Primary structure of v-raf: relatedness to the src family of oncogenes

A replication-defective, acute transforming retrovirus (murine sarcoma virus 3611) was isolated from mouse and molecularly cloned. The nucleotide sequence of 1.5 kilobases encompassing the transforming gene (v-raf) was determined. This sequence, which predicts the amino acid sequence of a gag-raf fusion protein, terminates 180 nucleotides from the 3' end of the acquired cellular sequence. Comparison of the predicted amino acid sequence of v-raf with the predicted amino acid sequences of other oncogenes reveals significant homologies to the src family of oncogenes. There is a lack of homology within the sequence of the tyrosine acceptor domain described for the phosphotyrosine kinase members of the src family of transforming proteins. Phylogenetic arrangement of this family of oncogenes suggests that tyrosine-specific phosphorylation may be a recently acquired activity.     

Activation of ras oncogenes preceding the onset of neoplasia

The identification of ras oncogenes in human and animal cancers including precancerous lesions indicates that these genes participate in the early stages of neoplastic development. Yet, these observations do not define the timing of ras oncogene activation in the multistep process of carcinogenesis. To ascertain the timing of ras oncogene activation, an animal model system was devised that involves the induction of mammary carcinomas in rats exposed at birth to the carcinogen nitrosomethylurea. High-resolution restriction fragment length polymorphism analysis of polymerase chain reaction-amplified ras sequences revealed the presence of both H-ras and K-ras oncogenes in normal mammary glands 2 weeks after carcinogen treatment and at least 2 months before the onset of neoplasia. These ras oncogenes can remain latent within the mammary gland until exposure to estrogens, demonstrating that activation of ras oncogenes can precede the onset of neoplasia and suggesting that normal physiological proliferative processes such as estrogen-induced mammary gland development may lead to neoplasia if the targeted cells harbor latent ras oncogenes.     

Activated oncogenes in B6C3F1 mouse liver tumors: implications for risk assessment

The validity of mouse liver tumor end points in assessing the potential hazards of chemical exposure to humans is a controversial but important issue, since liver neoplasia in mice is the most frequent tumor target tissue end point in 2-year carcinogenicity studies. The ability to distinguish between promotion of background tumors versus a genotoxic mechanism of tumor initiation by chemical treatment would aid in the interpretation of rodent carcinogenesis data. Activated oncogenes in chemically induced and spontaneously occurring mouse liver tumors were examined and compared as one approach to determine the mechanism by which chemical treatment caused an increased incidence of mouse liver tumors. Data suggest that furan and furfural caused an increased incidence in mouse liver tumors at least in part by induction of novel weakly activating point mutations in ras genes even though both chemicals did not induce mutations in Salmonella assays. In addition to ras oncogenes, two activated raf genes and four non-ras transforming genes were detected. The B6C3F1 mouse liver may thus provide a sensitive assay system to detect various classes of proto-oncogenes that are susceptible to activation by carcinogenic insult. As illustrated with mouse liver tumors, analysis of activated oncogenes in spontaneously occurring and chemically induced rodent tumors will provide information at a molecular level to aid in the use of rodent carcinogenesis data for risk assessment.     

The approaching era of the tumor suppressor genes

Genes that can inhibit the expression of the tumorigenic phenotype have been detected by the fusion of normal and malignant cells, the phenotypic reversion of in vitro transformants, the induction of terminal differentiation of malignant cell lineages, the loss of "recessive cancer genes," the discovery of regulatory sequences in the immediate vicinity of certain oncogenes, and the inhibition of tumor growth by normal cell products. Such tumor suppressor genes will probably turn out to be as, if not more, diversified as the oncogenes. Consideration of both kinds of genes may reveal common or interrelated functional properties.     

Induction of gene amplification by arsenic

Arsenic is a well-established carcinogen in humans, but there is little evidence for its carcinogenicity in animals and it is inactive as an initiator or tumor promoter in two-stage models of carcinogenicity in mice. Two arsenic salts (sodium arsenite and sodium arsenate) induced a high frequency of methotrexate-resistant 3T6 cells, which were shown to have amplified copies of the dihydrofolate reductase gene. The ability of arsenic to induce gene amplification may relate to its carcinogenic effects in humans since amplification of oncogenes is observed in many human tumors. The inability of arsenic to induce gene mutations may relate to the negative results of arsenic in long-term animal studies and suggests that these experiments may not detect some environmental agents that act late in the carcinogenic process in humans.     

