RESUMEN
The culture of tissue-derived human cells is an important aspect of tissue engineering. Prior to transplantation, the quality of the cultured cells/tissue should be routinely tested so that any enrichment of procarcinogenic cells can be excluded. On this account, a UVB-induced p53 fingerprint mutation would be a valuable quality control marker for skin cells cultured for use in tissue engineering. We developed an allele-specific real-time polymerase chain reaction assay based on SYBR Green which can quantitatively detect CC-TT transitions in the tumor suppressor gene p53. To analyze the transition 281/282, we used DNA from HaCaT cells, a keratinocyte cell line containing the investigated mutation, as a standard to determine the mutation frequency in cultured cutaneous cells. We analyzed a variety of skin cells grown in culture and found a notable decrease in the UVB fingerprint mutation in fibroblasts during proliferation. Furthermore, we quantified the total amount of mutated DNA in different cutaneous cells and detected a significantly higher level in melanocytes. These results are consistent with findings obtained in our laboratory concerning the common deletion, the most frequently reported mutation in the mitochondrial genome, which suggest a positive influence of prolonged in vitro cell proliferation on the quality of genomic DNA.
Asunto(s)
Genes p53/genética , Mutación/efectos de la radiación , Rayos Ultravioleta , Anciano , Southern Blotting , Línea Celular , Células Cultivadas , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Masculino , Melanocitos/citología , Melanocitos/metabolismo , Melanocitos/efectos de la radiación , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
The questions of T cell receptor (TCR) clustering and preferential pairing of TCR alpha- and beta-chains are discussed controversially. We here describe the rare case of a non-pairing TCR alpha-TCR beta combination detected in the murine T cell hybridoma Hy-E6. Of its two TCR alpha-chains (Valpha3.2, Vbeta17) and one Vbeta16-chain only the Valpha17/Vbeta16 TCR is exposed on the surface, despite intracellular expression of Valpha3.2 protein. The lack of Valpha3.2/Vbeta16 pairing was confirmed by TCR transfections. Surprisingly, however, the parental T cell clone CTL-E6 expressed both alpha-chains on its plasma membrane. Different size distribution of TCR clusters in CTL-E6 versus Hy-E6 and transfectants as determined by Blue-Native gel electrophoresis indicated differences in the supra-molecular TCR assembly as one possible reason for this phenomenon. Our data further reveal that the nominal specificity of CTL-E6 for the fully agonistic trinitrophenyl (TNP) modified peptide M4L-TNP was specifically mediated by the trimeric Valpha3.2/Valpha17/Vbeta16 TCR of CTL-E6. In contrast, the Valpha17/Vbeta16 combination in Hy-E6 only conferred specificity for the cross-reactive partial agonist O4-TNP. Both specificities are H-2Kb-restricted and, hence, appear to be positively selected. The differences in TCR clustering in CTL and hybridoma might indicate differences in the reception and transmission of TCR-signals between these two cell types.