Environmental Health Perspectives Vol. 75, pp. 81-86, 1987 The Role of Oncogenes in Chemical Carcinogenesis by S. Jill Stowers,* Robert R. Maronpot,t Steven H. Reynolds,* and Marshall W. Anderson* Proto-oncogenes are cellular genes that are expressed during normal growth and developmental pro- cesses. Altered versions of normal proto-oncogenes have been implicated in the development of human neoplasia. In this report, we show the detection of activated proto-oncogenes in various spontaneous and chemically induced rodent tumors. The majority of activated proto-oncogenes found in these tumors are members of the ras gene family and have been activated by a point mutation. Characterization of the activating mutation may be useful in determining whether this proto-oncogene was activated by direct interaction of the chemical with the DNA. Comparison of activating lesions in spontaneous versus chem- ically induced tumors should be helpful in determining whether the chemical acts via a genotoxic or a nongenotoxic mechanism. All of this information may be helpful in the assessment of potential carcin- ogenic hazards of human exposure to chemicals. Introduction myb, fos), and tyrosine kinases (scr, abl, ras). Thus, the encoded proteins appear to play a crucial role in Recent evidence suggests that neoplastic develop- normal cellular growth and/or differentiation. ment, at least in part, is the result of the abnormal The activation ofproto-oncogenes in spontaneous and activation of a small set of cellular genes. These genes, chemically induced tumors has been extensively studied termed proto-oncogenes, were originally discovered as during the past several years. Although oncogenes such the transduced genes of acute transforming retrovi- as ras and myc can complement each other in the ma- ruses (1-3). Subsequent studies have established that lignant transformation of a cell in vitro (2), the number these proto-oncogenes can also be activated as onco- of proto-oncogenes that must be activated in the mul- genes by mechanisms independent of retroviruses (4). tistep process of carcinogenesis is unclear at present. Mechanisms for the conversion of proto-oncogenes to Also, new evidence from several laboratories suggests activated oncogenes include point mutations, gene am- that in addition to the activation of positive factors (on- plification, chromosomal rearrangements, and promoter cogenes), the loss of negative regulatory functions (tu- insertion (Fig. 1). The activation of proto-oncogenes by mor suppressor genes) may also be a necessary but genetic alterations results in altered levels ofexpression distinct step in neoplastic development (5). This paper of the normal protein product, or in normal or altered will discuss the detection of activated oncogenes in ro- levels of expression of an abnormal protein. dent tumors and the implication of oncogenes in risk Proto-oncogenes are expressed during regulated analysis of carcinogen-induced rodent tumor data. growth such as embryogenesis, regeneration of dam- aged liver, and stimulation of cell mitosis by growth factors. Proto-oncogenes are highly conserved. They Detection of Activated Oncogenes are detected in species as divergent as yeast, Droso- phila, and humans. Proto-oncogenes include genes that in Tumors encode for growth factors (sis), growth factor receptors Detection of activated oncogenes in neoplasia can be (neu, erbB, fms), regulatory proteins in signal trans- achieved by using several different techniques depend- duction (ras family), nuclear regulatory proteins (myc, ing on how the particular oncogene might be activated. Abnormal expression of oncogenes in tumors due to *Laboratory of Biochemical Risk Analysis, National Institute of amplification of the gene may be detected by dot blot Environmental Health Sciences, P.O. Box 12233, Research Triangle or Southern blot analysis. Increased expression due to Park, NC 27709. tChemical Pathology Branch, National Toxicology Program, Na- deregulation of the gene may be detected by dot blot tional Institute of Environmental Health Sciences, P.O. Box 12233, as well as Northern blot analysis. Chromosomal trans- Research Triangle Park, NC 27709. locations may be detected by cytogenetic analysis. Ex- 82 STOWERS ET AL. Activation Proto-Oncogene Oncogene Examples c-onc Retrovirus v-onc Acutely Transforming Transduction Leukemia/Sarcoma Virus Murine Plasmacytoma c-myc Chromosomal Ig/myc Human Burkitt's Translocation bcr/abl Lymphoma c-abl CML N-myc Gene Neuroblastoma c-myc Amplification DM/HSR Small Cell Lung Carcinoma Ki-ras Adrenocortical Tumor of Mice Colon, Carcinoma c-Ha-ras 12th, 13th, 61st Lung Carcinoma c-Ki-ras Point Codon AML c-N-ras Mutation Mutation Chemical Induced Rodent Tumors myc myb Promoter/Enhancer P LTR/c-onc Chronic, Non- erb B LTR/Common mos Insertion Transforming int-1 Domain Leukemia Virus int-2 FIGURE 1. Mechanisms of proto-oncogene activation. amples of abnormal expression of oncogenes detected gene. The selected cells are then injected SC into the in human tumors and tumor cell lines are shown in Table immunocompromised mice. The tumors that develop 1. from the nude mice are then analyzed using the tech- A number of oncogenes present in human tumors as niques described earlier to characterize the activated well as animal tumors have been detected by the NIH/ oncogenes. 