Suppression of apoptosis in C3H mouse liver tumors by activated Ha-ras oncogene
Steffen Frey,
Albrecht Buchmann,
Wilfried Bursch1,
Rolf Schulte-Hermann1 and
Michael Schwarz2
Institut für Toxikologie, Universität Tübingen, Wilhelmstraße 56, 72074 Tübingen, Germany and
1 Institut für Tumorbiologie-Krebsforschung, Universität Wien, Austria
 |
Abstract
|
---|
Liver tumors were induced in male C3H mice by a single injection of N-nitrosodiethylamine and characterized with respect to the presence of base substitutions in the hot-spot position at codon 61 of the Ha-ras proto-oncogene. An increase in Ha-ras mutation prevalence was found with time after induction of tumors, suggesting that the activated ras gene provides a selective growth advantage. However, no significant differences in 5-bromodeoxyuridine labeling indices were evident between ras mutated and ras wild-type tumors, demonstrating that cell division rates in the two tumor populations were very similar. Apoptotic indices were determined by counting eosinophilic apoptotic bodies. The frequency of occurrence of apoptotic bodies was found to be approximately five times lower in tumors with Ha-ras mutations when compared with tumors not showing the mutation. This demonstrates that the activated p21Ras protein has anti-apoptotic activity in transformed mouse hepatocytes in vivo and suggests that the preferential outgrowth of Ha-ras-mutated hepatoma cells is mediated by suppression of apoptosis rather than by stimulation of cell division.
Abbreviations: BrdUrd, 5-bromodeoxyuridine; DEN, N-nitrosodiethylamine; H&E, hematoxylin and eosin; NF
B, nuclear factor
B; PI3 kinase, phosphatidylinositide 3'-OH kinase.
 |
Introduction
|
---|
p21Ras proteins play an important role in cellular transduction pathways that mediate signals from transmembrane receptors to the nucleus (15). The functional Ras signaling pathway is essential for many signals initiated by growth factors and is also required for the expression of the transforming potential of oncogenic proteins upstream of p21Ras. The cellular p21Ras protein is active as a signal transducer in its GTP-complexed form and is inactivated as a signal transducer by an intrinsic GTPase activity which can be strongly stimulated by GTPase activating proteins. Point mutations at one of the three mutational hot-spot positions in codons 12, 13 and 61 lead to the expression of a Ras oncoprotein locked in its GTP-complexed form as a result of a defective GTPase activity. There is good evidence to suggest that Ras has multiple effectors and that many of these are required for oncogenic transformation. Direct interaction with GTP-complexed p21Ras has been demonstrated for some proteins, including members of the Raf kinase family and phosphatidylinositide 3'-OH kinase (PI3 kinase), suggesting that these proteins represent downstream effectors of Ras (6). Aberrant signaling to the nucleus by constitutive activation of Ras oncoproteins may lead to increased cell proliferation (3,5), one of the hallmarks of neoplastically transformed cells.
Neoplasias in mouse liver are characterized by a high frequency of activating mutations in either the Ha-ras or the Ki-ras gene (7,8). Interestingly, which of the two genes is affected by mutation appears to depend, at least in part, on the carcinogen used for induction of the neoplasias. While codon 13 Ki-ras mutations occur in tumors induced by polycyclic aromatic hydrocarbons (911), tumors induced by N-nitrosodiethylamine (DEN) almost exclusively harbor mutations in codon 61 of the Ha-ras gene (7,1012). The available evidence strongly suggests that mutation of ras represents an initial genetic alteration during mouse hepatocarcinogenesis and that hepatoma cells containing a mutated ras gene have a selective growth advantage (1114). We have now comparatively analyzed the rates of cell division and apoptosis in liver tumors with and without detectable Ha-ras codon 61 mutations. Our results show that there exists a selection for Ha-ras mutated tumors which appears to be primarily driven by suppression of apoptosis of hepatoma cells mediated by the oncoprotein. This is, to our knowledge, the first report demonstrating anti-apoptotic activity of activated p21Ras in primary tumors.
