Institut für Krebsforschung der Universität Wien, Borschkegasse 8a, A-1090 Vienna, Austria,
1 Fred Hutchinson Cancer Research Center, Public Health Sciences Division, MP-665, 1124 Columbia Street, Seattle, WA 98104, USA and
2 Department of Mathematics and Computer Science, Free University of Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands
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Abstract |
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Abbreviations: 2D, two-dimensional; 3D, three-dimensional; AB, apoptotic body; AI, apoptotic body index; CI, confidence interval; G+, placental glutathione S-transferase-positive; G, placental glutathione S-transferase-negative; GLDH, glutamate dehydrogenase; GST-P, placental glutathione S-transferase; LI, labeling index; NNM, N-nitrosomorpholine.
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Introduction |
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In rat liver the expression of placental glutathione S-transferase (GST-P) is a useful marker for most (pre)neoplastic lesions (4,5). Studies on the very first stages of hepatocarcinogenesis have become feasible after the discovery of selective staining of single rat hepatocytes for GST-P [placental glutathione S-transferase-positive (G+) and placental glutathione S-transferase-negative (G) cells], which appear after administration of various genotoxic hepatocarcinogens, but not after tumor promoters and other non-genotoxic agents (6). Formation of G+ single cells increased with the dose of the genotoxic initiator and decreased when metabolic activation of the initiator to the ultimate carcinogen was inhibited (68). Mutated Ha-ras was found in small preneoplasias of mice and rat liver a few weeks after initiation, suggesting that the mutation occurred early in the carcinogenic process (9,10). Furthermore, the incidences of single cells with the G+ phenotype or with a mutation in the albumin locus were nearly identical, which supports the hypothesis that the formation of G+ cells results from a mutational event (11). When initiation is followed by treatment with a tumor promoter a small percentage of G+ single cells develop to G+ foci, which may give rise to G+ tumors (6,12). Based on these findings, most investigators assume that single G+ hepatocytes are initiated and capable of evolving into hepatic neoplasia.
The number of initiated cells within an organ is a major determinant of the risk of cancer formation. In previous studies the dose of the initiating carcinogen, extent of genotoxic damage, size or frequency of putative preneoplastic liver foci and incidence and multiplicity of tumors have been used as the main parameters of mathematical models developed for the description and prediction of carcinogenesis (1320). In the present work a quantitative analysis has been performed on the formation and further fate of G+ single cells using both experimental and mathematical methods. To compute the number and the three-dimensional (3D) size distributions of G+ lesions we first applied stereological procedures, used in previous studies (2123). However, these procedures assume a spherical shape of preneoplasias, a condition not met by many small G+ lesions. Therefore, a novel stereological approach was developed and used that is based on a discrete computer model for the variable 3D aggregation of G+ cells into lesions (24; E.G.Luebeck and M.de Gunst, manuscript in preparation). Combined with a temporal stochastic growth model for these lesions it allows for the quantitative estimation of the number and sizes of clones (i.e. the number of G+ single cells, of G+ foci and of G+ cells within foci) in the liver as a function of time (16,25). The parameters of the growth model are estimated from transectional observations; they provide quantitative information on the rate of initiation as well as of division and death of G+ cells, which can be determined only partly from experimental observations. Thus, model-derived estimates are essential for understanding the cell kinetics of initiated cells in the first period after carcinogenic insult.
The crucial property of initiated cells is considered to be their potential for selective growth under the influence of growth-promoting stimuli. Whether this property is acquired during initiation and is already expressed by single initiated cells has not been investigated experimentally. Studies on more advanced stages of hepatocarcinogenesis revealed that preneoplasias and neoplasias exhibit higher replicative activities than normal liver, leading to preferential expansion (2628). Apoptotic activity also increases from normal liver, to foci, to adenomas and to carcinomas (27,29). As a result, cell turnover is accelerated in the course of hepatocarcinogenesis. However, at all stages rates of cell replication were higher than those of apoptosis, allowing a net gain of (pre)neoplastic cells (27,29). Tumor promoters suppress apoptotic activity and thus further accelerate growth of the lesions. In the case of withdrawal of the promoter, the high rate of apoptosis in (pre)neoplasias may increase even further, leading to a net loss of cells and to selective regression of tumors and tumor prestages while the surrounding unaltered tissue is little affected (27,2931).