Sequencing the erbA gene of avian erythroblastosis virus reveals a new type of oncogene

Avian erythroblastosis virus (AEV) contains two distinct oncogenes, erbA and erbB . The erbB oncogene, which is homologous to a portion of the epidermal growth factor receptor, is related to the src family of oncogenes and efficiently transforms erythroblasts, whereas erbA potentiates the effects of erbB by blocking the differentiation of erythroblasts at an immature stage. This "potentiator" was sequenced; the amino acid sequence deduced from it was clearly different from the sequences of other known oncogene products and was related to carbonic anhydrases. These enzymes participate in the transport of carbon dioxide by erythrocytes, the precursors of which are main targets of avian erythroblastosis virus. A src-related oncogene such as erbB in synergy with an activated specific cell-derived gene such as erbA can profoundly affect early erythroid differentiation.     

Transformation and plasmacytoid differentiation of EBV-infected human B lymphoblasts by ras oncogenes

The biological effects of ras oncogene activation in B cells were studied by using amphotropic retroviral vectors to introduce H- or N-ras oncogenes into human B lymphoblasts immortalized by Epstein-Barr virus. Expression of both H- and N-ras oncogenes led to malignant transformation of these cells, as shown by clonogenicity in semisolid media and tumorigenicity in immunodeficient mice. In addition, terminal differentiation into plasma cells was detectable as specific changes in morphology, immunoglobulin secretion, and cell surface antigen expression. This combined effect, promoting growth and differentiation in human lymphoblasts, represents a novel biological action of ras oncogenes and has implications for the pathogenesis of terminally differentiated B-lymphoid malignancies such as multiple myeloma.     

Independent molecular pathways in initiation and loss of hormone responsiveness of breast carcinomas

These studies were set up to determine whether those oncogenes participating in the initiation of mammary carcinogenesis (for example, ras oncogenes) play a direct role in the outcome of events associated with the late stages of tumor development such as loss of hormone dependency. Mammary carcinomas induced by a single carcinogenic insult in pubescent rats was selected as an in vivo model system with direct relevance to human breast cancer. Acquisition of hormone-independent growth in these carcinogen-induced tumors was found to be independent of the activation of ras oncogenes during the early stages of carcinogenesis. In agreement with these observations, introduction of a human ras oncogene into human MCF-7 breast carcinoma cells did not abrogate their hormonal dependency for growth in vivo. These findings suggest that those events responsible for the critical stages of breast cancer development occur independently and in an uncoordinated manner.     

Transformation by oncogenes encoding protein kinases induces the metastatic phenotype

Oncogenes encoding serine/threonine or tyrosine kinases were introduced into the established rodent fibroblast cell line NIH 3T3 and tested for tumorigenic and metastatic behavior in T cell-deficient nude mice. Transforming oncogenes of the ras family were capable of converting fibroblast cell lines to fully metastatic tumors. Cell lines transformed by the kinase oncogenes mos, raf, src, fes, and fms formed experimental metastases and (in some cases) these genes were more efficient at metastatic conversion than a mutant ras gene. In contrast, cells transformed by either of two nuclear oncogenes, myc or p53, were tumorigenic when injected subcutaneously but were virtually nonmetastatic after intravenous injection. These data demonstrate that, in addition to ras, a structurally divergent group of kinase oncogenes can induce the metastatic phenotype.     

Sustained loss of a neoplastic phenotype by brief inactivation of MYC

Pharmacological inactivation of oncogenes is being investigated as a possible therapeutic strategy for cancer. One potential drawback is that cessation of such therapy may allow reactivation of the oncogene and tumor regrowth. We used a conditional transgenic mouse model for MYC-induced tumorigenesis to demonstrate that brief inactivation of MYC results in the sustained regression of tumors and the differentiation of osteogenic sarcoma cells into mature osteocytes. Subsequent reactivation of MYC did not restore the cells' malignant properties but instead induced apoptosis. Thus, brief MYC inactivation appears to cause epigenetic changes in tumor cells that render them insensitive to MYC-induced tumorigenesis. These results raise the possibility that transient inactivation of MYC may be an effective therapy for certain cancers.     

Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes

An accelerated rate of glucose transport is among the most characteristic biochemical markers of cellular transformation. To study the molecular mechanism by which transporter activity is altered, cultured rodent fibroblasts transfected with activated myc, ras, or src oncogenes were used. In myc-transfected cells, the rate of 2-deoxy-D-glucose uptake was unchanged. However, in cells transfected with activated ras and src oncogenes, the rate of glucose uptake was markedly increased. The increased transport rate in ras- and src-transfected cells was paralleled by a marked increase in the amount of glucose transporter protein, as assessed by immunoblots, as well as by a markedly increased abundance of glucose transporter messenger RNA. Exposure of control cells to the tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) for 18 hours had a similar effect of increasing the rate of glucose transport and the abundance of transporter messenger RNA. For ras, src, and TPA, the predominant mechanism responsible for activation of the transport system is increased expression of the structural gene encoding the glucose transport protein.     

