Fundamental and Molecular Mechanisms of Mutagenesis
333 (1995) El-87
Apoptosis and multistage carcinogenesis
in rat liver
*, W. Bursch, B. Grasl-Kraupp, B. Ruttkay-Nedecky
Abstract Apoptosis is a type of active cell death. It is involved in the homeostasis of cell number in tissues and is controlled by the growth regulatory network in the organism. It is also involved in the active removal of damaged cells. We have studied the role of apoptosis in cancer pre-stages and overt cancer in vivo, using rat liver as our main model system. Quantitative determination of apoptosis in histological specimens revealed that the rate of apoptosis tends to increase from normal to (preh-reoplastic to malignant cells. Thereby active cell death largely counterbalances the increasing rephcative activity in developing malignancy. Tumor promoters shift the balance in favor of cell replication, whereas promoter withdrawal, fasting or TGF-/? 1 favor apoptosis (anti-promotion). Preneoplastic cells are more susceptible than normal liver cells to stimulation of both cell replication or cell death. Consequentially (prekteoplastic tissue may preferentially grow or die during the appropriate treatment. Regimens that favor apoptosis and lower cell replication are shown to result in the elimination of preneoplastic cell clones from the liver (anti-initiation) and to reduce the cancer risk of the animal. Keywords: Apoptosis;
Apoptosis is a type of active cell death (ACD). The occurrence and biological relevance of ACD was first recognized by developmental biologists. During the embryonal development of mammalian organisms, cell death was found to be a necessary event to remove excessive tissue such as interdigital webs, the Millerian duct in males, and excessive neurons. Likewise, during metamorphosis in invertebrates and lower vertebrates massive tissue involution and cell elimination were found to be a physio-
* Corresponding author. Borschkegasse Sa, A-1090 Vienna, Austria. Tel.: +43 (1) 40154/345. Fax: +43 (1) 4060790. 0027-5107/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0027-5 107(95)00 134-4
logical event (‘programmed cell death’) (Nussbaum, 1901; Gliicksmann, 1930; Lockshin and Williams, 1965; Schweichel and Merker, 1973). The widespread occurrence of ACD in different situations including normal cell turnover, organ regression and after certain types of injury in adult organisms was recognized by Kerr et al. (1972) who coined the term ‘apoptosis’ (from Greek: falling down). Apoptosis was defined as an active, genetically encoded self-destruction of cells characterized by a specific morphology. These authors (Kerr et al., 1972) re-defined the traditional term ‘necrosis’ to indicate the type of cell death occurring after massive tissue injury associated with rapid incapacitation of major cell functions such as gene expression, ATP synthesis and membrane potential. At the same time,
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E. Farber and his colleagues developed a similar concept of an ‘active’ or ‘suicide’ type of cell death that depended on protein synthesis and appeared after treatment with genotoxic drugs (Liebermann et al., 1970; Farber et al., 1971). The morphological characteristics of apoptosis as defined by Kerr et al.. 1972 and Wyllie et al., 1980 are condensation of the cytoplasm, condensation of chromatin at the nuclear membrane and fragmentation of the nucleus and of the cell into apoptotic bodies, which are then phagocytosed by neighboring cells. On the other hand, in developing embryos Schweichel and Merker ( 1973) discovered the occurrence of 3 morphologically different types of ACD (type I is probably identical to apoptosis, type II is characterized by extensive autophagy, and type III by non-lysosomal disintegration). ACD can be inhibited by tissue-specific mitogens (‘mitogen rescue’); this provides a valuable functional criterion for discrimination from degenerative or necrotic types of cell death (Bursch et al., 1992a; Bursch et al., 1992b). ACD serves at least two different functions: it helps to maintain homeostasis of cell number in tissues and it may eliminate damaged cells. Its occurrence is under the control of the growth regulatory network in the organism and can be induced by growth regulating factors via specific receptors. ACD can also be induced by cell or DNA damage after radiation or after treatment with genotoxic agents. How DNA damage is monitored within cells and translated into activation of the suicide program is not well understood; the tumor-suppressor gene, ~53, seems to be involved in the control of damage-induced ACD, but not of receptor-mediated ACD (Yonish-Rouach et al., 1991; Clarke et al., 1993: Lowe et al., 1993; Lane, 1993). Also it is not clear how these different pathways to ACD. namely via receptors and via damage, are coordinated. 2. ACD in multistage
