RESUMEN
Transforming growth factor-beta (TGF-beta) inhibits growth and induces apoptosis of colon epithelial cells. Binding of TGF-beta to its receptor induces phosphorylation of the Smad proteins Smad2 and Smad3, which then form heteromeric complexes with Smad4, translocate to the nucleus, and activate gene transcription. Smad4 function has been considered an obligate requirement for TGF-beta signaling, and Smad4 mutations present in some cancers have been considered sufficient to inactivate TGF-beta signaling. In this work, we describe studies with a nontransformed human colon epithelial cell line that is mutant for Smad4 but remains growth-inhibited by TGF-beta. The colon cell line VACO-235 has lost one of its Smad4 alleles via a chromosome 18q deletion. The remaining allele bears two missense point mutations located in regions important for Smad4 trimer formation, which is thought necessary for Smad4 function. As expected, pSBE4-BV/Luc, a Smad4-activated transcriptional reporter, was inactive in VACO-235. Nonetheless, VACO-235 demonstrated 80% growth inhibition in response to TGF-beta, as well as retention of some TGF-beta-mediated activation of the p3TP-Lux transcriptional reporter. Transient transfection of the VACO-235 Smad4 mutant allele into a Smad4-null cell line confirmed that this allele is functionally inactive as assayed by both the pSBE4-BV and p3TP-Lux reporters. The simplest explanation of these results is that there is a non-Smad4-dependent pathway for TGF-beta-mediated signaling and growth inhibition in VACO-235 cells.
Asunto(s)
Adenoma/genética , Adenoma/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Proteínas de Unión al ADN/genética , Inhibidores de Crecimiento/farmacología , Mutación , Transactivadores/genética , Factor de Crecimiento Transformador beta/farmacología , División Celular/efectos de los fármacos , División Celular/genética , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Proteína Smad4 , Activación Transcripcional/genética , Células Tumorales CultivadasRESUMEN
We previously demonstrated that mutational inactivation of transforming growth factor beta type II receptors (RIIs) is very common among the 13% of human colon cancers with microsatellite instability. These mutations principally cluster in the BAT-RII polyadenine sequence repeat. Among microsatellite stable (MSS) colon cancers, we now find that non-BAT-RII point mutations inactivate RII in another 15% of cases, thus doubling the known number of colon cancers in which RII mutations are pathogenetic. Functional analysis confirms that these mutations inactivate RII signaling. Moreover, another 55% of MSS colon cancers demonstrate a transforming growth factor beta signaling blockade distal to RII. The transforming growth factor beta pathway and RII in particular are major targets for inactivation in MSS colon cancers as well as in colon cancers with microsatellite instability.
Asunto(s)
Neoplasias del Colon/genética , Repeticiones de Microsatélite , Mutación , Receptores de Factores de Crecimiento Transformadores beta/genética , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales CultivadasRESUMEN
Fourteen colorectal cancer cell lines, categorized according to the presence or absence of microsatellite instability, were further analysed for chromosomal stability by karyotyping. NonRER (microsatellite stable) cell lines typically displayed highly aberrant karyotypes with alterations not only of chromosome number but also of chromosome structure including chromosomal deletions, inversions, and translocations. RER (microsatellite unstable) cell lines, in contrast, displayed significantly fewer alterations of chromosome number. Moreover, RER cell lines also displayed significantly fewer cytogenetically evident alterations of chromosome structure. Compared to NonRER colon cancers, RER colon cancers are significantly less likely to have undergone chromosomal gain, loss, or breakage. Characterization of p53 gene status by gene sequencing was performed in an attempt to determine if p53 gene status correlated with the chromosomal stability of the RER cancers. Gene mutations in p53 were present in all of the NonRER colon cancers. However, p53 gene mutations were also found present in four of nine of the RER colon cancers. Unexpectedly, RER colon cancers bearing mutant p53 demonstrated the same stability of chromosome number, and the same stability of chromosome structure, as the RER colon cancers with wild-type p53. Therefore, in RER colon cancers specific p53 independent mechanisms actively maintain the stability of both chromosome number and structure.
Asunto(s)
Cromosomas/genética , Neoplasias Colorrectales/genética , Replicación del ADN , ADN de Neoplasias/genética , Mutación , Proteína p53 Supresora de Tumor/genética , Aberraciones Cromosómicas , Neoplasias Colorrectales/clasificación , Humanos , Cariotipificación , Repeticiones de Microsatélite/genética , Células Tumorales CultivadasRESUMEN
We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.
Asunto(s)
Neoplasias del Colon/genética , Reparación del ADN/genética , Hipoxantina Fosforribosiltransferasa/genética , Mutación , Composición de Base/genética , Secuencia de Bases , Línea Celular , Humanos , Mutación Puntual , Eliminación de Secuencia , Células Tumorales CultivadasRESUMEN
Inactivation of DNA-mismatch repair underlies the genesis of microsatellite unstable (MSI) colon cancers. hPMS2 is one of several genes encoding components of the DNA-mismatch repair complex, and germline hPMS2 mutations have been found in a few kindreds with hereditary nonpolyposis colorectal carcinoma (HNPCC), in whom hereditary MSI colon cancers develop. However, mice bearing null hPMS2 genes do not develop colon cancers and hPMS2 mutations in sporadic human colon cancers have not been described. Here we report that in Vaco481 colon cancer the hPMS2 gene is inactivated by somatic mutations of both hPMS2 alleles. The cell line derived from this tumor is functionally deficient in DNA mismatch repair. This deficiency can be biochemically complemented by addition of a purified hMLH1-hPMS2 (hMutLalpha) complex. The hPMS2 deficient Vaco481 cancer cell line demonstrates microsatellite instability, an elevated HPRT gene mutation rate, and resistance to the cytotoxicity of the alkylator MNNG. We conclude that somatic inactivation of hPMS2 can play a role in development of sporadic MSI colon cancer expressing the full range of cancer phenotypes associated with inactivation of the mismatch repair system.