To study the growth characteristics of the very first stages of hepatocarcinogenesis we applied N-nitrosomorpholine (NNM), one of the nitrosamines occurring in tobacco smoke and in a variety of foods and alcoholic beverages, which likely contributes to the development of human cancer (32,33). NNM causes damage to and death of hepatocytes, activation of p53, inflammation, regeneration of the liver and, finally, the development of numerous preneoplastic lesions that evolve into tumors under various promoting stimuli (3437). Similar events are associated with human hepatocarcinogenesis. Therefore, NNM-induced changes may provide a useful model to elucidate the mechanisms of carcinogenesis in the mammalian liver.
In the present study application of NNM generated numerous G+ single cells in the livers of rats; a subfraction of these cells developed into larger clones. In single G+ cells replication was low but in G+ mini-foci replication and death of cells were increased. This indicates that the defects in growth control, previously found in more advanced (pre)neoplasias, are acquired during initiation or soon thereafter and become manifest in the first stages of carcinogenesis. These studies provide direct experimental evidence that initiated cells have a selective growth advantage. In addition, their increased rate of apoptosis suggests the potential to undergo selective elimination. By this mechanism clones carrying the irreversible `initiation effect' may become extinct under certain conditions. This would lead to reversal of the biological effects of initiation.
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Materials and methods |
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Four animals each were analyzed at days 0.5, 1.5, 2.5, 17.5, 20.5, 24.5, 27.5 and 31.5 post-NNM treatment; the number of animals analyzed at days 0, 3.5, 4.5, 7.5, 13.5 and 107.5 were 9, 6, 2, 3, 3 and 10, respectively.
The determination of hepatic DNA content was as given elsewhere (38). The extent of cell lysis was estimated by determining serum glutamate dehydrogenase (GLDH) activity, applying commercial test kits (Merck, Darmstadt, Germany).
Histology and morphology.
Liver weight was recorded and specimens of liver tissue were fixed and processed as described (27). Sections 2 µm thick were stained either with hematoxylin and eosin or by immunohistochemistry, which was accomplished by the unlabeled antibody peroxidaseanti-peroxidase technique according to previous descriptions (39). The following antisera were used (dilutions in parentheses): anti-GST-P (1:320) (Biotrin International, Dublin, Eire); anti-rabbit immunoglobulin and horseradish peroxidaseanti-peroxidase complex (both 1:50) (Dakopatts, Glostrup, Denmark). G+ single cells and G+ multicellular foci were identified by the anti-GST-P stain. Autoradiography for evaluation of labeling indices was performed as described (27).
For 3D reconstruction of G+ single cells 30 serial sections, 4 µm thick, were stained for GST-P and subjected to autoradiography. Any G+ single cells, with or without [3H]thymidine label, were identified in serial section no. 15. Individual G+ cells were then followed in the consecutive serial sections (nos 1630 and 141) using two microscopes linked by a bridge for overprojection (Zeiss, Germany). Whether a G+ single cell was actually single or whether there was immediate contact with one or more G+ cells in one of the serial sections was recorded. This allowed 3D reconstruction of the actual size of lesions of 1, 2 or >2 G+ cells.
Areas of evaluated tissue sections or areas of necrotic liver tissue were measured by means of a semi-automatic image analyzer (VIDS IV; Ai-Tektron GmbH, Meerbusch, Germany).
Experimental determination of DNA synthesis and of apoptosis per day.
Implantation of osmotic minipumps may lead to significant shifts in proliferation kinetics in liver regenerating after toxic injury (W.Bursch, personal communication). We therefore used the following protocol that allows for the determination of rates of cell replication and apoptosis per day. Animals were adapted to rhythmic feeding (from 9 a.m. to 2 p.m.) from 4 weeks before NNM treatment until the end of the experiment. This procedure synchronizes DNA synthesis and, presumably, apoptosis in unaltered liver and in preneoplastic lesions to single waves per day, which are ~12 h apart (27,40). At the peak of DNA synthesis, replicating cells were labeled by [3H]thymidine application. In order not to disturb their eating behavior, animals were killed 36 h later, allowing determination of apoptosis at its daily maximum.