 |
Materials and methods
|
---|
Induction of tumors
In this study, tumors and corresponding normal liver tissue which was available to us from a previous experiment performed in the laboratory in Vienna (15) were analyzed. Tumors were induced in 5-week-old male C3H-mice by a single i.p. injection of DEN (90 µg/g body wt). A single 5-bromodeoxyuridine (BrdUrd) pulse (100 mg/kg body wt) was given by i.p. injection 14 h before death. Mice were killed after induction periods of 40, 52 or 75 weeks and visible liver tumors were carefully removed and embedded in paraffin. A total of 56 tumors from DEN-treated mice and five tumors from untreated controls were collected. Consecutive paraffin sections were prepared with a microtome and used for the analyses described below.
ras mutation analysis
For analysis of Ha-ras mutations in liver tumors, 10 µm paraffin sections were mounted on dialysis bags. Sections were stained with haemalaun to allow identification of tumors. Tissue samples with a weight of ~530 µg were then taken from macroscopically visible tumors and from normal parts of the liver using punching cannuli with diameters of 0.71.5 mm. The tissue samples were collected into Eppendorf vials and used for in vitro amplification without prior isolation of DNA. The PCR reaction was performed essentially as recently described (12). The samples were subsequently screened for the presence of base substitutions (AAA, CGA, CTA and CAT; wild-type = CAA) at codon 61 of the Ha-ras gene by allele-specific oligonucleotide hybridization using the oligonucleotide probes previously described (16). All results were confirmed by at least one independent experiment.
Determination of BrdUrd labeling indices
BrdUrd incorporation into nuclei of DNA-synthesizing cells was determined immunohistochemically in hematoxylin and eosin (H&E) counterstained paraffin sections (2 µm) as recently described (17). Because of the presence of contaminating surrounding normal liver tissue, the outer limits of the tumor transections were marked at low magnification with a thin marker pen. BrdUrd-positive nuclei in tumor transections were counted in a mean total area of 8.4 mm2 (range 1.916.5 cm2), equivalent to an average of ~20 300 cells. Directly neighboring BrdUrd-positive nuclei (see Figure 1
) were counted as single cell division events. Counts of BrdUrd-positive nuclei were subsequently related to the area of the counted parts of each tumor transection which was determined by use of a computer assisted digitizer system. The resulting values were corrected for differences in cell density (determined in a representative area of 0.142 mm2/tumor) to yield final values on the number of labeled cells per 1000 nucleated hepatocytes.

View larger version (70K):
[in this window]
[in a new window]
|
Fig. 1. BrdUrd labeling indices of mouse liver tumors. BrdUrd-positive nuclei were counted in immunohistochemically stained tissue sections. (A) Detection of BrdUrd incorporation into nuclei of cells synthesizing DNA (indicated by arrows). Liver sections were counterstained with H&E. Isolated BrdUrd-positive hepatocellular nuclei (1) as well as directly neighboring BrdUrd-positive nuclei (2) were counted as single cell division events. (B) Quantitative determination of BrdUrd labeling indices in Ha-ras codon 61 mutated liver tumors and tumors not harboring the mutation (means ± SD). Numbers of tumors analysed in each group are indicated. There were no significant differences between both tumor types (P = 0.36).
|
|
Determination of apoptotic indices
Apoptotic bodies within tumor tissues were identified in H&E stained liver sections by fluorescence microscopy (17) using a Leitz Laborlux D microscope (Leitz, Wetzlar, Germany) equipped with an epifluorescence unit (3
Ploemopak) containing the fluorescence filter set N2 (BP530-560, RKP580, LP 580). Identification of each apoptotic body was confirmed by transmitted light microscopy by switching between fluorescent and transmitted light. In accordance with data obtained in rat liver (18), most of the apoptotic bodies observed were of round or oval shape and exhibited classical morphological features, with a subpopulation containing chromatin. Apoptotic bodies in tumor transections were counted in a mean total area of 11.4 mm2 (range 3.239.4 mm2), equivalent to an average of ~27 500 cells. If the tumors were only small, multiple parallel sections were analyzed, which were taken at sufficient distance to avoid double counting of apoptotic bodies (diameter of hepatocyte apoptotic body ~35 µm). If single cells or clusters of directly neighboring cells contained multiple apoptotic bodies, these were assumed to be derived from one and the same apoptotic cell and were therefore counted as one single event. Total counts of apoptotic events were used to calculate the number of apoptotic cells per 1000 hepatocytes as described above for BrdUrd labeling indices. To avoid personal bias, all tumors were scored by only one observer. Re-examination of a subpopulation of tumors by a second independent observer yielded essentially the same results.
