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[Cancer Research 62, 6297-6303, November 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Bcl-2 Determines Susceptibility to Induction of Lung Cancer by Oncogenic CRaf1 ,,2

Lev M. Fedorov, Oleg Yu. Tyrsin, Thomas Papadopoulos, Guadalupe Camarero, Rudolf Götz and Ulf R. Rapp3

Institut für Medizinische Strahlenkunde und Zellforschung, Bayerische Julius-Maximilians-Universität, D-97078 Würzburg, Germany [L. M. F., O. Y. T., R. G., U. R. R.]; Pathologisches Institut Histologisches Labor, Friedrich-Alexander-Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany [T. P.]; and Instituto de Investigaciones Biomédicas Alberto Sols, Madrid 28029, Spain [G. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The efficiency of tumor induction by oncogenes is influenced by modifier genes that determine individual susceptibility. We have used a transgenic mouse model to examine the role of a candidate susceptibility gene, bcl-2, for development of Raf oncogene-induced lung adenomas. Loss of bcl-2 greatly retarded tumor development without affecting tumor phenotype. Tumor tissues from bcl-2 positive and negative mice were compared for the fraction of S phase cells by staining for proliferating cell nuclear antigen and for the fraction of apoptotic cells by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. The data indicate that the increased tumor latency in the absence of bcl-2 results primarily from an increased apoptotic rate but also involves a decrease in tumor cell proliferation. Both effects can be rescued by breeding with H2K-bcl-2 transgenic mice demonstrating that loss of bcl-2 was the major genetic factor determining tumor resistance. These findings suggest that bcl-2 is a major susceptibility gene for development of lung cancer in mice and perhaps in humans.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Lung cancer in humans is today the leading cause of cancer death in the world (1) . To facilitate development of new cancer drugs transgenic mouse models have been developed in attempts to recapitulate human lung cancer by introducing the most frequently occurring genetic changes (2, 3, 4, 5, 6, 7) . The genetic changes in lung cancer include point mutations in Raf activating oncogenes, mainly ras (8) , in addition to overexpression of bcl-2 (9) , CRaf (10) , the epidermal growth factor, and bombesin receptors (8) .

We have generated previously a transgenic mouse model to evaluate the ability of Raf oncogenes to induce lung tumors alone (2) or in conjunction with loss of the tumor suppressor p53 and cell cycle regulator p21WAF1/CIP1.4 Raf protein serine/threonine kinases are critical mediators of signaling by growth factor receptors and function at the helm of the classic mitogenic cascade, Raf-mitogen-activated protein/ERK kinase-ERK1/2,5 which regulates cell growth, differentiation, proliferation, and survival (11 , 12) . Experiments with a hormone-inducible form of activated CRaf kinase (CRaf BXB-ER) in cell culture demonstrated that depending on signal intensity, the mitogenic cascade can either promote proliferation (low intensity) or late G1 arrest and differentiation (high intensity; Ref. 13 ). In both settings induction of CRaf kinase activity endows cells with increased survival activity, as the CRaf kinase inducing artificial hormone, 5-hydroxytamoxifen, can replace growth factors present in serum in short term cell culture assays (13) . There are three Raf isozymes, A-, B-, and CRaf, that overlap in the control of the mitogenic cascade. Genetic and biochemical data indicate that they also have unique functions that derive from specific interactions with metabolic enzymes, proteasome regulator (14) , and bcl-2 family proteins (15, 16, 17) . We have evidence that multiple signaling pathways are required for transformation by Raf oncogenes. One example of an auxiliary pathway is Raf-MAPK kinase kinase-inhibitor of NF{kappa}B kinase-NF{kappa}B (18) . Activation of NF{kappa}B accompanies transformation of NIH 3T3 cells by Raf oncogenes, and dominant-negative mutants that block NF{kappa}B activation also block transformation by oncogenic CRaf (18) . A third signaling pathway connects CRaf with bcl-2 (15, 16, 17) and several bcl-2 interacting proteins such as BAG-1 (17) , and the major pore protein in the outer mitochondrial membrane, VDAC (19) . CRaf physically interacts with these proteins, shares their localization at the outer mitochondrial membrane (20) , and cooperates with bcl-2 in the suppression of cytochrome c release and apoptosis (21) . These findings suggested that CRaf is an effector kinase of bcl-2 in the outer mitochondrial membrane reminiscent of its role as a Ras effector in the plasma membrane. However, the ability of CRaf to suppress apoptosis does not absolutely require bcl-2, as mitochondria-targeted oncogenic CRaf also has survival activity in embryonic fibroblasts from bcl-2 knockout mice (22) . Nevertheless, the combined data on bcl-2/CRaf cooperation suggest that bcl-2 may be an important regulator of Raf oncogenesis in vitro and in vivo.