Determination of cell replication.
The labeling index (LI) is defined as the number of labeled nuclei/100 nucleated hepatocytes; LIs were determined in at least 2000 hepatocyte nuclei/liver and in all nucleated cells of individual G+ cell clones.
We assumed that all labeled hepatocytes proceeded through mitosis, as shown previously (41). We tested whether or not a fraction of labeled hepatocytes underwent a second cell division during the 36 h interval between [3H]thymidine application and death. [3H]thymidine was injected 24 h post-NNM; there was no significant difference between the LIs of animals killed either 12 [9.43, 95% confidence interval (CI) 8.8210.1] or 36 h later (10.56, 95% CI 9.9811.2) and hence no evidence for a second replication cycle within 36 h.
To study whether rhythmic feeding synchronizes DNA synthesis of G+ single cells, as shown for (pre)neoplastic lesions (27,40), a subgroup of four animals received three consecutive injections of [3H]thymidine at 1, 6 and 11 p.m on day 19 post-NNM treatment. The percentage of labeled nuclei of G+ single cells obtained with this protocol was 2.2% (95% CI 0.466.38), as opposed to 1.94% (95% CI 0.54.96) after a single injection at 8 p.m. This result indicates that rhythmic feeding also synchronizes DNA synthesis of G+ single cells.
Determination of cell death by apoptosis.
Apoptotic bodies (ABs) were identified in hematoxylin and eosin stained sections according to previously established criteria (30). The apoptotic body index (AI) indicates the number of ABs per 100 intact hepatocytes. AIs were determined in >4000 unaltered cells/liver, in all cells of individual G+ clones and in all unaltered cells immediately surrounding G+ cells.
In untreated livers of full-grown rats with balanced gain and loss of hepatocytes, LI is on average ~3-fold higher than AI (27,40). In order to estimate the actual loss of hepatocytes per day we multiplied the AI by a correction factor of 3. The discrepancy between LI and AI was expected for the following reasons. ABs and hepatocytes are of approximately spherical shape with a mean diameter of 4 and 28 µm, respectively, as schematically depicted in Figure 1 (W.Bursch, personal communication; 42). According to Fullman's formula the relative probability of cutting hepatocytes and ABs in a section 2 µm thick is (4 + 2)/(28 + 2) = 0.2 (21); thus the probability of detection is 5-fold lower for ABs than for hepatocytes. Furthermore, one hepatocyte undergoing apoptosis usually breaks into several ABs, which are supposed to be randomly grouped in three dimensions; in two dimensions two ABs are seen on average. In summary, the probability of finding at least one residue of a dying hepatocyte may be lower by a factor of 23 than of seeing an intact hepatocyte.
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m is the number of ABs within G+ cells and in hepatocytes immediately surrounding G+ cells, , Nsurr is the number of hepatocytes immediately surrounding G+ cells, AIunalt is the index of ABs in unaltered G hepatocytes and NG+ is the number of G+ cells.
Modeling growth and regression of G+ single cells and multicellular foci
The clonal growth model
. The G+ cell clones observed in this experiment were analyzed quantitatively via a stochastic growth model which provides explicit expressions for the number and size distributions of the clones. To be specific, let X be the number of normal hepatocytes that are at risk of acquiring the G+ phenotype. This alteration may occur either spontaneously or in response to genotoxic exposures such as the NNM exposure described in the present experiment. Let v be the rate (per cell) at which normal hepatocytes are transformed into G+ cells. Clones are then assumed to arise according to a non-homogeneous Poisson process with rate vX. In the context of chemical carcinogenesis this process is referred to as initiation. Subsequent clonal expansion of the initiated cells is described in terms of a simple birth and death process. Initiated cells either divide with cell division rate or disappear with rate ß. The model, however, does not distinguish between cell loss due to apoptosis and loss of the G+ enzyme marker. Only cells or clones of cells that show the G+ phenotype are evaluated by the mathematical analyses here.
Based on these assumptions formulae for the number and size distribution of initiated cells were derived by Dewanji et al. (16) and are explicitly evaluated here for the case of piecewise constant parameters assuming (mean) exponential growth of the clones (see Appendix).