All microscopic pictures were taken by use of a video camera system (KAPPA, Gleichen, Germany).
Akt and P(Ser473)-Akt protein determination
Akt and P(Ser473)-Akt proteins were quantified by immunoprecipitation and western analysis. Total Akt proteins were immunoprecipitated from tissue homogenates (2.5 mg protein total) using a polyclonal rabbit antibody (New England Biolabs, Beverly, MA) that detects Akt in both its unphosphorylated and its phosphorylated form [P(Ser473)-Akt] according to the protocol of the manufacturer. Each of two halves of the immunoprecipitates were separated on a denaturing 10% SDSpolyacrylamide gel at 200 V, 30 mA for 3 h. Following transfer of the proteins to a PVDF membrane (Millipore, Bedford, MA), non-specific binding was blocked by incubation in a blocking buffer (0.1% Tween-20, 10% non-fat dry milk, 0.02% NaN3). The membrane was then incubated with either polyclonal rabbit anti-Akt (1:2000 in blocking buffer), which detects Akt irrespective of its phosphorylation status, or polyclonal rabbit anti-P(Ser473)-Akt antibody (1:2000 in blocking buffer; New England Biolabs, Beverly, MA), which detects exclusively the activated P(Ser473)-Akt. After extensive washing, an alkaline phosphatase-conjugated goat anti-rat IgG secondary antibody (1:10 000 in blocking buffer; Dianova, Hamburg, Germany) was used with CDP-Star (Tropix, Bedford, MA) as a substrate for chemiluminescence detection according to the manufacturer's protocol. Chemiluminescence was detected by use of a CSC camera system.
Statistical analysis
To test for differences between group means Student's t-test (two-sided) was used.
 |
Results
|
---|
In the present study, a total of 56 liver tumors from mice killed at 40, 52 and 75 weeks after a single injection of DEN and five tumors that occurred spontaneously in untreated controls were screened for the presence of point mutations at codon 61 of Ha-ras. To allow for the analysis of ras mutations, tumor material was punched out from tissue sections as previously described (1214). A summary of the mutation analysis is given in Table I
. Activating point mutations in Ha-ras codon 61 were detected in 21 tumors. In accordance with results obtained in a previous study (14), the prevalence of mutations was found to increase from 40 to 75 weeks after induction of tumors by DEN. The most prevalent types of base substitutions were C
A transversions and A
G transitions at the first and second positions of codon 61, respectively. A
T transversions at the third position of codon 61 were not detected. In 14 cases, where multiple independent samples were taken at different positions within one and the same Ha-ras mutated tumor, the identical tumor-specific mutational patterns were present.
Cell division of hepatocytes in liver tumors was estimated by determination of BrdUrd incorporation into nuclei of DNA-synthesizing cells. For this purpose mice received a single injection of the DNA precursor 14 h prior to death and BrdUrd incorporation was determined immunohistochemically. A representative example of a tumor section is shown in Figure 1A
. In theory, one round of S phase and subsequent cell division should result in two labeled nuclei, which should be positioned within directly neighboring cells. For stereological reasons, however, often only one of the two labeled nuclei is observable in the tumor transection, because the other is out of plane (see Figure 1A
). Since both single nuclei and doublets represent principally the same entity, we have counted doublets as only one event. As shown in Figure 1B
, the mean BrdUrd labeling index of the Ha-ras mutated tumors was slightly lower than that of the tumors without the genetic alteration, but this effect was not statistically significant.
