RESUMO
Telomere maintenance is necessary to maintain cancer cell unlimited viability. However, the mechanisms maintaining telomere length in colorectal cancer (CRC) have not been extensively investigated. Telomere maintenance mechanisms (TMM) include the re-expression of telomerase or alternative lengthening of telomeres (ALT). ALT is genetically associated with somatic alterations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes. Cells displaying ALT present distinctive features including C-circles made of telomeric DNA, long and heterogenous telomeric tracts, and telomeric DNA co-localized with promyelocytic leukemia (PML) bodies forming so-called ALT-associated PML bodies (APBs). Here, we identified mutations in ATRX and/or DAXX genes in an extensive collection of CRC samples including 119 patient-derived organoids (PDOs) and 232 established CRC cell lines. C-circles measured in CRC PDOs and cell lines showed low levels overall. We also observed that CRC PDOs and cell lines did not display a significant accumulation of APBs or long telomeres with no appreciable differences between wild-type and mutated ATRX/DAXX samples. Overall, our extensive analyses indicate that CRC is not prone to engage ALT, even when carrying genetic lesions in ATRX and/or DAXX, and support the notion that ATRX/DAXX genomic footprints are not reliable predictors of ALT.
Assuntos
Neoplasias Colorretais , Deficiência Intelectual , Telomerase , Talassemia alfa , Humanos , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , Homeostase do Telômero/genética , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Telomerase/genética , Telomerase/metabolismo , Mutação/genética , Linhagem Celular , Telômero/genética , Telômero/metabolismo , Organoides/metabolismo , Neoplasias Colorretais/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMO
A major challenge in the development of a gene therapy for hemophilia A (HA) is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells (LSECs) are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets-1/Est-2 combination, while Ets2 alone was ineffective. Moreover, Ets-1/Ets-2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carrying Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of GFP or FVIII cassettes driven by the shortened promoters led to GFP expression mainly in endothelial cells in the liver and to long-term FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in HA gene therapy.