Carnosol, a diterpene present in rosemary, increases ELP1 levels in familial dysautonomia patient-derived cells and healthy adults: a possible therapy for FD.

Recent research on familial dysautonomia (FD) has focused on the development of therapeutics that facilitate the production of the correctly spliced, exon 20-containing, transcript in cells and individuals bearing the splice-altering, FD-causing mutation in the elongator acetyltransferase complex subunit I (ELP1) gene. We report here the ability of carnosol, a diterpene present in plant species of the Lamiaceae family, including rosemary, to enhance the cellular presence of the correctly spliced ELP1 transcript in FD patient-derived fibroblasts by upregulating transcription of the ELP1 gene and correcting the aberrant splicing of the ELP1 transcript. Carnosol treatment also elevates the level of the RNA binding motif protein 24 (RBM24) and RNA binding motif protein 38 (RBM38) proteins, two multifunctional RNA-binding proteins. Transfection-mediated expression of either of these RNA binding motif (RBMs) facilitates the inclusion of exon 20 sequence into the transcript generated from a minigene-bearing ELP1 genomic sequence containing the FD-causing mutation. Suppression of the carnosol-mediated induction of either of these RBMs, using targeting siRNAs, limited the carnosol-mediated inclusion of the ELP1 exon 20 sequence. Carnosol treatment of FD patient peripheral blood mononuclear cells facilitates the inclusion of exon 20 into the ELP1 transcript. The increased levels of the ELP1 and RBM38 transcripts and the alternative splicing of the sirtuin 2 (SIRT2) transcript, a sentinel for exon 20 inclusion in the FD-derived ELP1 transcript, are observed in RNA isolated from whole blood of healthy adults following the ingestion of carnosol-containing rosemary extract. These findings and the excellent safety profile of rosemary together justify an expedited clinical study of the impact of carnosol on the FD patient population.

CPF impedes cell cycle re-entry of quiescent lung cancer cells through transcriptional suppression of FACT and c-MYC.

Blockade of cell cycle re-entry in quiescent cancer cells is a strategy to prevent cancer progression and recurrence. We investigated the action and mode of action of CPF mixture (Coptis chinensis, Pinellia ternata and Fructus trichosanthis) in impeding a proliferative switch in quiescent lung cancer cells. The results indicated that CPF impeded cell cycle re-entry in quiescent lung cancer cells by reduction of FACT and c-MYC mRNA and protein levels, with concomitant decrease in H3K4 tri-methylation and RNA polymerase II occupancy at FACT and c-MYC promoter regions. Animals implanted with quiescent cancer cells that had been exposed to CPF had reduced tumour volume/weight. Thus, CPF suppresses proliferative switching through transcriptional suppression of FACT and the c-MYC, providing a new insight into therapeutic target and intervention method in impeding cancer recurrence.

Development of a Screening Platform to Identify Small Molecules That Modify ELP1 Pre-mRNA Splicing in Familial Dysautonomia.

Familial dysautonomia (FD) is an autonomic and sensory neuropathy caused by a mutation in the splice donor site of intron 20 of the ELP1 gene. Variable skipping of exon 20 leads to a tissue-specific reduction in the level of ELP1 protein. We have shown that the plant cytokinin kinetin is able to increase cellular ELP1 protein levels in vivo and in vitro through correction of ELP1 splicing. Studies in FD patients determined that kinetin is not a practical therapy due to low potency and rapid elimination. To identify molecules with improved potency and efficacy, we developed a cell-based luciferase splicing assay by inserting renilla (Rluc) and firefly (Fluc) luciferase reporters into our previously well-characterized ELP1 minigene construct. Evaluation of the Fluc/Rluc signal ratio enables a fast and accurate way to measure exon 20 inclusion. Further, we developed a secondary assay that measures ELP1 splicing in FD patient-derived fibroblasts. Here we demonstrate the quality and reproducibility of our screening method. Development and implementation of this screening platform has allowed us to efficiently screen for new compounds that robustly and specifically enhance ELP1 pre-mRNA splicing.

Concordant and opposite roles of DNA-PK and the "facilitator of chromatin transcription" (FACT) in DNA repair, apoptosis and necrosis after cisplatin.

BACKGROUND: Platinum-containing chemotherapy produces specific DNA damage and is used to treat several human solid tumors. Tumors initially sensitive to platinum-based drugs frequently become resistant. Inhibition of DNA repair is a potential strategy to enhance cisplatin effectiveness. After cisplatin treatment, a balance between repair and apoptosis determines whether cancer cells proliferate or die. DNA-dependent protein kinase (DNA-PK) binds to DNA double strand breaks (DSBs) through its Ku subunits and initiates non-homologous end joining. Inhibition of DNA-PK sensitizes cancer cells to cisplatin killing. The goal of this study is to elucidate the mechanism underlying the effects of DNA-PK on cisplatin sensitivity. RESULTS: Silencing the expression of the catalytic subunit of DNA-PK (DNA-PKcs) increased sensitivity to cisplatin and decreased the appearance of γH2AX after cisplatin treatment. We purified DNA-PK by its Ku86 subunit and identified interactors by tandem mass spectrometry before and after cisplatin treatment. The structure specific recognition protein 1 (SSRP1), Spt16 and γH2AX appeared in the Ku86 complex 5 hours after cisplatin treatment. SSRP1 and Spt16 form the facilitator of chromatin transcription (FACT). The cisplatin-induced association of FACT with Ku86 and γH2AX was abrogated by DNase treatment. In living cells, SSRP1 and Ku86 were recruited at sites of DSBs induced by laser beams. Silencing SSRP1 expression increased sensitivity to cisplatin and decreased γH2AX appearance. However, while silencing SSRP1 in cisplatin-treated cells increased both apoptosis and necrosis, DNA-PKcs silencing, in contrast, favored necrosis over apoptosis. CONCLUSIONS: DNA-PK and FACT both play roles in DNA repair. Therefore both are putative targets for therapeutic inhibition. Since DNA-PK regulates apoptosis, silencing DNA-PKcs redirects cells treated with cisplatin toward necrosis. Silencing FACT however, allows both apoptosis and necrosis. Targeting DNA repair in cancer patients may have different therapeutic effects depending upon the roles played by factors targeted.