For determination of apoptotic indices of hepatoma cells, liver sections were screened for the presence of apoptotic bodies. For prescreening, we made use of the fact that apoptotic bodies show strong eosin fluorescence in H&E stained sections (17). Identification of each apoptotic body was subsequently confirmed by switching to transmitted light. A representative example is given in Figure 2A
. As demonstrated in Figure 2B
, there was a 5-fold decrease in the apoptotic index of Ha-ras mutated tumors compared with tumors without detectable Ha-ras mutation. The difference in apoptotic indices was statistically highly significant (P < 5x105). Similar results were obtained when apoptotic indices were compared separately for each of the three time points of the experiment (data not shown). A plot showing the frequency distribution of tumors stratified into different apoptotic index classes is shown in Figure 2C
. All of the 17 tumors with a Ha-ras codon 61 mutation had apoptotic indices in the interval 00.5
. In contrast, only two of the 23 tumors without the Ha-ras mutation were categorized in this interval. The maximum in the distribution of Ha-ras wild-type tumors was located in the interval 11.5
(eight of 23 tumors). There were no significant differences in apoptotic indices between tumors harboring a CGA, AAA or CTA mutation in Ha-ras codon 61 (data not shown).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 2. Apoptotic indices of mouse liver tumors. Apoptotic bodies were counted in haemalaun/eosin stained tissue sections. (A) Representative example of an eosinophilic apoptotic body (indicated by arrow) which was phagozytosed by a surrounding tumor cell. (B) Quantitative determination of apoptotic indices in Ha-ras codon 61 mutated liver tumors and tumors not harboring the mutation (means ± SD). If single cells or clusters of directly neighboring cells contained multiple apoptotic bodies, these were counted as one single event. Numbers of tumors analysed in each group are indicated. The difference between Ha-ras codon 61 mutated and non-mutated tumors was highly significant (P < 5x105). (C) Frequency distribution of Ha-ras codon 61 mutated and non-mutated liver tumors stratified into the indicated apoptotic index classes.
|
|
Since rodent liver tumors generally show increases in both the rate of cell division and the rate of apoptosis (17,19,20), we investigated the possibility of a mechanistic link by comparing apoptotic and BrdUrd labeling indices for each individual tumor. This comparison was possible for the majority of tumors, where data on both parameters were available. The results of this analysis, however, demonstrated no significant correlation between the two parameters (Figure 3
).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3. Relationship between BrdUrd labeling and apoptotic indices. Each point represents an individual tumor with its apoptotic and its labeling index. Note that ras mutated and ras wild-type tumors represent distinct subpopulations, there is, however, no correlation between apoptotic and BrdUrd labeling indices.
|
|
One of the Ras downstream effectors is PI3 kinase, which activates Akt/protein kinase B and thereby potentially mediates anti-apoptotic signals (2124). We have investigated this pathway by making use of a phospho-specific antibody against P(Ser473)-Akt which detects the kinase in its activated form. The results of the immunoprecipitation/western analysis demonstrated only very weak increases in P(Ser473)-Akt levels in liver tumors compared with surrounding normal liver tissue. In addition, there were no significant differences in the P(Ser473)-Akt to total Akt ratios between ras mutated and ras wild-type tumors (Figure 4
).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 4. Detection of phosphorylated and total Akt in homogenates from normal liver and liver tumors. Total Akt protein was immunoprecipitated from homogenates and separated by SDSPAGE. The levels of total Akt and of Ser473 phosphorylated Akt were subsequently determined by western analysis using anti-Akt and anti-P(Ser473)-Akt specific antibodies. Numbers indicate individual samples from Ha-ras wild-type and Ha-ras mutated tumors and from normal tissues. A lysate from PDGF-treated NIH 3T3 cells served as a positive control (P).