3T3 transfection assay. The NIH/3T3 transfection tech- The majority of activated genes in human tumors de- nique involves the ability of the NIH/3T3 mouse fibro- tected by the NIH/3T3 assay have been members ofthe blast to accept and express genes from donor tumor ras gene family: the H-ras, the K-ras, and the N-ras. DNA, resulting in the formation of transformed cells. Early studies using the NIH/3T3 assay showed only ras Only a few years ago, Shih et al. (6) were the first to gene activation in a low percentage ofthe human tumors show that DNA from carcinogen-transformed cell lines (approximately 10%). Later studies have shown that could cause transformation ofNIH/3T3 cells after trans- other oncogenes can also be detected in the human tu- fection. This transformation was characterized by a mors by this assay, including the Ica (8), hst (9), and change in the morphology of the NIH/3T3 cells and by the trk (10) oncogenes. In addition, the percentage of anchorage-independent growth. Other investigators us- certain tumor types that test positive for activated ras ing this technique were then able to show that dominant oncogenes are higher than 10%. For example, Verlaan- transforning genes or oncogenes were present in hu- deVries et al. (11) detected activated ras genes in the man tumors and in carcinogen-induced animal tumors. 27% of acute myeloid leukemia examined. Ananthas- An extension of the NIH/3T3 transfection assay that wamy et al. (12) detected Ha-ras genes in four of six affords greater sensitivity is the nude mouse tumori- human squamous cell carcinomas examined. The addi- genicity assay (7). This involves cotransfection of NIH/ tion of the tumorigenicity parameter to the assay sys- 3T3 cells with tumor DNA and a selectable marker tem appears to improve the efficiency in detection of activated oncogenes in human tumors. Table 1. Abnormal expression of oncogenes in human tumors A variety of animal tumor model systems have also and tumor cell lines. been examined for activated genes using the NIH/3T3 assay. These include spontaneous tumors in rats and Mechanism Tumor type Oncogene Reference mice, tumors that arise after single or multiple doses Amplification Breast tumor neu (24) of carcinogen, and tumors that arise after long-tern Amplification Squamous cell c-erbB (25) exposure to a carcinogen. of the activated carcinoma Examples Amplification Small cell lung c-myc (26) genes in the different tumor model systems are shown carcinoma in Table 2. Like the human tumors, the majority of Amplification Small cell lung L-myc (27) activated oncogenes detected in the animal tumors are carcinoma members of the ras gene family. Other oncogenes have Amplification Neuroblastoma N-myc (28) Amplification Acute myelogenous c-myb (29) also been detected in animal tumors using the NIH/3T3 leukemia assay (Table 3). One example is the activated neu on- Translocation Chronic myelogenous c-abl (80) cogene found in nervous tissue tumors induced in rats leukemia by transplacental exposure to N-methyl-N-nitrosourea Translocation Burkitt's lymphoma c-myc (31) (MNU) or N-ethyl-N-nitrosourea (ENU). The c-raf on- ROLE OF ONCOGENES IN CHEMICAL CARCINOGENESIS 83 Table 2. Activated oncogenes in rodent tumor models. Number positive/ Model Tumor Number tested Oncogene Reference Spontaneous Mouse liver 17/27 H-ras (15)a, raf (1), unknown (1) (18,32) Single dose Rat 1/37 H-ras (1) (18) NMU Rat mammary 61/71 H-ras (61) (14) DMBA Rat mammary 6/29 H-ras (6) (15) Single dose, neonatal HO-AAFb Mouse liver 10/10 H-ras (10) (33) HO-DHE Mouse liver 11/11 H-ras (10), K-ras (1) (33) vC Mouse liver 10/10 H-ras (10) (33) DEN Mouse liver 14/33 H-ras (14) c Multiple doses DMN-OME Rat renal 10/35 K-ras (34) Aflatoxin B, Rat liver 10/11 K-ras (2), unknown (9) (35) Continuous dose TNM Rat and mouse liver 18/19, 10/10 K-ras (18), K-ras (10) (19) Furan Mouse liver 13/29 H-ras (10), raf (1), K-ras (2) (23) Furfural Mouse liver 13/16 H-ras (9), K-ras (1), unknown (3) (23) Benzidine-derived dyes Ratd 34/58 H-ras (31), N-ras (3) e DEN Rat liver 1/12 Unknown c Initiation-promotion DEN-Farber protocol Rat liver 0/20 (36) DEN + PB Rat liver 0/10 c DEN + EE2 Rat liver 2/19 non-ras (2) f DMBA + TPA Mouse skin 33/37 H-ras (37) Transplacental dose ENU Rat neuroblastomas 3/3 neu (3) (38) MNU Rat schwannomas 10/13 neu (10) (16) a Numbers in parentheses are the number of positive samples with that oncogene.