Susceptibility to Raf oncogenesis in vivo has been studied in the late 1980s by using retroviruses that carry the craf-related oncogene v-raf alone or in combination with v-myc (23) . The availability of transgenic mice with lung targeted expression of Raf oncogenes has prompted us to evaluate whether bcl-2 might behave as a major susceptibility gene in this model. In these transgenic mice, expression of a truncation activated form of CRaf kinase, CRaf BXB, was targeted by the SP-C promoter exclusively to pneumocyte type II cells in adult mice. Induction of cuboid cell adenomas occurred with short latency and at 100% incidence. The tumors were histologically very stable and eventually killed the mice by suffocation (2) .

To evaluate the role of bcl-2 in CRaf oncogenesis in mouse lungs, we have used bcl-2-deficient mice in crosses with SP-C-craf BXB transgenics and determined the effect on latency and tumor phenotype. The bcl-2-/- mice have not been examined previously in crosses with oncogene transgenic mice. The results indicate that bcl-2 is a strong susceptibility gene for induction of lung adenomas by oncogenic CRaf. These data suggest that tumors that involve CRaf may be sensitive to at least two types of drugs, those that inhibit Raf kinase activity and others that neutralize bcl-2 function.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Production and Genotyping of Transgenic Mice.
Transgenic mouse line SP-C-craf BXB-23 (Bl/6xD2 background) was described recently (2) . Bcl-2-deficient mice (24) were provided on a 129Sv background by Michael Sendtner (University of Würzburg, Germany). H2K-bcl-2 transgenic mice expressing human Bcl-2 (Ref. 25 ; C57Bl6 background) were a gift from Irving L. Weissman (Stanford University, CA). All of the mice were housed in pathogen-free conditions and handled in accordance with institutional guidelines. To produce animals hemizygous for the transgene and heterozygous for bcl-2, the SP-C-craf BXB mice were mated with bcl-2+/- mice. Resultant compound mice were subsequently backcrossed with Bcl-2+/- mice to yield SP-C-craf BXB/bcl-2-/- and SP-C-craf BXB/bcl-2+/- animals. To generate SP-C-craf BXB/bcl-2-/-/H2K-bcl-2 triple transgenic mice the following approach was taken. The SP-C-craf BXB and H2K-bcl-2 transgenic mice were independently crossed with heterozygous bcl-2+/- mice. Progeny heterozygous for the bcl-2-targeted allele and hemizygous for the transgenes were interbred to produce bcl-2-deficient mice hemizygous for each transgene. Genotyping of animals was performed by PCR using mouse tail DNA. A 406-bp fragment specific for the SP-C-craf BXB transgene was detected with the following primers: SPC-5'-GAGAGGAGAGCATAGCAC-3' and the craf BXB primer 5'-ATCTCCGTGCCATTTACC-3' using the following PCR protocol: 95°C for 5 min followed by 35 cycles of 30 s at 95°C, 30 s at 55°C, and 1 min at 72°C followed by a 6-min extension at 72°C. For bcl-2 genotyping, the sense primer 5'-CAC CAG AAT CAA GTG TTC GGT G-3' and antisense primer 5'- GGT AGC GAC GAG AGA AGT CAT C-3' were used to amplify a 512-bp fragment of exon 2 of the wild-type allele, while a combination of the same sense primer 5'-CAC CAG AAT CAA GTG TTC GGT G-3' with a primer for lacZ 5'-CAT TCA GGC TGC GCA ACT GTT G-3' amplifies a 313-bp fragment of the targeted allele using the following PCR protocol: 95°C for 5 min followed by 30 cycles: 45 s at 95°C, 45 s at 57°C, and 1 min at 72°C followed by a 6-min extension at 72°C. A 200-bp fragment of the H2K bcl-2 transgene was detected using primers: H2K primer 5'- CGC GGA CGC TGG ATA TAA AGT C -3' and an antisense primer 5'- ACA TCT CCC GCA TCC CAC TC -3' using the following PCR protocol: 95°C for 5 min followed by 35 cycles: 30 s at 95°C, 30 s at 62°C, and 1 min at 72°C followed by a 6-min extension at 72°C.