|
|
 |
Discussion
|
---|
Numerous studies have demonstrated that point mutations leading to the activation of ras protooncogenes occur at a high frequency in liver tumors of mice, mostly at codon 61 of the Ha-ras gene (for a recent review see ref. 7), while they are very infrequent in hepatocellular cancers from rats or humans (25). These mutations are detectable in some of the earliest precancerous liver lesions and in those cases where this has been investigated, data suggest that all cells of a mutated liver tumor carry the identical type of Ha-ras point mutation (14; this paper). The types of base substitutions observed are characteristically different between carcinogens of different classes (7,10,26). While Ha-ras mutations are not detectable, even with sensitive methods, in normal mouse liver, ~50% of all liver tumors in C3H mice carry Ha-ras mutations and the frequency of occurrence of Ha-ras mutated tumors is increased ~300-fold in mice treated with a single injection of 10 mg/kg body wt DEN on day 15 after birth in comparison with untreated controls (14). In summary, these data suggest that ras mutations may be induced directly by liver carcinogens and represent an initial event in mouse hepatocarcinogenesis (1214). We have, on the other hand, repeatedly observed that the relative fraction of ras mutated mouse liver tumors increases with time after their induction (14; this paper). There are several possible explanations for this finding. Increased cell proliferation could render cells more prone to mutational events which could result in additional Ha-ras mutations occurring spontaneously in some of the tumors at a later stage of carcinogenesis. In addition, confluence of larger ras mutated and ras wild-type tumors could lead to increased counts of mutated tumors. In both instances, only a certain subpopulation of tumor cells would harbor the mutation. In accordance with data obtained in an earlier study (14), however, multiple samples taken from one and the same mutated tumor always yielded the identical type of base substitution and the radioactivity signals of mutated and wild-type sequences in the allele-specific oligonucleotide hybridization analysis were almost identical, strongly suggesting that the tumors were clonal in origin with all cells carrying a mutated and a wild-type allele. In fact, we have previously shown that Ha-ras mutated mouse liver tumors express both alleles in a similar ratio (10). Based on these findings we therefore assume that ras mutations do not, in general, occur within subpopulations of tumor cells at a later stage of hepatocarcinogenesis. Taking the observed increase in mutation prevalence with time, this implies that ras mutated hepatocytes have a proliferative advantage over their non-ras mutated counterparts. In this context it is necessary to point out that all mutational analyses are, for technical reasons, biased, in that not all detectable tumors (or transections from tumors) but only those exceeding a certain size limit are subject to screeening for the presence of base substitutions. Consequently, if the mutated tumors, in this case the tumors harboring a Ha-ras codon 61 mutation, grow faster than wild-type clones, there will be an apparent increase in the prevalence of the mutated clones with time even though the actual ratio may stay largely constant. If only subtle differences in growth exist between ras mutated and ras wild-type tumors the selection for mutated clones is likely to become increasingly pronounced with length of observation period. This may explain why the prevalence of ras mutations in spontaneous mouse liver tumors (which generally occur later) is often higher than the prevalence seen in carcinogen-induced tumors (which occur earlier) (7) and why the frequency of mutations was found to be inversely related to carcinogen dose in a long-term carcinogenicity study (27,28).
Given a selective growth advantage of the Ha-ras codon 61 mutated liver tumors over their non-mutated counterparts, one might tentatively assume that the mutated hepatocytes must show an increased rate of cell division. In fact, a significant increase in labeling indices was detectable in very small glucose 6-phosphatase-negative, presumably preneoplastic, liver lesions in C3H mice, when analyzed within the first weeks after their induction by DEN (14). Surprisingly, however, no significant differences in labeling indices between ras mutated and ras wild-type lesions were detectable when analyzing tumors at later stages of the carcinogenic process (10,14; this study). An increase in tumor size not only depends on the rate of tumor cell division but also on the rate of death of initiated cells and mathematical modeling of carcinogenesis shows that failure of apoptosis, as seen in the ras mutated liver tumors, can fully explain exponential growth in tumor cell number (29). Growth rates of rat liver foci calculated on the basis of observed labeling indices clearly exceeded those found experimentally, demonstrating loss of cells during the development of the focal lesions (30). Treatment of animals with the tumor promoter phenobarbital inhibited disappearance of the focal cells and, thereby, induced enlargement of the foci (30). This led us to speculate that the activated p21Ras protein may act as an `endogenous tumor promoter' (25) by blocking apoptosis of transformed mouse hepatocytes in vivo (10). Our experimental data now demonstrate a 5-fold reduction in the frequency of apoptotic bodies in liver tumors with Ha-ras codon 61 mutations in comparison with tumors not showing this alteration. Although the exact rates at which cell elimination occurs in the tumor tissue cannot be directly deduced from the counts of apoptotic bodies, because the time window of observability of the apoptotic bodies within the mouse liver tumors is not known, our data suggest that ras mutated hepatocytes undergo apoptosis less frequently than their non-mutated counterparts. Since cell division rates were not significantly different between mutated and wild-type tumors, suppression of apoptosis appears to be the driving force for the selective outgrowth of the ras mutated clones. We cannot exclude the possibility that those tumors not showing the Ha-ras codon 61 mutation harbor a mutation at one of the other hot-spot loci of the gene or in other potentially oncogenic ras genes, although mutations other than in Ha-ras codon 61 appear to be very infrequent in mouse liver tumors induced by DEN (7,10). Possibly, liver tumors without detectable ras mutations harbor genetic aberrations in other yet unknown genes within ras-dependent signal transduction pathways. Irrespective of the nature of these genetic changes, however, they do not appear to affect apoptosis of the transformed hepatocytes as the Ha-ras codon 61 mutations do.
