Medical Hypotheses Hdcd njpchm8 (1992) 39.7.67-270 OLoqLnm!Gl9UpUKlAdl9?2

Activation of Oncogenes and/or Inactivation of Anti-oncogenes by Reactive Oxygen Species H. WEI Division of Toxicology, Department of Environmental Birmingham, AL 352944008, USA


Sciences, University of Alabama at Birmingham,

Abstract-Abundant evidence indicates that reactive oxygen species (ROS) are involved in mutagenesis and carcinogenesis. These chemical-generated or phagocyte-released ROS are known to cause a variety of genetic alterations which lie at the heart of the carcinogenic process. ROS have also been shown to cause malignant transformation of normal cells, and to increase expression of certain proto-oncogenes such as c-fos and c-jun. It is known that certain proto-oncogenes and anti-oncogenes may serve as the targets of carcinogens of various sorts. I hypothesize that ROS-mediated DNA damage may cause mutations and/or deletions in certain specific coding regions of tumor-related genes, and could be responsible for subsequent activation of oncogenes and/or inactivation of anti-oncogenes.


Reactive oxygen species (ROS) are ubiquitously present, and mainly produced by incomplete reduction of oxygen during respiration, by exposure to radiation or to oxidation-reduction active chemicals, or by release from phagocytic cells in response to bacterial invasion and other stimuli, such as opsonized zymosan and tumor promoters (14). These chemicalgenerated and phagocyte-released ROS are represented by a family of oxygen-centered derivatives, including superoxide anion radicals (Qz-), hydrogen peroxide (Hz%) hydroxyl radicals (OH), hypochlorous acid (HOCI) and, sometimes singlet oxygen (1%) (l-7). Activation of phagocytes is an important source of ROS in vivo, and characterized by a rapid consumption of molecular oxygen with an almost concomitant formation of .@- (1, 5, 6). These Date received 31 March 1992 Dale accepted 14 May 1992


anion radicals can be dismutated both spontaneously and enzymatically to Hz@, one of the relatively stable species. Hz4 freely diffuses through cellular membranes and is a precursor of .OH, an actual DNA damaging species, as well as of other potentially damaging agents such as HOC1 and 1% (5,6,7). Excessive or unnecessary formation of those ROS causes a number of destructive processes, including necrosis and breakdown of surrounding tissue, lipid peroxidation of cellular membranes, lysis of red blood cells and injury to epithelial cells, which lead to a variety of diseases including cancer (l-7). What is known about oncogenes and oncogene activation?

A growing number of vertebrate genes have been implicated in tumorigenesis. In their normal guise,

268 these genes are called ‘proto-oncogenes’, which are thought to be substrates for carcinogens of various sorts. Abnormal alleles of these genes can be found in retroviruses or in tumors, they are generally called ‘oncogenes’. Proto-oncogenes, when activated by either chemical carcinogens or viruses, will be converted to the malefactors known as oncogenes that play an important role in tumorigencsis (8). So far more than 60 pmto-oncogene/oncogenes have been identified (9, 10). The mechanisms of the oncogeneencoded proteins in tumorigenesis have been shown to mainly function through: 1) phosphorylation of proteins and polyphosphoinositides (e.g. sis gene); 2) regulation of adenylate cyclase (e.g. rus gene); 3) regulation of transcription (e.g. myc gene) and 4) regulation of DNA replication (8). Mechanisms of oncogene activation remain unclear. Prom-oncogenes are known to be activated in the following ways: point mutation by base substitution, provirus insertion, chromosomal translocation, gene rearrangement, and gene amplification (8, 9, 10). A wide range of carcinogens including viruses can cause DNA damage and certain genetic alterations. Most, if not all, cancer cells contain genetic damage that appears to lie at the heart of carcinogenesis. One of the genetic alterations has been identified as damage to cellular proto-oncogenes because mutant or activated forms of these proto-oncogenes have been detected in a variety of human and animal malignancies (8, 9, 10). Activation of proto-oncogenes may depend on the nature of carcinogens or on the origins of tumor. Some oncogenes only show one specific genetic alteration when activated (such as c-rus activated by a single base substituted mutation), whereas others may exhibit several forms of genetic alterations (such as c-myc activated by provirus insertion, chromosomal translocation and/or gene amplification) (g-11).

What is known aboul anti-oncogenes and antioncogene inaclivation? The recent evidence shows that carcinogenesis de-

pends not only on activation of ‘dominant’ protooncogenes but also on inactivation of ‘recessive’ antioncogenes (also known as tumor suppressor genes), suggesting that cancer development is a complicated balance between genes (10. 11). Anti-oncogenes are genes which appear to be involved in providing negative signals for cell proliferation and behavior (12, 13). These signals may operate in quite different ways, either to regulate temporary withdrawal from the cell cycle or differentiation. The products of antioncogenes are normally involved in inhibition of cell


growth. Loss of or alterations in structure of these anti-oncogene products would lead to disorders in the interactions between cells and their surroundings, and eventually tumor formation arises as a consequence of the altered control of cell proliferation and differentiation. The precise biochemical mechanisms by which any of the anti-oncogene products regulates cell behavior remains unknown. However, the biochemical functions of anti-oncogene proteins have been shown as follows (13): 1) interact with DNA tumor viral proteins, which might regulate initiation of DNA synthesis (e.g. Rb. ~53); 2) act as a transcription factor (e.g. WTI); 3) interact with p21 r-as, and thereby regulate cell proliferation through signal transduction pathways that involve p21 ras, (e.g. NFI). Like protooncogenes, anti-oncogenes may serve as possible targets of various carcinogens. However, unlike oncogene activation which exhibits the gain-of-function mutations, the anti-oncogene inactivation is characterized by the loss-of-function genetic alterations. So far only a few anti-oncogenes (less than 10) have been identified. The well-characterized anti-oncogene include Rb, ~53, WTl , NFI, FAP, MEN-l and DCC (10, 13). The mechanism of anti-oncogene inactivation, though unclear, appears to be related to point mutations and deletions in the region-coding sequences of these genes (12, 13). How are ROS associated with carcinogenesis?