RNA Preparation and RT-PCR Analysis.
Total RNA was prepared from lung tissues using the TRIzol LS Reagent followed by amplification grade DNase I treatment (Invitrogen). For semiquantitative PCR of RNA, cDNA was prepared by reverse transcription of 5 µg of each RNA sample using Moloney murine leukemia virus reverse transcriptase (Invitrogen). PCR amplifications were performed in a 50 µl reaction volume containing 5 µl of each cDNA. Primers for SP-C detection were sense 5'-GCC TAT AAG CCA GCT CCA GG-3' and antisense 5'-TTC TAC CGA CCC TGT GGA TG -3', and for {alpha}-actin were sense 5'-GTC GTA CCA CAG GCA TTG TGA TGG-3' and antisense 5'-GCA ATG CCT GGG TAC ATG GTG G-3'. The conditions for amplification were as follows: 95°C denaturation for 2 min followed by 95°C for 30 s, 56°C for 30 s, 72°C for 1 min for 22 cycles with SP-C, and 25 cycles with {alpha}-actin primers followed by a 6-min extension at 72°C. Cycle curve studies confirmed that for the amounts of cDNA being amplified, the reactions had not reached the plateau of the amplification curve with either primer pair. PCR control reaction without reverse transcription yielded no detectable fragments with all of the primer pairs.

Western Blot Analysis.
Mice were sacrificed, the left auricle was transected, the right ventricle and correspondingly lungs perfused with PBS lacking calcium and magnesium, and lung tissues were frozen in liquid nitrogen. Frozen tissue samples were homogenized in radioimmunoprecipitation assay buffer containing 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1% NP40, 0.5% desoxycholate, 0.01% SDS, 10 mM Na-PPi, 25 mM glycerophosphate, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml aprotinin using an Ultra Turrax blender. After centrifugation at 13,000 x g for 10 min the protein content of the supernatant was determined using the Dc Protein Assay kit (Bio-Rad). The protein lysates were separated by electrophoresis in 12% SDS polyacrylamide gel and blotted onto nitrocellulose membranes. Proteins were detected by rabbit anti-bcl-2 antiserum (sc-492; Santa Cruz Biotechnology) diluted 1:200; rabbit anti-bcl-xL antiserum (#2762; Cell Signaling Technology) diluted 1:1,000, and rabbit anti-ERK 2 antiserum (sc-154; Santa Cruz Biotechnology) polyclonal antiserum diluted 1:500 for loading control. Donkey antirabbit immunoglobulins linked to horseradish peroxidase (Amersham) diluted 1:1,000 were used as secondary antibodies. Bound antibodies were detected by chemiluminescence (ECL; Amersham).