The role of activated ras oncogenes in apoptosis of cells in culture has been subject to intense investigation by various groups. In the majority of cases, p21Ras appears to deliver an anti-apoptotic signal but, depending on the cell line and experimental protocol used, expression of the activated oncogene may also induce pro-apoptotic signaling (for a recent review see ref. 31). In most of these studies the activated oncogene was overexpressed in cells by transfection of the gene or microinjection of the respective protein. Mouse liver tumors, however, do not exhibit any change in expression level of ras genes, irrespective of the presence or absence of ras mutations (10). The mechanism by which the activated p21Ras protein mediates suppression of apoptosis of hepatocytes from primary mouse liver tumors is not clear. Several ras-dependent anti-apoptotic signaling pathways are known. The Raf kinase/Erk pathway has been shown to mediate cell survival signals as opposed to signaling via p38 kinase (32). Raf kinase may also play some role in anti-apoptotic signaling at the mitochondrial membrane (33,34). However, we recently demonstrated that Raf kinase is constitutively activated in mouse liver tumors, irrespective of whether the tumors show ras mutations or not (10), suggesting that the selective proliferative advantage mediated by the activated ras gene is not due to anti-apoptotic signaling via Raf kinase in transformed mouse hepatocytes. Cell survival signals are also transmitted via PI3 kinase and its downstream effector protein kinase B/Akt, a transduction pathway which may also be initiated by activated p21Ras (2124). Activation of Akt is associated with phosphorylation at Ser473, which can be detected by use of phospho-specific antibodies. We were not able, however, to detect significant differences in the concentration of P(Ser473)-Akt between ras mutated and ras wild-type tumors, suggesting that this pathway may also not be responsible for decreased apoptosis in ras mutated mouse liver tumors. Finally, ras transformation of cells depends on the function of the nuclear transcription factor
B (NF
B) (35). In the absence of functional NF
B, Ras may induce apoptosis which is effectively suppressed in the presence of the activated transcription factor (3639). Activation of p21Ras leads to specific binding of NF
B to DNA resulting in the activation of transcription of yet unidentified proteins mediating cellular survival (40). Whether NF
B signaling is important in mouse hepatocarcinogenesis is presently under investigation in our laboratory.
 |
Acknowledgments
|
---|
The excellent technical assistence of Mrs E.Zabinski (Tübingen) and Mrs K. Bukowski (Vienna) is greatly acknowledged. This work was partly supported by grant Bo 306/14-4 from the Deutsche Forschungsgemeinschaft.
 |
Notes
|
---|
2 To whom correspondence should be addressedEmail: michael.schwarz{at}uni-tuebingen.de

 |
References
|
---|
-
McCormick,F. (1994) Raf: the Holy Grail of Ras biology. Trends Cell Biol., 4, 347350.
-
McCormick,F. (1994) Activators and effectors of ras p21 proteins. Curr. Opin. Genet. Dev., 4, 7176.[Medline]
-
Marshall,C. and Wyllie,A. (1996) Oncogenes and cell proliferation. Curr. Opin. Genet. Dev., 6, 13.[ISI][Medline]
-
Mittnacht,S., Paterson,H., Olson,M.F. and Marshall,C.J. (1997) Ras signalling is required for inactivation of the tumour suppressor pRb cell-cycle control protein. Curr. Biol., 7, 219221.[ISI][Medline]
-
Marshall,C.J. and Nigg,E.A. (1998) Oncogenes and cell proliferation cancer genes: lessons from genetics and biochemistry. Curr. Opin. Genet. Dev., 8, 1113.[ISI][Medline]
-
Marshall,C.J. (1996) Ras effectors. Curr. Opin. Cell Biol., 8, 197204.[ISI][Medline]
-
Maronpot,R.R., Fox,T., Malarkey,D.E. and Goldsworthy,T.L. (1995) Mutations in the ras proto-oncogene: clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology, 101, 125156.[ISI][Medline]
-
Dragan,Y., Klaunig,J., Maronpot,R. and Goldsworthy,T. (1998) Meeting overviewmechanisms of susceptibility to mouse liver carcinogenesis. Fundam. Appl. Toxicol., 41, 37.