The development of cancer through the stages of initiation, promotion and progression has been generally accepted, and mutation and growth of mutated cell clones appear to be the key factors in carcinogenesis. Cell proliferation and cell death determine the growth rate of cell clones. Numerical growth
Research 333 (1995) S/-K7
results from rates of birth ((~1 minus rates of death ( p 1 of cells in the population ( CY- p ). In addition, LYand p also determine clone survival. If initiated cells undergo cell death, they are extinguished and cannot develop into a preneoplastic clone or cancer. The probability of extinction of a clone depends on the ratio of rates of cell death and birth (/3 : a). clone size and observation period (Moolgavkar, 1986; Luebeck et al., 1991). In order to investigate the role of ACD for growth and survival of (pre)neoplastic cell clones, quantitative determination of incidence and rates are required. This can only be achieved by histological procedures. ‘Classical’ techniques such as staining with hematoxylin and eosin are very useful to identify and discriminate cells dying by apoptosis or necrosis. The fluorescent dye Hoechst 33258 is valuable to reveal the morphology of nuclear chromatin, at least in vitro. Antibodies to proteins specifically associated with apoptosis may become available in the future. A promising example is provided by the precursor of transforming growth factor p 1 (TGFp 1) which appears in hepatocytes preparing for apoptosis and can be detected by specific antibodies (Bursch et al., 1993). On the other hand, the TUNEL assay and similar procedures to detect DNA fragmentation in situ on histological sections are not specific for apoptosis, contrary to wide-spread assumptions. A positive response in this test is not only seen in apoptotic hepatocytes, but also in necrotic liver cells dying after toxic injury (Ccl,. nitrosamines) and even after tissue autolysis (GraslKraupp et al., 1995). Morphological analysis also allows identification of the fype of ACD. Studies on the human mammary cancer cell line MCF 7 show that estrogen antagonists predominantly activate the autophagic type of ACD (type II, see above) rather than apoptosis (type nature of auI). The active, receptor-mediated tophagic cell death in MCF 7 cells was confirmed by the observation that estrogen addition prevented cell death (‘rescue’) (Bursch et al., 1995). Discrimination between different types of ACD is important because the underlying biochemical events obviously are different; unawareness of ACD type may lead to confusion when different models are compared. Furthermore, the various types of ACD may require specific. different strategies for therapeutic intervention.
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Information prerequisites for quantification of rates of ACD are incidence in histological sections and duration of the process. We used the inhibition of ACD by mitogens (‘rescue’) to estimate the duration of apoptosis in rat liver. From the kinetics of disappearance of apoptotic bodies an average length of 3 h was calculated. Based on this estimate we found apoptosis rates of 0.5% and 2-5% per hour in rat liver during regression of enlargement and in preneoplastic liver foci, respectively (Bursch et al., 1990). 2.1. Apoptosis
and tumor promotion
Most known non-genotoxic carcinogens appear to be tumor promoters and promotion of ‘spontaneously’ appearing initiated cells appears to be an important mechanism of carcinogenesis by these 1983; Schulte-Hermann, 1985; agents (Ward, Kraupp-Gras1 et al., 1991). The importance of ACD during tumor promotion was first described by Bursch and associates. These authors found that putative preneoplastic cell foci in rat liver showed high rates of apoptosis, which partially antagonized the effect of cell replication in these lesions. Tumor promoting agents such as phenobarbital or cyproterone acetate (CPA) inhibited cell death, thereby accelerating growth of foci and cancer development (SchulteHermann et al., 1982; Bursch and Lauer, 1983; Bursch et al., 1984; Columbano et al., 1984; Schulte-Hermann et al., 1990). A similar inhibition of apoptosis in altered cell foci was seen with TCDD (Schwarz et al., this volume). On the other hand, promoter withdrawal resulted in a massive increase of apoptosis in putative preneoplastic foci, suggesting the possibility of regression of tumor promotion in the liver (Bursch et al., 1984; Schulte-Hermann et al., 1990; Grasl-Kraupp et al., 1995). These findings show that tumor promoting non-genotoxic carcinogens act as survival factors for preneoplastic cells. A dramatic enhancement of apoptosis in foci was also obtained by restricted feeding (Grasl-Kraupp et al., 1994) and by treatment with S-adenosyl-methionine (SAM) (Garcea et al., 1989). Both regimens resulted in a reduction of size of altered foci and of tumor formation indicating an anti-promoting effect (Grasl-Kraupp et al., 1994; Pascale et al., 1992). These studies confirmed and extended the original