Immunohistochemistry and TUNEL Assays.
For the immunochistochemical detection of mouse bcl-2 protein, paraffin-embedded 6-µm thick sections were deparaffinized, rehydrated, and microwaved for 6 min in 10 mM sodium citrate buffer (pH 5.5). Subsequently the slides were incubated for 6 min in peroxidase blocking solution (3% H2O2 in PBS). After antigen retrieval, slides were rinsed in distilled water, incubated in 20% sucrose in PBS at +4°C for 30 min, washed in PBS, and placed in blocking buffer (2.5% goat serum in PBS) for 40 min, and slides were incubated with an antiserum against mouse bcl-2 (sc-492; Santa Cruz Biotechnology) diluted 1:150 in blocking buffer at +4°C overnight. To avoid nonspecific binding, this antiserum had been preincubated with acetone powders of lung and liver proteins of bcl-2 -/- mouse (26) . Antigen-antibody complexes were detected with immunoperoxidase system (Vectastain ABC kit; Vector). The sections were then counterstained with hematoxylin. To identify expression of human bcl-2 oncoprotein and PCNA the deparaffinized tissue sections (5 µm) were rehydrated and microwaved twice for 10 min in 10 mM sodium citrate buffer (pH 5.5), and incubated with primary antibodies: mouse antihuman bcl-2 monoclonal antibody (DAKO) diluted 1:100 or mouse anti-PCNA monoclonal antibodies (PharMingen) diluted 1:300 in 50 mM Tris-buffer (pH 7.4) overnight at room temperature. Subsequently the slides were rinsed with 50 mM Tris-buffer (pH 7.4), incubated with biotinylated secondary rabbit-antimouse antibodies (DAKO), diluted 1:50 in 50 mM Tris buffer (pH 7.4) for 30 min at room temperature, rinsed with Tris-buffer, and incubated with streptavidin-biotinylated alkaline phosphatase-complex (Strept AB Complex; DAKO) for 30 min at room temperature, followed by Fast-red reaction for 20 min at room temperature and counterstaining with hemalaun. All of the immunohistochemical reactions were carried out in parallel with reactions lacking primary antibodies to ensure the specificity of the observed staining. TUNEL assay was performed on 6-µm thick paraffin sections according to the manufacturer’s protocol using In Situ Cell Death Detection, POD kit (Roche). 3'-OH DNA groups in the apoptotic cells were labeled by fluorescein-labeled nucleotides with terminal deoxynucleotidyl transferase followed by direct immunoperoxidase labeling of fluorescein-labeled genomic DNA. A TUNEL-negative control was obtained by omitting terminal deoxynucleotidyl transferase from labeling mix; positive sections included weaning-stage mouse mammary gland tissue and also lung tissue preincubated with 1 mg/ml DNase I for 10 min at room temperature to induce 3'-OH strand breaks detected by the TUNEL method. The sections were counterstained with hematoxylin. Positive nuclei were scored both on the basis of 3,3'-diaminobenzidine labeling and morphological features of apoptosis including nuclear versus cytoplasmic staining, death of single cells, marked condensation of chromatin and cytoplasm, uniformly dense chromatin, intracellular chromatin fragmentation (micronuclei), and halo effect around the nucleus at x400. Apoptotic and PCNA indices were determined by counting 10 randomly chosen fields per 3–4 sections and determining the percentage of apoptotic or proliferating cells per 2000 cells at x400. Statistical analyses for PCNA and TUNEL labeling were performed by the Student t test, and differences were considered significant when P < 0.05.

Histopathological Evaluation.
Groups of 1.5, 2.5, 4, 6, and 8.5 month-old mice were subjected to complete autopsy, and both gross and microscopic examinations were conducted. Organs were fixed in 3.7% formaldehyde in PBS, embedded in paraffin, sectioned at 6 µm, and stained with H&E. For morphometric studies the three to four pieces of the lungs from 1.5 month-old SP-C-craf BXB, bi-, or triple-transgenic mice were serially sectioned in their entirety. Every tenth section was stained with H&E and evaluated by light microscopy for the presence of neoplasia. The average number of foci for 1-mm2 area of the lungs of transgenic mice was counted by recording the number of individual foci on each section in several frames. Statistical analysis was performed by the Student t test using a P of <0.05. To compare the tumor growth (lung weight) as a function of time we used a linear regression assay and calculated the regression coefficients.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Loss of bcl-2 Interferes with Tumor Formation in SP-C-craf BXB Transgenic Mice.
Bcl-2-negative mice have normal lungs and live for up to 1 year when they die presumably from failure of the immune system and polycystic kidney disease (27 , 28) . Transgenic mice that express a human bcl-2 transgene under the control of the general promoter H2K show no apparent effect on the rate of lung cancer (25) . To control for the different genetic backgrounds, which were used to establish the bcl-2-deficient and H2K-bcl-2-transgenic animals, we assessed tumor data from mice that belong to individual litters. In the course of transformation by oncogenic CRaf, levels of endogenous bcl-2 RNA (data not shown) and protein were detectable at 1.5 months in the lung but did not increase significantly at 6 months (Fig. 1ECitation ; Fig. 3DCitation ). When one allele of bcl-2 was removed, no effect on tumor latency was observed (Fig. 1A)Citation . Loss of the second allele causes a pronounced 6–8-fold delay in tumor incidence. The analysis of foci numbers and lung weight from several litters obtained from intercrossing SP-C-craf BXB/bcl-2+/- and bcl-2+/- (mixed C57Bl/6xBDA-2 and 129/Sv backgrounds) yielded essentially similar values for SP-C-craf BXB or SP-C-craf BXB/bcl-2-/- animals showing that no unknown tumor modifier had been derived from the 129/Sv strain. These findings are in line with data in our original SP-C-CRaf transgene research(2) , where tumor induction was observed at 100% penetrance in three different genetic backgrounds (C57Bl/6xDBA-2, C57Bl/6, and 129/Sv). In triple transgenic mice that express bcl-2 from the H2K promoter (Fig. 1E)Citation , rapid tumor development was almost completely restored (Fig. 1BCitation ; Supplementary Fig. 1Citation ). Introduction of the H2K-bcl-2 transgene on a wild-type background increased the level of bcl-2 in pneumocyte type II cells already at day 15.5 postcoitum embryos (Fig. 1F)Citation with no consequence for tumor incidence or latency in SP-C-craf BXB mice (Fig. 1A)Citation .