The control of the metabolic switch in cancers by oncogenes and tumor suppressor genes

Cells from some tumors use an altered metabolic pattern compared with that of normal differentiated adult cells in the body. Tumor cells take up much more glucose and mainly process it through aerobic glycolysis, producing large quantities of secreted lactate with a lower use of oxidative phosphorylation that would generate more adenosine triphosphate (ATP), water, and carbon dioxide. This is the Warburg effect, which provides substrates for cell growth and division and free energy (ATP) from enhanced glucose use. This metabolic switch places the emphasis on producing intermediates for cell growth and division, and it is regulated by both oncogenes and tumor suppressor genes in a number of key cancer-producing pathways. Blocking these metabolic pathways or restoring these altered pathways could lead to a new approach in cancer treatments.     

An oncogene-induced DNA damage model for cancer development

Of all types of DNA damage, DNA double-strand breaks (DSBs) pose the greatest challenge to cells. One might have, therefore, anticipated that a sizable number of DNA DSBs would be incompatible with cell proliferation. Yet recent experimental findings suggest that, in both precancerous lesions and cancers, activated oncogenes induce stalling and collapse of DNA replication forks, which in turn leads to formation of DNA DSBs. This continuous formation of DNA DSBs may contribute to the genomic instability that characterizes the vast majority of human cancers. In addition, in precancerous lesions, these DNA DSBs activate p53, which, by inducing apoptosis or senescence, raises a barrier to tumor progression. Breach of this barrier by various mechanisms, most notably by p53 mutations, that impair the DNA damage response pathway allows cancers to develop. Thus, oncogene-induced DNA damage may explain two key features of cancer: genomic instability and the high frequency of p53 mutations.     

Microinjected c-myc as a competence factor

While a number of oncogenes are expressed in a cell cycle-dependent manner, their role in the control of cell proliferation can only be established by a direct functional assay. The c-myc protein, upon microinjection into nuclei of quiescent Swiss 3T3 cells, cooperated with platelet-poor plasma in the stimulation of cellular DNA synthesis. This suggests that c-myc protein, like platelet-derived growth factor (PDGF), may act as a competence factor in the cell cycle to promote the progression of cells to S phase. The presence in the medium of an antibody against PDGF abolished DNA synthesis induced by microinjected PDGF; however, the microinjected c-myc protein stimulated DNA synthesis even when its own antibody was present in the medium. The c-myc protein may act as an intracellular competence factor, while PDGF expresses its biological activity only from outside the cells.     

Mitogens and oncogenes can block the induction of specific voltage-gated ion channels

The mechanisms underlying the ontogeny of voltage-gated ion channels in muscle are unknown. Whether expression of voltage-gated channels is dependent on mitogen withdrawal and growth arrest, as is generally true for the induction of muscle-specific gene products, was investigated in the BC3H1 muscle cell line by patch-clamp techniques. Differentiated BC3H1 myocytes expressed functional Ca2+ and Na+ channels that correspond to those found in T tubules of skeletal muscle. However, Ca2+ and Na+ channels were first detected after about 5 days of mitogen withdrawal. In order to test whether cellular oncogenes, as surrogates for exogenous growth factors, could prevent the expression of ion channels whose induction was contingent on mitogen withdrawal, BC3H1 cells were modified by stable transfection with oncogene expression vectors. Expression vectors containing v-erbB, or c-myc under the control of the SV40 promoter, delayed but did not prevent the appearance of functional Ca2+ and Na+ channels. In contrast, transfection with a Val12 c-H-ras vector, or cotransfection of c-myc together with v-erbB, suppressed the formation of functional Ca2+ and Na+ channels for greater than or equal to 4 weeks. Potassium channels were affected neither by mitogenic medium nor by transfected oncogenes. Thus, the selective effects of certain oncogenes on ion channel induction corresponded to the suppressive effects of mitogenic medium.     

Normal cells of patients with high cancer risk syndromes lack transforming activity in the NIH/3T3 transfection assay

Oncogenes capable of transforming NIH/3T3 cells are often present in human tumors and tumor cell lines. Such oncogenes were not detected in normal fibroblast lines derived from patients with several clinical syndromes associated with greatly increased cancer risk. Thus, germ-line transmission of these oncogenes does not appear to be the predisposing factor responsible for these high cancer risk syndromes.