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observation (Schulte-Hermann et al., 1982; Bursch et al., 1984) that phenotypically altered cell foci in the liver not only exhibit enhanced cell proliferation, but also show enhanced apoptotic activity that largely counterbalances the proliferative activity. As a result, the turnover of cells in foci is much higher than in normal liver, yet the net growth rate of foci (a - fl> may be low. 2.2. Apoptosis
Irreversibility of initiation is considered a cardinal principle of the multistage concept of carcinogenesis; it is supported by numerous experimental findings. However, if initiated cells occasionally undergo cell death it follows that a certain percentage of these cells are extinguished. In fact, on the basis of mathematical models it was predicted that as many as 80% of initiated cells in the liver may become extinct (Moolgavkar et al., 1989; Luebeck et al., 1991). The validity of this prediction was experimentally supported by counting single putative initiated cells (detected by glutathione transferase P antibodies). These cells, almost absent in untreated young rat liver, dramatically increased after administration of genotoxic carcinogens, but were found to disappear subsequently, with evidence of apoptosis of some cells (Grasl-Kraupp, Wagner, Low-Baselli, Huber, Schulte-Herrnann, unpublished). Similar observations were made in another model of hepatocarcinogenesis (Satoh et al., 1989). Elimination of initiated cell clones of spontaneous origin was studied in aged rats. Animals were subjected to food restriction (60% of normal) for 3 months. This resulted in a decline of hepatic DNA synthesis, a tendency to enhanced apoptosis, and a loss of approximately 20% of cells in the liver. Qualitatively similar effects occurred in (spontaneous) foci, but were much more pronounced, so that foci number and volume declined by 85%. Subsequent treatment with the potent liver tumor promoter, nafenopin, induced significantly less tumors than in animals steadily fed ad libitum, indicating that many initiated (promotable) cell clones had been eliminated during the fasting period (Grasl-Kraupp et al., 1994). It may be concluded that initiation can be reversed, at least partially, by apoptosis. These findings show for the first time that food restriction enhances apoptosis preferentially in pre-
neoplastic lesions and may provide a new explanation for the well-known protection from cancer by low calorie diets in both experimental animals and in humans. Furthermore they show that the carcinogenic efficacy of non-genotoxic carcinogens and tumor promoters may depend on the number of spontaneous initiated cells or cell clones present in the liver. 2.3. Apoptosis
and tumor growth
Adenomas and carcinomas induced in rat liver by nafenopin exhibited a dramatically enhanced cell turnover with higher rates of DNA replication and apoptosis than in normal liver. Somewhat unexpectedly, the incidence of apoptosis increased from normal liver to foci to adenomas to carcinomas. This shows that the enhanced susceptibility of early cancer prestages to undergo ACD is not eliminated by selection in the course of progression. Rather the enhanced rates of both birth and death of cells may be an inherent characteristic of increasing malignancy. From a biological point of view, the high cell turnover may enhance the chance of acquisition of additional genetic damage and may therefore provide a selection advantage to the population of (pre)neoplastic cells (Grasl-Kraupp et al., 1995). When treatment with nafenopin was stopped, most adenomas and even carcinomas showed rapid regression and disappearance. DNA synthesis ceased almost completely, while ACD further increased (Grasl-Kraupp et al., 1995). Thus, even carcinoma cells may still depend on the presence of a promoter as a survival factor. Likewise, in hormone-dependent carcinomas, massive apoptosis and rapid tumor regression can be induced by hormone withdrawal or anti-hormone treatment. It can be concluded from these findings that the balance between birth and death can be shifted towards cell death even in tumors (Bursch et al., 1991; Szende et al., 1989). This is of principal interest for tumor therapy since hormonal signals inducing ACD seem to preferentially enhance cell death in tumors.