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  		Fig. 1. Retardation of tumor development in SP-C-craf BXB transgenic mice lacking bcl-2. A, tumor incidence curves of SP-C-craf BXB/bcl-2-/- mice. B, mean number of foci/1 mm2 in lung sections of 1.5-month-old mice. See litter analysis in Supplementary Data. C, weight increase of lungs from SP-C-craf BXB/bcl-2-/- and SP-C-craf BXB/H2K-bcl-2 mice. Difference of SP-C-craf BXB/bcl-2-/- versus SP-C-craf BXB, SP-C-craf BXB/bcl-2+/-, and SP-C-craf BXB/H2K-bcl-2 mice is statistically significant (P < 0.001). D, weight increase of lungs from mice without SP-C-craf BXB transgene. E and F, Western blot analysis of bcl-2 expression in the lung of adult and embryonic mice, respectively; bars, ± SD.

 


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  		Fig. 3. Immunohistochemistry of lungs from 6-month-old mice. A and B, immunostaining for CRaf BXB. Cytoplasmic expression of human CRaf BXB protein in a solid cuboidal cell adenoma of SP-C-craf BXB (A) and a SP-C-craf BXB/bcl-2-/- mouse (B). C–F, immunostaining for mouse bcl-2. Intense cytoplasmic staining of bronchial epithelia (arrows) in the lung of wild-type (C) and moderate expression in the lung tumor from a SP-C-craf BXB mouse (D). Immunoperoxidase staining (brown); in addition sections were counterstained with hematoxylin (blue). Absence of staining in the lung of bcl-2-/- (E) and SP-C-craf BXB/bcl-2-/- mouse (F). Immunohistochemistry (red, alkaline phosphatase staining) with an antibody specific for human bcl-2 shows staining in SP-C-craf BXB/H2K-bcl-2 mice (H) but no staining in lung tissue from SP-C-craf BXB (G). Scale bars = 60 µm (A and B) and 30 µm (C–H).