-
Manam,S., Storer,R.D., Prahalada,S., Leander,K.R., Kraynak,A.R., Ledwith,B.J., van Zwieten,M.J., Bradley,M.O. and Nichols,W,W. (1992) Activation of the Ha-, Ki- and N-ras genes in chemically induced liver tumors from CD-1 mice. Cancer Res., 52, 33473352.[Abstract]
-
Kalkuhl,A., Troppmair,J., Buchmann,A., Stinchcombe,S., Buenemann,C.L., Rapp,U.R., Kaestner,K. and Schwarz,M. (1998) p21ras downstream effectors are increased in activity or expression in mouse liver tumors but do not differ between Ras-mutated and Ras-wild-type lesions. Hepatology, 27, 10811088.[ISI][Medline]
-
Gressani,K.M., Rollins,L.A., Leone-Kabler,S., Cline,J.M. and Miller,M.S. (1998) Induction of mutations in Ki-ras and INK4a in liver tumors of mice exposed in utero to 3-methylcholanthrene. Carcinogenesis, 19, 10451052.[Abstract]
-
Buchmann,A., Bauer-Hofmann,R., Mahr,J., Drinkwater,N.R., Luz,A. and Schwarz,M. (1991) Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: correlation with susceptibility to hepatocarcinogenesis. Proc. Natl Acad. Sci. USA, 88, 911915.[Abstract]
-
Buchmann,A., Mahr,J., Bauer-Hofmann,R. and Schwarz,M. (1989) Mutations at codon 61 of the Ha-ras proto-oncogene in precancerous liver lesions of the B6C3F1 mouse. Mol. Carcinog., 2, 121125.[ISI][Medline]
-
Bauer-Hofmann,R., Klimek,F., Buchmann,A., Müller,O., Bannasch,P. and Schwarz,M. (1992) Role of mutations at codon 61 of the c-Ha-ras gene during diethylnitrosamine-induced hepatocarcinogenesis in C3H/He mice. Mol. Carcinog., 6, 6067.[ISI][Medline]
-
Wastl,U.M., Rossmanith,W., Lang,M.A., Camus-Randon,A.M., Grasl-Kraupp,B., Bursch,W. and Schulte-Hermann,R. (1998) Expression of cytochrome P450 2A5 in preneoplastic and neoplastic mouse liver lesions. Mol. Carcinog., 22, 229234.[ISI][Medline]
-
Brown,K., Buchmann,A. and Balmain,A. (1990) Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression. Proc. Natl Acad. Sci. USA, 87, 538542.[Abstract]
-
Stinchcombe,S., Buchmann,A., Bock,K.W. and Schwarz,M. (1995) Inhibition of apoptosis during 2,3,7,8-tetrachlordibenzo-p-dioxin-mediated tumour promotion in rat liver. Carcinogenesis, 16, 12711275.[Abstract]
-
Bursch,W., Taper,H.S., Lauer,B. and Schulte Hermann,R. (1985) Quantitative histological and histochemical studies on the occurrence and stages of controlled cell death (apoptosis) during regression of rat liver hyperplasia. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol., 50, 153166.[ISI][Medline]
-
Schulte-Hermann,R., Bursch,W., Grasl-Kraupp,B., Oberhammer,F., Wagner,A. and Jirtle,R. (1993) Cell proliferation and apoptosis in normal liver and preneoplastic foci. Environ. Health Perspect., 101 (suppl. 5), 8790.