Stereology
. Several analytical methods are available that deal with the stereological problem, namely the problem of how to translate the number and size distribution of foci seen in two-dimensional (2D) tissue sections to the respective 3D quantities. Most parametric methods (i.e. methods that assume a parametric form of 3D size distribution like the clonal growth model described above) assume that foci are of spherical shape, an assumption that is clearly inadequate for small foci such as those observed in this experiment (see Results and Figure 2). In order to address this problem a discrete stereological method was used that deals with the stereology of randomly shaped cellular clusters and was applied to a subset of the data presented in this study (for details see ref. 24). By analogy with Wicksell (43) and the Saltykov method (22) for spheres this method consists of a discrete 3D to 2D transformation.
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Our analyses were restricted to foci showing no more than 20 cells. Two separate calcluations were performed. The first without a lower limit (n 1) and the second with a lower size limit (n
2). For computation of the transformation coefficients anm an upper size limit of M = 200 cells was selected.
Hepatocytes are described as densely packed cubes of fixed length. We chose a = 28 µm, a value that is close to that derived from the measured mean cellular volume of focal hepatocytes (42). For completeness, we provide the relevant expressions for analysis of the 2D data observed in this experiment.
Let the size distribution pm represent the probability that a clone which is observed at time t contains m cells. Since we assume an upper size limit at M = 200 cells, pm = 0 for m > M. Time of origin of the clone is integrated out. The number of non-extinct clones in a unit volume at time t, NV, is Poisson distributed with mean . We note that pm, NV and
and all quantities derived from them depend on t. For simplicity, we omit this dependence in our notation. The expressions for pm and
are derived in the Appendix.
The conditional probability qn of finding a transection that shows exactly n (20) cells at time t is given by
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with the denominator defined as
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Assuming that the clones are Poisson distributed in space, the number of transections observed at time t on a section of area A, NA, is also Poisson distributed with mean ENA = AVEZ.
Likelihood maximization.
We fitted the model to the data by maximizing the likelihood with respect to the model parameters , ß and vX on each of several time intervals, starting at the time of birth. The time intervals were selected to partition the entire period from birth of the animals to 51 days after NNM so that important changes in the initiation rate and cell kinetic parameters could be adequately captured. It is important that the time origin is chosen to coincide with the birth of the animal since foci may occur spontaneously prior to NNM exposure at day 56. Preliminary analyses, using up to four different time intervals with variable change points, showed that a partitioning of the time line into three intervals post-NNM was adequate to fit the data.
Phase I: expansion phase from 0 to 14 days after NNM.
Phase II: regression phase from 14 to 28 days after NNM.
Phase III: stabilization phase from day 28 after NNM onwards.
The likelihood of the data is given by a product of the probabilities over all sectional observations, i.e.
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where the index i runs over all sections observed, their area being Ai, and where nij denotes the number of cells seen on the jth transection of the ith section.
The likelihood was maximized using the DFP algorithm (44) and confidence intervals were obtained using the profile likelihood method (45). As will be illustrated in the next section, the results, using the maximum likelihood estimates, can either be expressed in terms of 3D quantities (size distribution, expected number of 3D clones per ml liver) or in terms of observable 2D quantities.
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Results |
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NNM-induced loss of liver tissue
After NNM application a wave of apoptosis occurred at the 12 h time point (Figure 3A). This was followed by the appearance of large areas of hepatocellular necrosis around the central venules at 24 h (not shown). Morphometric analysis at the maximal extension of damage 36 h after NNM demonstrated that 45 ± 7.9% of total liver area was affected by necrosis. On the border of the necrotic areas liver cell death through apoptosis was detected. Due to massive damage of the liver tissue a thorough quantification of apoptotic bodies at the 36 h time point was not feasible.
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While GLDH levels returned rapidly to pretreatment levels, the apoptotic activity remained elevated throughout the 31.5 days of observation (Figure 3A).
Regeneration of the liver
Regeneration of the liver started 48 h after NNM, as indicated by 10-fold increased replicative DNA synthesis (Figure 3B). As a result, total hepatic DNA content and absolute liver weights were back to their original level 24 days after NNM (Figure 3C
). Rates of replication and apoptosis were still elevated at day 31.5, indicating that cell turnover was increased for at least 1 month after NNM (Figure 3A
).