 
Bcl-2 might affect tumor initiation and/or growth. To distinguish between these possibilities, the number of foci per mm2 of lung section was determined at 1.5 months of age. There was no significant difference between mice that carried one or two alleles of endogenous bcl-2 or the H2K-bcl-2 transgene on a bcl-2 null background (triple transgenic mice). No foci were detected in lungs from bcl-2-negative SP-C-craf BXB mice indicating resistance of type II pneumocytes to CRaf transformation in the absence of bcl-2. At 2.5 months, when 40% of bcl-2-deficient SP-C-craf BXB mice were tumor positive (Fig. 1A)Citation , the number of foci/mm2 was still far below the bcl-2-positive mice. The strong retardation in tumor development was additionally examined by determining the increase in lung weight over time (Fig. 1, C and D)Citation , which shows that the late start tumors in bcl-2-negative mice never caught up with tumor mass in bcl-2-positive mice. Fig. 1CCitation also shows that raising the level of bcl-2 expression 3–5-fold above the level of endogenous bcl-2 by breeding with H2K-bcl-2 transgenic mice did not additionally increase the rate of tumor growth. CRaf overexpression requires bcl-2 for tumor induction, but overexpression of bcl-2 does not show a synergistic effect on tumor penetrance in SP-C-CRaf transgenic mice indicating that the level or activity of endogenous bcl-2 is not a rate limiting factor. Taken together these studies suggest that bcl-2 may function downstream of CRaf in these lung tumors. No compensatory up-regulation of the 30 kDa bcl-xL protein that is expressed in normal lung was detected in Western blots (data not shown). We had observed previously cooperation of CRaf and bcl-2 in the suppression of apoptosis in cell culture (15 , 29) . The lack of cooperation in tumor formation may be explained by the opposing effects of bcl-2 on cell cycle progression versus apoptosis. The degree of tumor suppression in the absence of bcl-2 was unexpected as bcl-xL and other BH4 domain-containing antiapoptotic bcl-2 members share the ability to interact with CRaf BXB and could potentially compensate for bcl-2 loss. Moreover, a variant of the CRaf kinase, which included a mitochondrial targeting sequence, was able to suppress apoptosis even in the absence of bcl-2 in embryonic fibroblast cells (22) , and active cytosolic CRaf was shown to induce survival via the induction of an autocrine loop involving activation of another apoptosis suppressor, protein kinase B (30) . Finally, apoptosis suppression was not thought to be the primary mechanism of CRaf transformation, the major driving force was expected to lie in its ability to promote cell cycle progression by activation of the mitogenic cascade, Raf-mitogen-activated protein/ERK kinase-ERK1/2 (11 , 12 , 31 , 32) . The data on bcl-2 dependence of lung tumor induction by CRaf BXB now suggest that suppression of apoptosis in cooperation with bcl-2 is a critical step in lung tumor induction by CRaf.

Histology and Immunohistochemistry of Lungs from SP-C-craf BXB Mice in Presence or Absence of bcl-2.
As bcl-2-deficient mice have reduced size we had to consider that lung development might be delayed. Conversion of the architecturally immature lung to the mature organ is developmentally regulated and involves the process of septation that occurs from day 5 to 14 in mice (33) . Histological examination of lungs from 9-day-old bcl-2-positive and -negative mice provided no evidence for significant differences (Fig. 2, A and B)Citation . To estimate the number of pneumocyte type II target cells in bcl-2 ablated versus wild-type mice, quantitative RT-PCR for SP-C RNA was performed, which showed similar levels (Fig. 2E)Citation indicating comparable relative numbers of target cells for Raf transformation. Comparison of lung tumors from 6-month-old mice shown in Fig. 2, C and DCitation , illustrates the difference in frequency and size of individual tumor foci that is still evident at this age. This figure also shows that tumor cell phenotype is unaffected by loss of bcl-2.



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  		Fig. 2. Histology of normal and tumor lung tissues derived from wild-type and transgenic mice. H&E staining of frontal sections through the left lung of 9-day-old SP-C-craf BXB mice wild-type for bcl-2 (A) or deficient for bcl-2 (B). Adenomas in the lungs of 6-month-old SP-C-craf BXB (C) and SP-C-craf BXB/bcl-2-/- mouse (D). Scale bar = 120 µm. E, RT-PCR of SP-C RNA from the lung of SP-C-craf BXB mice deficient or wild-type for bcl-2.

 
The fact that bcl-2 loss did not affect the morphology of tumor cells is also evident at higher resolution in Figs. 3, A, B, G, and HCitation . In this experiment lung sections were stained with either CRaf antibodies (Fig. 3, A and B)Citation , or antibodies specific for mouse (Fig. 3, C–F)Citation or human (Fig. 3, G and H)Citation bcl-2. Loss of endogenous bcl-2 was of no consequence for CRaf expression. Bcl-2 expression was not detectable in normal alveoli (Fig. 3 C and D)Citation but reached levels in the SP-C-craf BXB-induced adenoma that are readily detectable but still below those in bronchial epithelial cells in normal mice. Uniform staining for human bcl-2 was seen in lung tumors of SP-C-craf BXB/H2K-bcl-2 mice (Fig. 3, G and H)Citation , which illustrates that H2K-bcl-2 was expressed in pneumocyte type II cells, the target cells for CRaf BXB transformation.