3. Regulation of ACD Numerous genes and gene products. some of which are involved in recognition and transduction
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of positive or negative growth signals, have been implicated in the control of ACD in cell culture systems and, in a few studies in vivo (see recent reviews: McDonnell, 1993; Bursch, 1994, SchulteHermann et al., 1995). Some of our own studies were focused on the possible involvement of transforming growth factor p 1 (TGF-P l), a member of a peptide family with growth inhibitory functions (Roberts et al., 1988). Immunocytochemical studies on rat liver involuting after withdrawal of the hepatomitogen CPA suggested the presence of TGF-P 1 precursor specifically in apoptotic hepatocytes (Bursch et al., 1993). TGF-/3 1 induced apoptosis in vitro in cultured hepatocytes and in vivo when injected as a pulse a few hours before sacrifice (Oberhammer et al., 1992; Oberhammer et al., 1993). In vivo TGF-P 1 was particularly effective in inducing apoptosis in the liver after cessation of CPA treatment. Apparently it acts synergistically on cells already primed for apoptosis by unknown signals generated during mitogen treatment or by hyperplasia. We determined whether TGF-P 1 peptide increases in the liver after CPA treatment. The results suggest that neither in the growth period nor during involution following CPA withdrawal a detectable increase in the hepatic concentration of TGF-Pl occurred. On the other hand, after intoxication with Ccl, used as positive control a pronounced increase of TGF-P 1 was seen (Ruttkay-Nedecky, Grasl-Kraupp et al., 1995). Obviously, any changes in TGF-Pl levels during liver involution are localized to cells undergoing apoptosis and seem to be undetectable in homogenates from entire liver. Activin, another member of the TGF-P family, was also found to induce apoptosis in rat liver and in cultured hepatocytes. It appeared one-tenth as active as TGF-/3 1 and its presence for 24 h was required to induce apoptosis (Schwa11 et al., 1993). These studies support the view that TGF-P 1 and related peptides are involved in the regulation of the balance between cell proliferation and cell death in the liver. Injection of TGF-/31 into rats also induced apoptosis in cells of putative preneoplastic foci in the liver; repeated injections resulted in a preferential decline of foci number and volume. This suggests that putative preneoplastic foci are still sensitive to the apoptosis-inducing effect of TGF-P 1, at least at
R. Schulte-Hemuznn Table 1 Enhanced
levels of cell birth and death of (pre-hteoplastic
Initiation Promotion Progression Tumor growth
in stages of cancer development
Initiation can be reversed by elimination of initiated cells and cell clones Promoters may inhibit cell death and lead to accumulation of preneoplastic cells. Withdrawal of promoters tames excessive death in preneoplastic lesions and may reverse promotion Enhanced cell turnover in (pre-)neoplastic lesions may favor acquisition of genetic damage and accelerate appearance of malignant cells Mitogen withdrawal or treatment with anti-hormones may result in excessive cell death. This may lead to reeression and notential elimination of tumors
the early stage of foci development investigated (Mtillauer et al., in preparation). On the other hand, Jirtle et al. (1994) reported that in some foci, hepatocytes had lower levels of the M-6-P/IGF II receptor. This may result in less sensitivity of foci cells than of normal liver cells to TGF-P 1. Malignant cells including human hepatocellular carcinoma may overexpress TGF-P 1 and may or may not be sensitive to TGF-/31 (Itoh et al., 1991; Schulte-Hermann et al., 1995). It cannot yet be decided from these studies whether and to what extent the enhanced rate of apoptosis in preneoplastic and neoplastic liver lesions may be due to modified sensitivity to TGF-fl 1.
Research 333 (1995) 8I-87
for risk assessment
The major conclusion from our studies is that ACD may be a determinant factor of carcinogenesis at all major stages as summarized in Table 1. Since ACD is under the control of the growth regulatory network of the organism, alterations in the balance between positive and negative growth signals may affect carcinogenesis at multiple sites, by modifying either cell replication, or cell death, or both. Most or all non-genotoxic carcinogens interfere with growth regulatory mechanisms, probably through receptors, although these, with a few exceptions, have not been clearly identified. As shown in the present series of studies, a new mechanism of action of non-genotoxic carcinogens is their inhibitory effect (in other words, their survival factor activity) on ACD in normal, preneoplastic and neoplastic cells. Some possible implications of these findings are given below. Non-genotoxic carcinogens may increase the survival of initiated single cells or cell clones and
thereby enhance or stabilize the effect of initiation. So far, this hypothesis is supported only by feeding studies (Grasl-Kraupp et al., 1994); appropriate experiments with non-genotoxic chemical carcinogens are on the way. Non-genotoxic carcinogens accelerate accumulation of initiated, preneoplastic and, possibly, neoplastic cells and thereby will enhance carcinogenesis. The carcinogenic potency of the model compound nafenopin was shown to depend on the presence and number of (spontaneously) initiated cells in the liver (Grasl-Kraupp et al., 1994). Hypothetically, in a liver carrying no initiated cells nafenopin should produce no tumors. From a mechanistic point of view, the ability of non-genotoxic carcinogens to rescue initiated cells contrasts clearly to mutagenic effects by which genotoxic carcinogens may generate initiated cells. Underfeeding was shown to eliminate some of the initiated cells (initiated cell clones) in rat liver and to exert an anti-initiating and anti-promoting effect. Thereby, the tumor yield after treatment with a non-genotoxic carcinogen was significantly reduced. Conversely, feeding ad libitum or overfeeding may have co-initiating and co-promoting activity, in the liver and possibly also in other organs. Therefore, the need for nutritional balance should receive increased attention during the design and interpretation of long-term carcinogenesis tests of chemicals.
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