G+ single cells and multicellular foci after NNM treatment in histological sections (2D data)
Before treatment few G+ cells could be detected. After NNM G+ single cells increased dramatically in number reaching a maximum of ~150/cm2 section area at day 13 (Figures 2 and 4A). The increase in the number of lesions consisting of 2, 3 and more G+ cells per cross section was less steep, with maxima at days 13 and 17. Thereafter, numbers of G+ single cells per cm2 as well as of lesions <5 G+ cells per cross section declined. The number of G+ single cells dropped to one third of the maximum on day 34.5 (53/cm2) and later to one fifth on day 107.5 post-treatment (Figure 4A
). Lesions consisting of 2 or 35 G+ cells also leveled off to one third to one fifth of the peak value. The mean number of all lesions also slowly decreased to one third of the maximal value (200.2 ± 62.4/cm2 at day 13.5 versus 60.5 ± 26/cm2 at day 107.5, P < 0.01 according to Wilcoxon's test). A similar finding was reported by Satoh et al. (46).
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Rate of formation of G+ cell clones
Using the maximum likelihood estimates of the growth model the number of cells initiated per liver per day and the expected number of clones per liver were computed (see Appendix). According to these estimates, ~5200 clones were present in the liver at day 0. Between days 1 and 14 after NNM ~171 000 new clones developed (mean rate per liver per day = 12 200); the total clone number decreased by ~42 200 (23.9%) in phase II. In phase III the total number of G+ clones increased again, leading to the appearance of 11 350 new clones until day 51 (493 clones/liver/day). All phases together resulted in a net gain of ~140 150 G+ clones in the organ (for data per day see Figure 5). Due to the overwhelming numerical predominance of single G+ cells, gain or loss of all G+ cells, including those from larger foci, were within a similar range (approximate numbers: phase I, +231 000 G+ cells; phase II, 57 000 G+ cells; phase III, +52 000 G+ cells; total gain 226 000 G+ cells).
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DNA synthesis in single G+ cells and their precursors
DNA replication, which was determined by [3H]thymidine incorporation 36 h before death, was assayed on histological sections. At multiple time points after NNM LIs of single G+ cells were generally low and were consistently less than of G cells and of G+ cells in lesions of 2 cells (Figure 6
, upper panel). This suggests that the precursors of single G+ cells had a low rate of DNA synthesis 36 h before observation (killing). A similar result was seen when [3H]thymidine was injected only 12 h before death (Table II
).
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For stereological reasons some G+ cells appearing as single in histological sections (2D) are actually part of G+ foci with 2 cells. We therefore determined the true percentage of [3H]thymidine-labeled single G+ cells by 3D reconstruction in serial sections: 73% of all but only 26% of the labeled G+ single cells in two dimensions were truly single in three dimensions (Table IV
). This result indicates that the low LI of single G+ cells reported above are still overestimates. True rates of DNA synthesis of single G+ cells and of their precursors are even lower and may, on average, be only ~25% of rates calculated from histological counts.
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DNA synthesis and apoptosis in G+ foci
In G+ mini-foci the measured LIs for cell replication and AIs for apoptosis were initially high and then decreased in the course of the experiment (Figure 6). Replication was ~3- to 5-fold higher in the foci than in the surrounding normal tissue, which was seen after a 2, 12 and 36 h lag between [3H]thymidine injection and death (Figure 6
and Table II
). Rates of apoptosis in G+ foci were also 3- to 5-fold above the level of normal hepatocytes, indicating elevated cell turnover in the first stages of hepatocarcinogenesis (Figure 6
).
Estimation of cell kinetic parameters of initiated cells by mathematical analysis
The stochastic growth model combined with the stereological procedure served to estimate the division rate () and death rate (ß) of the G+ lesions. The growth model assumes that all G+ cells divide or disappear with the same rate regardless of clone size. Due to the overwhelming numerical predominance of G+ single cells in the data, the estimated
2 and ß2 values represent mainly single cells. Therefore, lesions of >1 G+ cell were subjected to a separate analysis to compute
3 and ß3. Table V
compares estimated
and ß values with experimentally derived rates for cell division and cell death among all G+ lesions, including G+ single cells, and among lesions with at least 2 G+ cells.
