N-Meier survival curves of aged Cc1-Cre KrasG12D mice and Cc1?Cre control mice cohorts. Naive (unimmunized) Cc1-Cre KrasG12D mice (n = 12) Ntrol (arrows) Sections on the right (iii, vi, ix) represent negative developed fatal T-cell lymphomas with a median latency of 125 days. ??Lung tumors were found incidentally at autopsy in both immunized Cc1-Cre KrasG12D (n = 7) and naive Cc1-Cre KrasG12D mice, while naive and ?immunized Cc1-Cre were healthy for the duration and had no lung adenomas at autopsy. B) PCR detection of KrasG12D allele recombination in naive Cc1-Cre KrasG12D mice with T-cell lymphomas. Recombination is detectable in unfractionated mononuclear splenic cells, consistent with infiltration of spleen by lymphoma cells. Recombination with loss of wild-type allele observed in unfractionated cells isolated from thymic tumor tissue. Results from two affected mice are shown. doi:10.1371/journal.pone.0067941.gAID-Cre-YFP KrasG12D Arf2/2 Mice Develop Fatal Epidermal Papillomas and Derivative CarcinomasWe reasoned that a lack of a detectable B-cell phenotype in Cc1Cre KrasG12D and AID-Cre-YFP KrasG12D mice was most likely due to a requirement for a cooperating “second hit” to induce cellular transformation. Therefore, to test the effects of a second mutation known to cooperate with KrasG12D, we crossed AID-Cre-YFP KrasG12D mice into a tumor-prone Arf-null background (Arf 2/2) (On of the partial differential equation describing the spreading process suggests Figure 1). 16985061 All AID-Cre-YFP KrasG12D Arf 2/2 mice developed rapidly progressive papillomas and by 13 wks, 66 of AID-CreYFP KrasG12D Arf 2/2 mice (n = 3) developed cutaneous sarcomas (Figure 7A), while AID-Cre-YFP Arf 2/2 control mice remained disease-free (Figure 7B). Histopathological sections of spleen from control mice show typical red pulp, white pulp and germinal center structures (Figure 7D); whereas AID-Cre-YFP KrasG12D Arf 2/2 spleen showed defacement of splenic architecture with loss of distinction between red and white pulp and a paucity of germinal centers (Figure 7C). Sections of sarcomas from AID-Cre-YFP KrasG12D Arf 2/2 showed characteristic undifferentiated spindle cells (Figure 7E), consistent with tumors previously described in Arf-deficient mice (10). The only abnormalities attributable to Bcells that we identified were small but significant increases in polyclonal antibody responses over time. The gamma proteinfraction by SPEP was higher in AID-Cre-YFP KrasG12D Arf 2/2 at 12 weeks compared to AID-Cre-YFP Arf 2/2 controls (Figure S4C), but none of the mice developed multiple myeloma or monoclonal gammopathy. AID-Cre-YFP KrasG12D Arf 2/2 and AIDCre-YFP KrasG12D Arf +/2 mice also showed significant differences in total serum gamma region protein levels between baseline and 12 weeks (Figure S4A). Serum ELISA of antibody subtypes from AID-Cre-YFP KrasG12D Arf 2/2, AID-Cre-YFP KrasG12D Arf +/2, and control AID-Cre-YFP Arf 2/2 also showed small but significant changes between baseline and 12 weeks in IgM and IgG isosubtypes (Figure S4B), perhaps related to infected, fungating papillomas in these mice. Flow cytometric immunophenotyping of bone marrow and splenic mononuclear cells failed to detect the abnormal growth in any B-cell populations in AID-Cre-YFP KrasG12D Arf 2/2 mice.DiscussionKras is the oncogene most frequently mutated in MM, yet its role in the pathogenesis of the disease has yet to be elucidated. Here, we used a mouse model of activated Kras to directly test the effect of activated Kras in post-germinal center B-cells using two