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Institut für medizinische Strahlenkunde und Zellforschung, Universität Würzburg, D-97078 Würzburg, Germany [E. K., L. M. F., R. S., A. O. W., U. R. R.], and Pathologisches Institut, Histologisches Labor, Friedrich-Alexander-Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany [T. P.]
Abstract
The c-Raf-1 kinase is a downstream effector of Ras signaling. Both proteins are highly oncogenic when they are mutationally activated, but only the Ras GTPase is frequently mutated in naturally occurring tumors. Although the c-Raf-1 protein was found to be amplified in different lung cancer cell lines, overexpression of the wild-type c-Raf-1 protein was shown to be insufficient to transform cultured cells. Here we have addressed the question of whether overexpression of the wild-type c-Raf-1 kinase can induce lung cancer in mice. We show that lung-targeted expression of oncogenically activated or wild-type c-Raf-1 proteins induces morphologically indistinguishable lung adenomas in transgenic mice. Compared with mice transgenic for the activated c-Raf-1-BxB, tumor development is delayed and occurs at a lower incidence in wild-type c-Raf-1 transgenic mice. Our studies show that the c-Raf-1 expression level is a critical parameter in tumor development and should be analyzed in more detail to evaluate its potential in the induction of cancer.
Introduction
Receptor tyrosine kinase signal transduction pathways are major regulators of cell proliferation and are frequently found to be mutated and activated during tumor development (1 , 2) . After ligand binding to receptor tyrosine kinases, the Ras GTPase is shifted from the GDP to the GTP loaded form (3) . Ras-GTP recruits the Raf protein to the cellular membrane, where the kinase becomes activated and transmits its signal via a cascade of successive phosphorylation events (4 , 5) . Mutations that lead to constitutive activation of the tyrosine kinase receptors are found frequently in naturally occurring tumors and have been shown to be highly transforming in cultured cells (1 , 2) . The Ras GTPase becomes oncogenically activated by single amino acid substitutions, which inhibit the GTPase activity and cause the Ras protein to persist in its activated GTP-bound status (1 , 6) . Of all oncogenes, the ras gene has the highest mutation rate in human tumors (6) . The Raf kinase is a downstream effector of Ras signaling (4 , 5) . The Raf protein consists of a NH2-terminal regulatory domain mediating the interaction with Ras and a COOH-terminal kinase domain (4) . Cell culture experiments have shown that deletion of the NH2-terminal regulatory domain leads to a constitutively active kinase that is highly oncogenic in animals as well as in cultured cells (7 , 8) . Single amino acid substitutions that activate Raf kinase for Mek3 phosphorylation have been found to be transforming in cell culture experiments (9 , 10) . Despite the high transforming potential of its upstream activators, mutationally activated Raf kinases could not be identified in naturally occurring tumors. Analysis of the expression levels of the wild-type c-Raf-1 protein in human lung tumor cell lines and biopsy material from malignant tissues revealed increased expression levels of the c-Raf-1 kinase in the majority of the transformed cells, raising the possibility that overexpression of the wild-type protein may contribute to oncogenic transformation (11) . In contrast to NH2-terminal deletion mutants or activated Ras proteins, overexpression of the wild-type c-Raf-1 kinase was shown to be insufficient to transform cultured cells (7 , 8) . Evidence for an oncogenic potential of the wild-type Raf kinase came from the observation that overexpression of the c-Raf-1 kinase sensitizes cells for Ras transformation (12) . Activated Ras expressed at low levels and wild-type c-Raf-1 overexpression were shown to cooperate in cell transformation in tissue culture experiments. Here we have addressed the question of whether lung-targeted overexpression of c-Raf-1 protein in transgenic mice is tumorigenic. We show that high expression levels of the c-Raf-1 protein lead to the development of lung adenomas within 1 year of age.
Results and Discussion
To evaluate a possible function of the c-Raf-1 protein in
tumorigenesis, we have generated transgenic mice
[(C57BL/6xDBA-2)F1] expressing the
wild-type human c-Raf-1 protein or the oncogenically activated
NH2-terminal deletion mutant c-Raf-1-BxB under
the control of human SP-C promoter (Fig. 1
). c-Raf-1-BxB is a constitutively active c-Raf-1 kinase and is highly
transforming in cell culture experiments
(13)
.4
In mice, the SP-C gene is expressed in type II
epithelial cells lining the lung alveoli (14, 15, 16)
. The
promoter region of the human SP-C gene has been shown to
direct the expression of chimeric genes in transgenic animals in a lung
epithelial cell-specific manner (15)
. The SP-C
promoter has been used frequently for lung-targeted expression of
transgenes (14, 15, 16, 17, 18, 19, 20, 21)
. Tumor formation could only be
observed in those transgenic mice in which the expression of an
oncogene was directed by the SP-C promoter (16
, 17)
. We have established 11 independent mouse strains carrying
the SP-C-c-raf-1 transgene and 2 mouse strains
carrying the SP-C-c-raf-1-BxB transgene (Table 1)
. Gene integration has been verified by Southern hybridization (data
not shown). In addition, we have generated mice transgenic for
sequences encoding the isolated NH2-terminal
regulatory domain of the c-Raf-1 protein (c-Raf-1-C4). The c-Raf-1-C4
protein acts as an inhibitor of Ras function by interacting with the
Ras protein without being able to transmit a signal (5
, 22)
. Although two SP-C-c-Raf-1-C4 founders were germ-line
transmitting, no transgene expression could be detected by
Northern hybridization (data not shown). This is consistent with the
absence of lung pathology in these mice (data not shown).
