 2-related factor 2 (Nrf2) is an essential component of cellular defense against a vast variety of endogenous and exogenous insults, including oxidative stress. Nrf2 acts as a master switch in the circuits upregulating the expression of various stress-response proteins, especially heme oxygenase-1 (HO-1). Paradoxically, however, recent studies have demonstrated oncogenic functions of Nrf2 and its major target protein HO-1. Levels of Nrf2 and HO-1 are elevated in many different types of human malignancies, which may facilitate the remodeling of the tumor microenvironment making it advantageous for the autonomic growth of cancer cells, metastasis, angiogenesis, and tolerance to chemotherapeutic agents and radiation and photodynamic therapy. In this context, the cellular stress response or cytoprotective signaling mediated via the Nrf2–HO-1 axis is hijacked by cancer cells for their growth advantage and survival of anticancer treatment. Therefore, Nrf2 and HO-1 may represent potential therapeutic targets in the management of cancer. This review highlights the roles of Nrf2 and HO-1 in proliferation of cancer cells, their tolerance/resistance to anticancer treatments, and metastasis or angiogenesis in tumor progression.     

Effect of sodium butyrate on the expression of genes transduced by retroviral vectors

We have studied the effects of sodium butyrate (NaBu) on the expression of genes transduced by retroviral vectors and stably expressed in two salivary gland-derived cell lines, A5-DAP and A5-BAG, established earlier. These cell lines were obtained by infecting A5 cells with the retroviral vectors DAP and BAG, respectively, and by selecting neomycin-resistant transduced cells. A5-DAP cells express human placental alkaline phosphatase (PLAP) and A5-BAG cells bacterial β-galactosidase, both under the control of the viral long terminal repeat (LTR) enhancer-promoter. NaBu in the concentration of 2–8 mM inhibited the growth of A5-DAP cells, and induced the expression of heat-stable PLAP. These effects of NaBu were dose-dependent. Induction of PLAP in clones of A5-DAP cells that express different basal levels of the enzyme was not correlated with the relative inducibilty by NaBu. Exposure to 4 mM NaBu for 48 h increased the PLAP mRNA level by 31%. A5-DAP cells released, in a time-dependent manner, PLAP into the culture medium. Cells treated with NaBu released more PLAP than untreated cells in proportion to their elevated level of the enzyme. The parent A5 cells also express a low level of tissue non-specific type alkaline phosphatase, which was also induced by NaBu. NaBu inhibited the growth of A5-BAG cells also, and increased the β-galactosidase level. These data indicate the genes transduced by retroviral vectors can be induced by NaBu, which most likely interacts with the viral LTR. J. Cell. Biochem. 69:201–210, 1998. © 1998 Wiley-Liss, Inc.     

Co-transfection of normal NIH/3T3 DNA and retroviral LTR sequences: a novel strategy for the detection of potential c-onc genes

[This corrects the article on p. 1121 in vol. 3, PMID: 6329737.].