-
Schulte-Hermann,R., Bursch,W., Grasl-Kraupp,B. and Ruttkay-Nedecky,B. (1995) Apoptosis and multistage carcinogenesis in rat liver. Mutat. Res., 333, 8187.[ISI][Medline]
-
Kauffman-Zeh,A., Rodriguez-Viciana,P., Ulrich,E., Gilbert,C., Coffer,P., Downward,J. and Evan,G. (1997) Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature, 385, 544548.[ISI][Medline]
-
Marte,B.M. and Downward,J. (1997) PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond. Trends Biochem. Sci., 22, 355358.[ISI][Medline]
-
Rubio,I., Rodriguez-Viciana,P., Downward,J. and Wetzker,R. (1997) Interaction of Ras with phosphoinositide 3-kinase gamma. Biochem. J., 326, 891895.[ISI][Medline]
-
Downward,J. (1997) Cell cycle: routine role for Ras. Curr. Biol., 7, R258R260.[ISI][Medline]
-
Schwarz,M., Buchmann,A. and Bock,K.W. (1995) Role of cell proliferation at early stages of hepatocarcinogenesis. Toxicol. Lett., 82/83, 2732.
-
Wiseman,R.W., Stowers,S.J., Miller,E.C., Anderson,M.W. and Miller,J.A. (1986) Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F1 mouse. Proc. Natl Acad. Sci. USA, 83, 58255829.[Abstract]
-
Chen,B., Liu,L., Castonguay,A., Maronpot,R.R., Anderson,M. and You,M. (1993) Dose-dependent ras mutation spectra in N-nitrosodiethylamine induced mouse liver tumors and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced mouse lung tumors. Carcinogenesis, 14, 16031608.[Abstract]
-
Johansson,E., Reynolds,S., Anderson,M. and Maronpot,R. (1997) Frequency of Ha-ras-1 gene mutations inversely correlated with furan dose in mouse liver tumors. Mol. Carcinog., 18, 199205.[ISI][Medline]
-
Tomlinson,I.P.M. and Bodmer,W.F. (1995) Failure of programmed cell death and differentiation as causes of tumors: some simple mathematical models. Proc. Natl Acad. Sci. USA, 92, 1113011134.[Abstract]
-
Schulte-Hermann,R., Timmermann-Trosiener,I., Barthel,G. and Bursch,W. (1990) DNA synthesis, apoptosis and phenotypic expression as determinants of growth of altered foci in rat liver during phenobarbital promotion. Cancer Res., 50, 51275135.[Abstract]
-
Downward,J. (1998) Ras signalling and apoptosis. Curr. Opin. Genet. Dev., 8, 4954.[ISI][Medline]
-
Xia,Z.G., Dickens,M., Raingeaud,J., Davis,R.J. and Greenberg,M.E. (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science, 270, 13261331.[Abstract]
-
Wang,H.G., Takayama,S., Rapp,U.R. and Reed,J.C. (1996) Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. Proc. Natl Acad. Sci. USA, 93, 70637068.[Abstract/Free Full Text]
-
Wang,H.G., Rapp,U.R. and Reed,J.C. (1996) Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell, 87, 629638.[ISI][Medline]
-
Finco,T.S., Westwick,J.K., Norris,J.L., Beg,A.A., Der,C.J. and Baldwin,A.S.Jr (1997) Oncogenic Ha-Ras-induced signaling activates NF-kappaB transcriptional activity, which is required for cellular transformation. J. Biol. Chem., 272, 41134116.
-
Mayo,M.W., Wang,C.Y., Cogswell,P.C., Rogers-Graham,K.S., Lowe,S.W., Der,C.J. and Baldwin,A.S.Jr (1997) Requirement of NF-kappaB activation to suppress p53-independent apoptosis induced by oncogenic Ras. Science, 278, 812815.
-
Baichwal,V.R. and Baeuerle,P.A. (1997) Activate NF-kappa B or die? Curr. Biol., 7, R94R96.[ISI][Medline]
-
Van Antwerp,D.J., Martin,S.J., Kafri,T., Green,D.R. and Verma,I.M. (1996) Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science, 274, 787789.[Abstract/Free Full Text]
-
Liu,Z.G., Hsu,H., Goeddel,D.V. and Karin,M. (1996) Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell, 87, 56576.[ISI][Medline]
-
Schulze-Osthoff,K., Ferrari,D., Riehemann,K. and Wesselborg,S. (1997) Regulation of NF-kappa B activation by MAP kinase cascades. Immunobiology, 198, 3549.[ISI][Medline]
Received August 20, 1999;
revised October 22, 1999;
accepted November 5, 1999.