BAG-1 heterozygosity impairs C-Raf driven tumorigenesis
In order to assess the functional role of BAG-1 on tumorigenesis, we have generated a null allele of BAG-1. To inactivate the BAG-1 gene, exons 1 and 2 were replaced with a neomycin resistance gene. This strategy was chosen to disrupt the expression of all known isoforms of BAG-1 which are generated by alternate translation initiation of a single mRNA; the start codons are present in exons 1 and 2. Western blot analysis of liver protein extracts of BAG-1 deficient embryos showed the complete loss of all BAG-1 protein isoforms. Embryos homozygous for this allele died at midgestation at around E13,5, but the heterozygous animals (BAG-1+/-) are normal. A comprehensive description of the BAG-1-/- phenotype is subject of another manuscript.
Previously, we had generated a lung cancer mouse model by targeting constitutively active C-Raf kinase (SP-C C-Raf BxB) to the lung [14
]. These mice develop multifocal adenomas early in adulthood. Based on the observation, that BAG-1 can activate C-Raf [2
], we asked whether heterozygosity for BAG-1 would affect C-Raf BxB driven adenoma growth. We observed that lung tumour initiation was reduced by half in 1, 2 and 4 months old BAG-1+/-
mice transgenic for SP-C C-Raf BxB compared to their BAG-1+/+
littermates. Tumour area was reduced by 75% in 4 month lungs of BAG-1 haploinsufficient mice compared to mice with two BAG-1 copies, see Figure . The histological picture emphasises the difference in adenoma formation between a representative SP-C C-Raf BxB/BAG-1+/+
and SP-C C-Raf BxB/BAG-1+/-
lung. The difference in the staining intensity of the two lung sections derives mainly from the observation that the adenoma cells have a tendency to bind more intensively hematoxylin and eosin compared to normal lung cells. Thus, reduction of the BAG-1 gene dosage impairs the oncogenic activity of C-Raf in vivo.
Figure 1 BAG-1 haplo-insufficiency delays C-Raf driven adenoma growth. (a) Adenoma initiation in SP-C C-Raf BxB mice (BAG-1+/+) and their Bag-1 haplo-insufficient littermates (BAG-1+/-) at 1, 2 and 4 months of age. Adenoma foci values represent mean ± (more ...)
Reduced BAG-1 expression in BAG-1 heterozygous lungs
Quantitative immunoblots demonstrated that the specific BAG-1 protein concentration in the lungs of BAG-1+/- mice was half the amount of BAG-1+/+ littermates, see Figure . Moreover, immunohistochemical staining showed that BAG-1 was expressed in adenoma cells, see Figure . There was no obvious difference in the BAG-1 immunohistochemistry of SP-C C-RafBxB/BAG-1+/+ and SP-C C-RafBxB/BAG-1+/- lungs.
Figure 2 BAG-1 expression in the lung of SP-C C-Raf BxB transgenic mice (a) Lanes 1–8 show immunoblotting data for expression of BAG-1 in the lungs of 8 month old SP-C C-Raf BxB transgenic mice heterozygous (+/-) or homozygous (+/+) for BAG-1 as indicated (more ...)
Tumour cells of BAG-1 heterozygous mice show increased apoptosis
Concerning the molecular mechanism how a reduction of the BAG-1 protein expression in the heterozygous mice would impair tumorigenesis, we determined the fraction of apoptotic cells. Staining for activated caspase-3 revealed indistinguishable apoptosis in healthy regions of the lung of 1 month old SP-C C-Raf BxB mice with either one or two BAG-1 alleles, in line with the unaltered, normal lung structure of BAG-1+/- mice. In the adenomas, however, we observed a significant increase of apoptotic cells in BAG-1+/- SP-C C-Raf BxB mice compared with their SP-C C-Raf BxB/BAG-1+/+ littermates, see Figure . This mechanism of action of BAG-1 on the regulation of cell survival is compatible with the phenotype of embryonic day 12,5 BAG-1 null embryos. Immunohistochemical staining for activated caspase-3 and trypan blue staining of dissociated cells showed hypocellularity and elevated levels of apoptosis in the livers of BAG-1-/- embryos (unpublished observations).
Figure 3 Increased apoptosis but no change in PCNA and p-ERK in tumor cells of SP-C C-RafBxB transgenic BAG-1 heterozygous mice (a-c) Quantification of immunohistochemical staining for apoptosis using an antibody that detects activated caspase-3 (a), PCNA (b) (more ...)
Proliferation and p-ERK signalling are unaffected in BAG-1 heterozygous mice
To exclude the alternative mechanism that the decreased level of BAG-1 expression in heterozygous animals would cause reduced cell proliferation in the adenomas, we performed proliferating cellular antigen (PCNA) staining. No significant differences were observed in the fraction of proliferating adenoma cells between SP-C C-Raf BxB animals heterozygous or wild type for BAG-1, see Figure . Also, the percentages of adenoma cells positive for Ki-67, another proliferation marker and Bmi-1, a chromatin-associated protein expressed in stem cells, were not affected by the BAG-1 heterozygosity (not shown). Furthermore, staining of lung sections for phosphorylated ERK revealed no quantitative differences in the adenomas of SP-C C-Raf BxB animals heterozygous or wild type for BAG-1, see Figure . Thus, signalling through the mitogenic cascade was not affected by the BAG-1 heterozygosity in the adenoma cells.