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|
. Confluent tumor development was observed
within 5–7 months, accompanied by an 8–10-fold increase in lung
tissue mass compared with wild-type mice. Histologically, tumors
consisted of cuboid epithelia comparable to alveolar type II cells
(Fig. 2
). The tumor cells showed a glandular proliferation, lining the alveolar
walls in a lepidic fashion. Sometimes an intra-alveolar micropapillary
growth pattern was observed. The grade of nuclear atypia and nuclear
polymorphism of the tumor cells was minimal. Single tumor cell mitoses
were detectable. Tumor invasion in intrapulmonary vessels or
infiltration of bronchial and pleural tissue has never been observed.
Extensive examination of extrapulmonary tissues provided no evidence of
metastasis. Thus far, the lesions were classified as multicentric
alveolar adenomas developed from alveolar type II cells. Analysis of
the transgene expression in SP-C-c-Raf-1-BxB mice shows a lung-specific
expression of the c-Raf-1-BxB protein (Fig. 3
A). Low expression of the oncogenic kinase could also be
detected in kidney (Fig. 3A)
. This is consistent with
several cases of hydronephrosis, which we have observed in SP-C-c-Raf-1
transgenic mice (Table 1)
. Comparison of transgene expression in
SP-C-c-Raf-1-BxB mice in the early stages of tumor development (3
months) and in the late stages of tumor development, when the tumor
cells have reached confluence (8 months), shows a strong amplification
of c-Raf-1-BxB protein expression at later stages, indicating that
cells expressing the transgene are amplified during tumor development
(Fig. 3B)
. This result has been confirmed by
immunohistochemical staining of the tumor tissue (Fig. 3C)
.
Backcrossing of SP-C-c-Raf-1-BxB-23 mice to C57BL6 mice (five
generations) or the transfer of the
SP-C-c-raf-1-BxB allele to 129/Sv mice by
crossing (three generations) results in 100% tumor penetrance,
indicating that the tumorigenesis is not dependent on the genetic
background of C57BL6xDBA-2 mice.
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. The incidence of transformation in the case of
SP-C-c-Raf-1 mice is much lower. Lungs of those mice exhibit several
isolated foci per lung, whereas the lungs of SP-C-c-Raf-1-BxB mice are
covered with numerous foci (Fig. 2)
. Six of 11 SP-C-c-Raf-1 transgenic
mouse strains did not develop lung cancer within 12–16 months of age
(Table 1)
. Within the strains that develop lung cancer, the percentage
of mice with lung adenomas ranged from 30–100% (Table 1)
. The delayed
onset as well as the variable incidence of the lung adenomas in the
different transgenic mouse strains strongly indicates that secondary
mutations are necessary for c-Raf-1 to induce cell transformation. As
in SP-C-c-Raf-1-BxB mice, the tumor tissue of SP-C-c-Raf-1 mice shows a
strong amplification of c-Raf-1 protein expression (Fig. 3B)
, underlining the oncogenic character of c-Raf-1
overexpression. The nature of the high transgene expression in the
tumor cells remains unclear. Future experiments analyzing the genomic
structure of the tumor cells may reveal whether the increases in
expression levels are due to gene or transgene amplification.
c-Raf-1 phosphorylates and activates the Mek protein, which
subsequently phosphorylates and activates the Erk kinases. To analyze
the activity of the Raf/Mek/Erk signal transduction pathway, we have
compared the degree of Erk phosphorylation in wild-type,
SP-C-c-Raf-1-87, and SP-C-c-Raf-1-BxB-23 mice (Fig. 4
). Mice expressing the activated c-Raf-1-BxB show an increased level of
Erk phosphorylation. Although the analyzed SP-C-c-Raf-1-87 transgenic
mouse (age, 10 months) expressed elevated levels of the c-Raf-1 protein
and developed a lung tumor, the level of the phosphorylated Erk was not
increased compared with the phosphorylation found in the wild-type
mouse (Fig. 4)
. The degree of Erk phosphorylation has been shown to
correlate with tumor development. Tumor progression is highly reduced
in the presence of agents inhibiting the Erk activator Mek
(23)
. In this respect, the lower Erk phosphorylation in
the SP-C-c-Raf-1-87 mouse may reflect the longer latency found in the
c-Raf-1 transgenic mice. The activation status of the c-Raf-1 kinase in
SP-C-c-Raf-1 mice remains to be analyzed in more detail. In particular,
the question of whether the c-Raf-1 transgenic mice exhibit a slightly
increased Raf activity compared with wild-type mice and whether such a
small difference can be sufficient for tumor development has to be
analyzed.
|
Because of the long delay phase in the onset of c-Raf-1-induced tumorigenesis, the transgenic SP-C-c-Raf-1 mice provide a unique system to study environmental influences in lung cancer development and can now be used for the preclinical testing of novel cancer drugs.