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Estimation of death rates revealed the highest ß2 and ß3 values of G+ cells in regression phase II, when G+ cells disappear at a rate of ~6 or 20% per day, respectively, leading to a net loss of G+ cells (ß2 > 2; ß3 >
3). In phases I and III, however, the model predicts a net gain of G+ cells (
2 > ß2;
3 > ß3), which is consistent with the experimental data.
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Discussion |
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Clonal evolution of hepatocarcinogenesis
The consecutive development of G+ single cells into G+ foci consisting of 2, 35 and >5 cells (Figure 4) supports the concept that liver preneoplasias are of monoclonal origin and that expression of the G+ phenotype is heritable by daughter cells. Mathematical analysis of the experimental data confirmed the clonal nature of liver preneoplasias and the persistent phenotype of the component cells. This also agrees with previous reports, e.g. homogeneous expression of isoenzymes in early preneoplasia (11,47).
Is DNA synthesis necessary for subsequent formation of G+ single cells?
Theoretically, G+ single cells may emerge from G cells without preceding DNA replication or via asymmetrical division into a G and a G+ cell. In the latter case expression of the G+ phenotype in one of the two daughter cells might follow immediately after DNA replication or after a certain time interval. If expression of the G+ phenotype was preceded by DNA synthesis a high proportion of G+ single cells, emerging in the first few days after NNM, should be labeled. [3H]thymidine was injected 0, 24 and 48 h after NNM treatment. At killing 36 h after label injection the G+ single cells newly formed within 24 h should carry the label. In sharp contrast to this prediction, LIs of G+ single cells in two dimensions were extremely low at death, i.e. 0% on days 1.5 and 2.5 and ~4% on the following days (Figure 6 and Table III
). Furthermore, after stereological reconstruction only 26% of the labeled G+ single cells in histological sections (2D) turned out to be a true single G+ cell in three dimensions (Table IV
). Thus, no more than ~1% of the G+ single cells seem to replicate DNA within 36 h of their formation.
In conclusion, a high proportion of G+ single cells, at least in the first days after NNM treatment, are formed without preceding DNA synthesis. These findings appear to contradict the classical concept of initiation, i.e. that the generation of an `initiated' phenotype requires a genetic alteration which is fixed by replication. Since the molecular mechanisms of induction of the G+ phenotype are not known, the appearance of G+ hepatocytes could be due to epigenetic rather than genetic events.
Experimental and mathematical determination of DNA synthesis in G+ single cells and small G+ foci
In several experimental approaches (see Figure 6 and Tables II and III
) replication of G+ single cells was found to be considerably lower than in lesions consisting of >1 G+ cell. Independent mathematical estimates confirmed the low rate of single cells entering the 2 cell stage (Table V
). The reasons for this finding are unclear. It appears unlikely that G+ single cells suffer from the genotoxic and cytotoxic effects of NNM, since a low replication rate was still observed on day 107.5 post-NNM treatment. Rather, it may be an inherent property of G+ single cells to be less prone to proliferation than small G+ clones.
The model also confirmed an elevated replication rate in multicellular lesions. This is further evidence for altered growth regulation from the very beginning of hepatocarcinogenesis onwards.
Experimental and mathematical determination of apoptosis in G+ single cells and small G+ foci
In the stochastic growth model ß does not distinguish between death of G+ cells and disappearance of the G+ phenotype. Comparison of data determined experimentally and mathematically revealed that in phases I and III ß values and the percentage of ABs were within a similar range; thus ß represents mainly loss of G+ cells, at least under the present experimental conditions (Table V).
The highest apoptotic activities of G+ cells were found in regression phase II. For this phase the model predicts that G+ single cells die at a rate of ~0.06/day (Table V). With current experimental procedures it is not possible to detect apoptotic events associated with the death of single G+ cells. Thus, a direct comparison between the mathematical and experimental data is not feasible. In addition, the molecular mechanisms of the appearance of single G+ hepatocytes are not known and could be due to unstable epigenetic rather than genetic changes. Then transient expression only of the G+ phenotype in single cells could be the cause of the disappearance of G+ cells at the later time points of the experiment.
