RESUMEN
Background: Aneuploidy and chromosomal instability (CIN) are common features of human malignancy that fuel genetic heterogeneity. Although tolerance to tetraploidization, an intermediate state that further exacerbates CIN, is frequently mediated by TP53 dysfunction, we find that some genome-doubled tumours retain wild-type TP53. We sought to understand how tetraploid cells with a functional p53/p21-axis tolerate genome-doubling events. Methods: We performed quantitative proteomics in a diploid/tetraploid pair within a system of multiple independently derived TP53 wild-type tetraploid clones arising spontaneously from a diploid progenitor. We characterized adapted and acute tetraploidization in a variety of flow cytometry and biochemical assays and tested our findings against human tumours through bioinformatics analysis of the TCGA dataset. Results: Cyclin D1 was found to be specifically overexpressed in early but not late passage tetraploid clones, and this overexpression was sufficient to promote tolerance to spontaneous and pharmacologically induced tetraploidy. We provide evidence that this role extends to D-type cyclins and their overexpression confers specific proliferative advantage to tetraploid cells. We demonstrate that tetraploid clones exhibit elevated levels of functional p53 and p21 but override the p53/p21 checkpoint by elevated expression of cyclin D1, via a stoichiometry-dependent and CDK activity-independent mechanism. Tetraploid cells do not exhibit increased sensitivity to abemaciclib, suggesting that cyclin D-overexpressing tumours might not be specifically amenable to treatment with CDK4/6 inhibitors. Conclusions: Our study suggests that D-type cyclin overexpression is an acute event, permissive for rapid adaptation to a genome-doubled state in TP53 wild-type tumours and that its overexpression is dispensable in later stages of tumour progression.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Colorrectales/genética , Ciclina C/genética , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Aminopiridinas/farmacología , Bencimidazoles/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Ciclina C/biosíntesis , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citocalasina B/análogos & derivados , Citocalasina B/farmacología , Diploidia , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Genes p53 , Células HCT116 , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Tetraploidía , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The human herpesvirus 8 (HHV8/KSHV), along with certain other herpesviruses, encodes a gene with cyclin homology. Although the functional significance of the encoded cyclin is not clear at present, various lines of evidence propose a role for this cyclin in latently infected cells and possibly in the induction of tumors that arise in HHV8-infected individuals. We provide evidence here that the cyclin protein is expressed in HHV8 positive primary effusion lymphoma (PEL)-derived cell lines and that its level of expression varies greatly between different lines. Our analysis indicates that the level of cyclin protein expression in different PEL cell lines may correlate with the level of transcript expression during latency but not in cells induced to undergo lytic replication. In highly expressing BC-3 cells the cyclin is complexed with cdk6, cdk4, cdk2, and cdk5 under both latent and lytic conditions, although subtle changes in the level of cdk association are seen after induction of the lytic cycle. Altogether our findings support the notion that the cyclin is a latency-associated gene product expressed in PEL tumor cells. They furthermore indicate that after lytic cycle induction, the level of cyclin transcript expression may not be a reliable indicator for the level of cyclin protein expression.
Asunto(s)
Ciclinas/genética , Herpesvirus Humano 8/genética , Linfoma de Células B/virología , Proteínas Virales/genética , Anticuerpos Antivirales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/biosíntesis , Ciclinas/inmunología , Herpesvirus Humano 8/enzimología , Humanos , Linfoma de Células B/química , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Proteínas Virales/biosíntesisRESUMEN
A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.