ROS are known to play important roles in mutagenesis and carcinogenesis (14, 15, 16). The known co-carcinogenic effect of wounding and inflammation in a variety of human organs, including skin, stomach, colon, bone and bladder, could be due to the action of ROS generated by infiltrating polymorphonuclear leukocytes (PMNs) (16). One of the typical characteristics of the carcinogenic processes is genetic alterations in cellular DNA. ROS are known to induce certain types of DNA damage in a way whereby hydroxy radicals (OH.) generated as intermediates are responsible for that damage (15). These OH. are highly reactive, and responsible for a variety of genetic alterations, including DNA strandbreaks, oxidative base modifications, chromosomal abnormalities, and cellular transformation (14, 15, 16). A number of studies have shown that in the presence of some transition metal ions, Hz& causes nucleoside oxidation in co-incubated bacterial DNA and in cellular DNA of co-incubated cells, all of these oxidized DNA bases are also formed by the action of ionizing radiation (15). When removal of abnormal bases from DNA is not complete or timely, they have the potential to exert their deleterious effect.



Evidence shows that thymidine glycol (dTG) blocks DNA replication, while &hydroxy-2’-deoxyguanosine (8-OHdG) causes mispairing and miscoding in either paired or neighboring bases. S-hydroxy-methyl2’-deoxyuridine (HMdU), which has been shown to be incorporated into cellular DNA, is mutagenic and cytoroxic to a number of mammalian cells (17). Recently, ROS were shown to activate certain protooncogenes in vitro. For example, an alteration of cabl methylation was induced in cells transformed by 12-0-teuadecanoyl phorbol-13-acetate (TPA)-stimulated neuwphils (18). ROS generated by the xanthine/xanthine oxidase system induced TPA-like expression of proto-oncogenes, such as c-fos and cmyc in mouse epidermal cells (19). The induction of c-fos and c-myc was also observed in mouse skin treated with TPA in vivo (20). H-rus was found to be activated in the uninitiated skin tumors of SENCAR mice induced by TPA alone (21), or by a free radical-generating agent-benzoyl peroxide (22). Recently, we have shown that TPA can significantly increase formation of oxidized DNA bases in mouse skin (23), and that skin tumor tissues contain signilicantly higher 8-OHdG (about 4-fold) than tumor surrounding tissues (unpublished data). These findings provide major pieces of evidence that free radicals generated indirectly (as the case of TPA) and directly (as the case of benzoyl peroxide) might be involved in tumor-related gene activation/inactivation. Hypotheses ROS are shown to be involved in mutagenesis and carcinogenesis, in which oxidatively damaged DNA is a characteristic event. Oxidative DNA damage is found to cause miscoding and mispairing in mammalian DNA (24), which may lead to base substitutions in DNA. If these genetic alterations fall within the specific coding sequence of proto-oncogenes and/or anti-oncogenes, the corresponding biological consequence may occur. Since both increased oxidative DNA damage and oncogene activationlantioncogene inactivation have been observed in human and animal tumors, a quantitative correlation between both events will strengthen our hypothesis that ROS-mediated DNA damage plays an important role in oncogene activation and/or anti-oncogene inactivation. Abundant evidence indicates that prolooncogenes/anti-oncogenes may serve as the targets of DNA damaging agents and carcinogens of various sorts. The question addressed here is: Is there any correlation between ROS-mediated oxidative DNA damage and proro-oncogene activation andlor anli-oncogene inactivalion? We hypothesize that if some proto-



oncogenes/anti-oncogenes were targets of ROS, treatment of these genes with ROS would cause certain genetic alterations which may fall within the specific coding regions of these genes. Mutations due to DNA repair or deletions due to severe DNA damage might lead to activation and/or inactivation of these genes. Activation of oncogene would be characterized by production of mutated or abnormal oncoproteins or overexpression of normal oncoproteins. Inactivation of anti-oncogene should exhibit a loss of normal gene products. Consequence of both events would lead to cellular transformation, and subsequently to tumor formation. In order to prove that ROS are directly responsible for gene activation or inactivation, epigenetic effects caused by ROS or tumor promoters in animal or cell models must be excluded. Therefore, transfection of the ROS-damaged gene fragments to cell lines provides a practical strategy towards this hypothesis. Furthermore, oncogene activation and/or anti-oncogene inactivation can be identified by detecting the presence and abnormal forms of gene products, observing the transformed foci, and analyzing molecular alterations in gene structure. Unveiling of the role of ROS-mediated oncogene acthation and/or anti-oncogene inactivation should have a major impact on our understanding of ROS-induced carcinogenesis, and contribute to development of the therapeutic and preventive strategties against human cancers associated with ROS exposure and chronic inllammation. References






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or inactivation of anti-oncogenes by reactive oxygen species.

Abundant evidence indicates that reactive oxygen species (ROS) are involved in mutagenesis and carcinogenesis. These chemical-generated or phagocyte-r...
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