Materials and Methods
Plasmids.
The SP-C-c-raf-1 vector was generated by
releasing a 3-kb DNA human c-raf-1 cDNA fragment from
pUC13-c-raf-1 (25)
by digestion with the
EcoRI restriction endonuclease and insertion of the
c-raf-1 cDNA into the SP-C-3.7-SV40 plasmid
(16)
, which was linearized by EcoRI digestion.
SP-C-3.7-SV40 contains the 3.7-kb promoter region of the
human SP-C and a SV40 small T intron and polyadenylation
signal. The SP-C-c-raf-1-BxB vector was generated
by releasing a 1.4-kb c-raf-1-BxB cDNA fragment lacking
sequences encoding the NH2-terminal regulatory
domain of the c-Raf-1 kinase from the pKRSPA-c-raf-1-BxB
vector (22)
by ClaI/XbaI digestion.
The c-raf-1-BxB fragment was inserted into the
SP-C-3.7-SV40 plasmid, which was linearized by digestion
with the EcoRI restriction endonucleases. The sticky ends of
the vector and insert DNAs were blunt-ended before ligation.
Generation of Raf Transgenic Mice.
The expression cassette of the SP-C-raf-1 and
SP-C-c-raf-1-BxB vectors was released by digestion with the
NdeI and NotI restriction endonucleases. The
resulting 7.1-kb (SP-C-raf-1) and 5.5-kb
(SP-C-raf-1-BxB) fragments were purified by preparative
agarose gel electrophoresis. Transgenic mouse lines were generated by
injection of recombinant DNA fragments into fertilized eggs of
C57BL/6xDBA-2 F1 mice from Harlan Winkelmann
(Borchen, Germany). Live-born animals were analyzed for
transgene integration by standard Southern blot and PCR techniques.
Histopathological Evaluation.
Animals were sacrificed and subjected to complete autopsy. Dissected
tissue samples were fixed in 3.7% formaldehyde in PBS. Specimens were
embedded in paraffin, sectioned at 4 µm, and stained with H&E.
Immunohistochemistry.
Paraffin-embedded sections were deparaffinized, rehydrated, and
microwaved in 10 mM sodium citrate buffer (pH 5.5). After
antigen retrieval, the samples were washed with water, incubated in
20% sucrose in PBS at 4°C for 30 min, washed with PBS, placed in
blocking buffer (2.5% goat serum in PBS) for 40 min, and subsequently
incubated in the presence of an anti-c-Raf-1 rabbit polyclonal
antiserum (30K; Rapp Laboratory) at room temperature overnight.
Antigen-antibody complexes were detected with an immunoperoxidase
staining system (Vectastain ABC Kit; Vector Laboratories). The sections
were counterstained with hematoxylin.
Immunoblot.
Frozen organs were homogenized in protein sample buffer [60
mM Tris-HCl (pH 6.8), 10% glycerol, 3% SDS, 5%
2-mercaptoethanol, and 0.005% bromphenol blue) with the help of an
Ultra-Turrax blender. The protein lysates were separated by SDS-PAGE
and blotted onto nitrocellulose membranes. The immunodetection was
performed as described previously (26)
. The following
primary antibodies were used: (a) c-Raf-1, SP-63 (rabbit
polyclonal antibody, 1:1000, Rapp Laboratory); (b)
phospho-Erk, phospho-p44/p42 mitogen-activated protein kinase
antibodies (rabbit polyclonal antibody, affinity purified, 1:400, New
England Biolabs); and (c) Cdk4, C-22 (rabbit
polyclonal antibody, affinity purified, 1 µg/ml, Santa Cruz
Biotechnology).
Acknowledgments
We thank J. Whitsett for providing the SP-C-3.7/SV40 vector. We also thank A. Ganscher, O. Sakk, and E. V. Chernigovskaya for technical assistance and advice and S. Pfränger for excellent photographic reproduction.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by grants from the Bundesministerium
für Bilding, Wissenschaft, Forschung, und Technologie,
Bonn, Germany (Interdisziplinäres Zentrum für Klinische
Forschung, Erlangen, Teilprojekt B20; Vorhaben 01KV9504/7) and
the Wilhelm Sander-Stiftung. E. K., L. M. F., and R. S. contributed
equally to this work. 
2 To whom requests for reprints should be
addressed, at Institut für medizinische Strahlenkunde und
Zellforschung, Universität Würzburg, Versbacher Strasse 5,
D-97078 Würzburg, Germany. Phone: 49-931-201-5141; Fax:
49-931-201-3835.
3 The abbreviations used are: Mek,
mitogen-activated protein/extracellular signal-regulated kinase kinase;
SP-C, surfactant protein C; Erk, extracellular signal-regulated kinase;
Cdk, cyclin-dependent kinase.
4 J. Troppmair and U. R. Rapp, unpublished
observations.
Received for publication 11/ 8/99. Revision received 2/ 8/00. Accepted for publication 2/12/00.
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