Alteration of Death versus Growth Rates of bcl-2-positive and -negative Tumor Cells.
Lung sections from 6-month-old mice were examined by TUNEL assay and staining with antibody against PCNA to assess the effect of bcl-2 on tumor cell growth and survival. Fig. 4Citation shows representative pictures and their quantification. The increase in the number of apoptotic cells (Fig. 4, A, B, and E)Citation was pronounced but not massive, consistent with data on bcl-2-independent suppression of apoptosis by CRaf (22) . The fraction of S phase cells as judged from PCNA staining was significantly lower in bcl-2-negative as compared with positive tumor tissues. This effect was more striking when comparison was made with wild-type rather than H2K-bcl-2 transgenic mice. We conclude that low levels of bcl-2 protein are essential for optimal tumor cell growth and cannot be compensated by constitutive CRaf kinase. High levels of bcl-2 on the other hand may negatively affect cell cycle progression (34) thereby counteracting to some degree a positive influence on accumulation of tumor mass by cooperation with CRaf BXB on suppression of apoptosis (35) .



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  		Fig. 4. TUNEL and PCNA staining of lung adenomas of bcl-2-positive and null mice at the age of 6 months. TUNEL staining of tumor from SP-C-craf BXB (A) and SP-C-craf BXB/bcl-2-/- (B) mice. Arrows indicate apoptotic cells. PCNA-labeled cells in a lung tumor from SP-C-craf BXB (C) and SP-C-craf BXB/bcl-2-/- (D) mice. Scale bars = 30 µm (A–D). Comparison of apoptotic indices in lung tumors (E). Difference of SP-C-craf BXB/bcl-2-/- mice versus mice of other genotypes is statistically significant (P < 0.03). Comparison of PCNA labeling indices in lung tumors (F). Difference of SP-C-craf BXB/bcl-2-/- mice versus all others is statistically significant (P < 0.046).

 
Despite extensive efforts in many laboratories, the precise function of bcl-2 has remained elusive. Many facets have been described such as its ability to form ion channels (36) to modulate the activity of VDAC in the outer mitochondrial membrane and thereby cytochrome c release (37) , to act as a protein translocator, or to induce the level of antioxidants such as reduced glutathione by suppressing its transporter (38) . Because of the multiple effects that bcl-2 has on cellular physiology, it is difficult to pinpoint the process most relevant to transformation by CRaf. The simplest model would be that CRaf and bcl-2 can no longer interact in membranes, most prominently the mitochondrial membrane in which bcl-2 and CRaf share other binding partners such as BAG-1 (17) and VDAC, a protein that forms part of the permeability transition pore (36) . Future experiments that use interaction site mutants of bcl-2 and CRaf should help to settle this question.

Expression of bcl-2 in human tumors has been determined frequently in efforts to evaluate this oncoprotein as a potential prognostic marker (36) . Opposing findings were reported for tumor recurrence for example in colorectal cancer, which was favored by bcl-2 loss (39) , and metastatic progression, which was promoted by increased expression of bcl-2 (40) . We propose based on the findings reported here that bcl-2 be evaluated as a susceptibility factor for individual cancer risk.


   ACKNOWLEDGMENTS
 
We thank Alla Ganscher and Lena Sakk for expert technical assistance; Elena V. Chernigovskaya for help with immunohistochemistry; Uwe Maeder for help with the statistical analysis; and Bruce Jordan, Albrecht Müller, and Jakob Troppmair for critical reading of the manuscript.


   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 in part by grants from M. Scheel Stiftung (10–1793-Ra7) and Deutsche Forschungsgemeinschaft (SFB 465 and SP 1109). Back

2 Supplementary data for this article is available at Cancer Research Online (http://cancerres.aacrjournals.org). Back

3 To whom requests for reprints should be addressed, at Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Universität Würzburg, Versbacher Str. 5, D-97078 Würzburg, Germany. Phone: 49-931-201-45141; Fax: 49-931-201-45835; E-mail: rappur{at}mail.uni-wuerzburg.de Back

4 L. M. Fedorov, T. Papadopoulos, O. Y. Tyrsin, T. Twardzik, R. Götz, and V. R. Rapp. Loss of p53 in craf-induced transgenic lung adenoma leads to tumor acceleration and phenotypic switch, manuscript in preparation. Back

5 The abbreviations used are: ERK, extracellular signal-regulated kinase; NF{kappa}B, nuclear factor {kappa}B; VDAC, voltage-dependent anion channel; SP-C, surfactant protein-c; RT-PCR, reverse transcription-PCR; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PCNA, proliferating cell nuclear antigen. Back

Received 5/23/02. Accepted 9/ 4/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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