In phase II the mathematically estimated death rate of 0.20 for G+ cells in multicellular foci (ß3) exceeds the experimentally determined rate of ~0.035 (Table V). This discrepancy may, at least in part, be due to a sampling bias, i.e. large lesions with a probably lower apoptotic activity have a higher probability of being hit by random cuts than small lesions with a presumably high apoptotic activity. Furthermore, there was considerable heterogeneity in the cell kinetic parameters in the foci.
Changes in replication and death of G+ cells in the course of liver regeneration after NNM
Both cell replication and cell death by apoptosis determine the fate of G+ clones. In any case, in phase II ß values exceeded values, resulting in a net loss of G+ cells. Conversely, in phases I and III the model predicts a net gain of G+ cells, i.e.
2 > ß2;
3 > ß3. These independent calculations are consistent with our experimental data (Table V
).
The change in rates of replication and death of G+ cells observed in phases IIII may reflect alterations in the concentrations of growth factors in the liver (48,49). In phase I regeneration signals, released in response to severe damage to the liver, may act on G+ cells and are probably responsible for the dramatic expansion of all G+ clones. In phase II regeneration of the liver and release of growth factors slows down. This probably results in deprivation of G+ cells of factors required for survival and/or growth. As a result, G+ clones stop growing and lose component cells.
Similar phenomena may occur in virus-induced hepatocarcinogenesis in humans, which is further enhanced by repeated aflatoxin B1 or alcohol intoxication. It appears that sustained cell damage or inflammatory responses in the liver release growth-promoting stimuli that are necessary for the development of (pre)neoplasias (50).
Characteristics of initiated cells
Elevated cell turnover is one of the hallmarks of the advanced stages of carcinogenic development. (Pre)neoplastic lesions exhibit enhanced sensitivity towards growth regulating factors which results in preferential growth or regression of the lesion (27,51). The present study shows that G+ single cells do not meet this criterion and that this important characteristic does not become manifest before lesions consist of at least 2 G+ cells. Thus early hepatocarcinogenesis seems to involve a kind of intermediary cell population that gives rise to a cell population undergoing preferential expansion or regression. It would be important to elucidate the mechanisms regulating this complex development. Then it might become feasible to induce preferential loss of initiated cells and thereby to antagonize the process of tumorigenesis at the very beginning.
Preferential growth or elimination of G+ cells
The findings described above allow for the following conclusions, as schematically depicted in Figure 7. In healthy tissues a low cell turnover (
1, ß1) protects against dramatic alterations in cell numbers. Initiation (µ1) generates persistent and irreversible cellular changes leading to altered
2 and ß2 values in G+ single cells. Elevated cell turnover (
3 and ß3) becomes evident in lesions of >1 G+ cells, which renders G+ cells more sensitive to induction of both proliferation and extinction. An elevation of ß may result in two different biological consequences. (i) The smaller the G+ clone the greater the probability of its complete elimination. By this mechanism the biological consequences of initiation may be reversed. Evidence for at least partial elimination of G+ clones was gained in phase II of the present study, but also in previous studies on food restriction or withdrawal of tumor-promoting agents (27,40). Thus, the early stages of cancer may be extinguished under appropriate conditions. (ii) The larger the G+ clone the smaller the probability of complete extinction. The G+ clone will be reduced in size but will not be eradicated, which reverses the effect of tumor promotion.
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Appendix |
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Assume k intervals Ii = [ti, ti + 1], (i = 1,K, k) with t1 = 0 and tk + 1 = t. Let the cell division rate be i, the cell death rate be ßi and the initiation rate (per unit volume) be vi on the ith interval Ii. Define the functions
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with
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The auxiliary rate variables i and
i can be obtained recursively from the hierarchy
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M
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Then,
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with
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With these definitions the expected number of clones (per unit volume), ENV, can be written as a sum over all time intervals prior to time t:
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and the size distribution, pm, as a sum (defining v0/0 = 0)
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Finally, the expected number of m cell clones (per unit volume) is simply pmENV. In the case of a separate analysis of lesions of >1 G+ cells replace 2 by
3 and ß2 by ß3. Please note that in the Results time intervals 1, 2 and 3 are designated phases IIII.
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Notes |
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Acknowledgments |
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References |
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