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ng in a subset of Burkitt lymphoma. Splenic B cellsfrom either precancerous λMyc transgenic mice or wildtypeC57BL6 littermates had been magnetically sorted making use of IgMspecificantibodies. These cells and palpable lymphomas harvested fromsick λMyc animals had been then applied to create protein lysates andRNA for protein gel blot and qRTPCR analysis. mk2206 Precancerouscells and all lymphomas exhibited high levels of Chek2 transcriptas compared with wildtype control cells. Nonetheless,analysis of Chk2 protein levels within the tumors revealed that thesewere comparable to wildtype and precancerous controls with theexception that a second band also was detectable. It isconceivable that this type represents an alternatively phosphorylatedform of Chk2.
Chk2 dimerization and autophosphorylationis essential for Chk2 activity,24 and has previously beenshown to give rise to such a band shift on SDS page.25 In orderto investigate if this type was phosphorylated, we treated lysatesof lymphomas from mk2206 the λMyc mouse with FastAPTM Alkalinephosphataseand compared these to untreated lysates fromthe identical tumor. Intriguingly, this treatment did not affect theband suspected to be the phosphorylated type of Chk2 but didreduce phosphorylation in the antiapoptotic Bcl2 family memberBad. Moreover, a cell line established from a tumorof a λMyc mouse did not display the reduced in the detected bands,suggesting that this alternate type of Chk2 is an effect of in vivotumor progression.Myc is deregulated in most human cancers because of indirect activationby upstream pathways.
Most colon cancer carries a mutationin the APC gene, giving rise to excessive Wntcatenin signalingand downstream cMyc activation.26 AP26113 We wanted to investigate iftumors arising in this setting regulate Chk2. As a way to answerthis question, we screened ApcMin mice that carry a mutation inthe adenomatus polyposis coligene. These mice developspontaneous adenomas within the colon and smaller intestine at around120 d of age.27 Comparing regular tissue with palpable adenomasof the smaller intestine, we detected an upregulation of Chek2 transcriptthat also correlated with Myc expression.Chk2 is dispensable for Myc induced colony formation.Chk2 is, as shown above, regulated by Myc in vitro and in vivo,suggesting that it could be crucial for Mycmediated transformation.As a way to investigate this, we genetically depleted Chek2mRNA making use of shRNA in Mycoverexpressing NIH 3T3 fibroblasts.
Clonogenic survival assays over 10 days showed thatremoval of Chek2 did not compromise the capability of Myc NSCLC to colonizethese plates, nor AP26113 did it affect Myc’s capability to transformcells in soft agar. Interestingly, nevertheless, the Chek2deficient fibroblasts appeared distorted in morphology. Several of these had been larger than controlinfected cells,and immunofluorescence analysis of mitotic cells making use of antibodiesagainst tubulin demonstrated a higher percentage of Chk2deficient cells stuck in mitosis. These data suggests adependency of these cells on Chk2 to properly execute mitosis.Recently, Chk2dependent BRCA1 phosphorylation wasimplicated as a crucial regulator of chromosomal instability.
28 BRCA1 localizes to mitotic centrosomes29 and isrequired for correct spindle assembly,30 thus Chk2 deficiencyresults in a failure to properly alignduplicated chromosomes, leading tolagging chromosomes mk2206 and increasedgenomic instability. Interestingly,when we introduced shRNA againstChek2 in a mouse lymphoma cell linederived from the λMyc transgenicmouse, these cells became severelypolyploid within a couple of passages. Although the cellstolerated this genomic instability, theirgeneration time was severely affectedcompared with control infected cells. Genomic instability hasbeen proposed to be an emerging hallmarkof cancer that drives tumor progression.31 Due to this, we wenton to transplant the Chk2deficientpolyploid lymphoma cells into recipientanimals and monitored these forvisible signs of disease.
The cells lackingChk2 expression had a significantlyslower disease progression thancontrolinfected cells, in line with all the slowergrowth phenotype observed in vitro.When sick, mouse tumor material wassnap frozen and prepared for protein gelblot AP26113 analysis. Interestingly, tumors didnot retain Chk2 knockdownbut remainedpolyploid, suggestingthat a selection against cells with lowChk2 expression had occurred in vivo.In addition, the tumors that emergedalso retained the band shift observedin the λMyc mice tumors; this bandwas not present within the parental cell lineinjected. Importantly, moribundmice transplanted with Chk2deficient cells did not exhibit a differentor additional invasive tumor spectra then control animals. Hence, the slower growth rate in the Chk2deficient cellswas dominant in vivo, and also the polyploidization induced by Chk2removal did not negatively affect disease progression.Chk2 is an crucial cell cycle regulator in response to DNAdamage, affecting both the Sphase32 and G2phase checkpoints.33Chk2targeted therapy is presently becoming pursued in order toaugment the effe