different Cre recombinases reported to be specific to germinal center B-cell.N-Meier survival curves of aged Cc1-Cre KrasG12D mice and Cc1?Cre control mice cohorts. Naive (unimmunized) Cc1-Cre KrasG12D mice (n = 12) developed fatal T-cell lymphomas with a median latency of 125 days. ??Lung tumors were found incidentally at autopsy in both immunized Cc1-Cre KrasG12D (n = 7) and naive Cc1-Cre KrasG12D mice, while naive and ?immunized Cc1-Cre were healthy for the duration and had no lung adenomas at autopsy. B) PCR detection of KrasG12D allele recombination in naive Cc1-Cre KrasG12D mice with T-cell lymphomas. Recombination is detectable in unfractionated mononuclear splenic cells, consistent with infiltration of spleen by lymphoma cells. Recombination with loss of wild-type allele observed in unfractionated cells isolated from thymic tumor tissue. Results from two affected mice are shown. doi:10.1371/journal.pone.0067941.gAID-Cre-YFP KrasG12D Arf2/2 Mice Develop Fatal Epidermal Papillomas and Derivative CarcinomasWe reasoned that a lack of a detectable B-cell phenotype in Cc1Cre KrasG12D and AID-Cre-YFP KrasG12D mice was most likely due to a requirement for a cooperating “second hit” to induce cellular transformation. Therefore, to test the effects of a second mutation known to cooperate with KrasG12D, we crossed AID-Cre-YFP KrasG12D mice into a tumor-prone Arf-null background (Arf 2/2) (Figure 1). 16985061 All AID-Cre-YFP KrasG12D Arf 2/2 mice developed rapidly progressive papillomas and by 13 wks, 66 of AID-CreYFP KrasG12D Arf 2/2 mice (n = 3) developed cutaneous sarcomas (Figure 7A), while AID-Cre-YFP Arf 2/2 control mice remained disease-free (Figure 7B). Histopathological sections of spleen from control mice show typical red pulp, white pulp and germinal center structures (Figure 7D); whereas AID-Cre-YFP KrasG12D Arf 2/2 spleen showed defacement of splenic architecture with loss of distinction between red and white pulp and a paucity of germinal centers (Figure 7C). Sections of sarcomas from AID-Cre-YFP KrasG12D Arf 2/2 showed characteristic undifferentiated spindle cells (Figure 7E), consistent with tumors previously described in Arf-deficient mice (10). The only abnormalities attributable to Bcells that we identified were small but significant increases in polyclonal antibody responses over time. The gamma proteinfraction by SPEP was higher in AID-Cre-YFP KrasG12D Arf 2/2 at 12 weeks compared to AID-Cre-YFP Arf 2/2 controls (Figure S4C), but none of the mice developed multiple myeloma or monoclonal gammopathy. AID-Cre-YFP KrasG12D Arf 2/2 and AIDCre-YFP KrasG12D Arf +/2 mice also showed significant differences in total serum gamma region protein levels between baseline and 12 weeks (Figure S4A). Serum ELISA of antibody subtypes from AID-Cre-YFP KrasG12D Arf 2/2, AID-Cre-YFP KrasG12D Arf +/2, and control AID-Cre-YFP Arf 2/2 also showed small but significant changes between baseline and 12 weeks in IgM and IgG isosubtypes (Figure S4B), perhaps related to infected, fungating papillomas in these mice. Flow cytometric immunophenotyping of bone marrow and splenic mononuclear cells failed to detect the abnormal growth in any B-cell populations in AID-Cre-YFP KrasG12D Arf 2/2 mice.DiscussionKras is the oncogene most frequently mutated in MM, yet its role in the pathogenesis of the disease has yet to be elucidated. Here, we used a mouse model of activated Kras to directly test the effect of activated Kras in post-germinal center B-cells using two different Cre recombinases reported to be specific to germinal center B-cell.
