DNA content was studied by staining with PI after ethanol fixation

DNA content was studied by staining with PI after ethanol fixation. of CRCs, in particular those lacking functional p53. Introduction Human colorectal cancer (CRC) is one of the most common cancers worldwide, with 1.2 million new cases annually diagnosed1. CRC often starts from premalignant lesions in the intestinal epithelium, that acquire mutations in tumor suppressor genes, including APC, SMAD4 and TP53, which consequently lead to malignant transformation2, 3. In spite of recent considerable advances in understanding of the molecular basis of CRC, metastatic and recurrent CRCs are still largely incurable4. Among the highly mutated genes in CRC is TP53, the guardian of the genome, that regulates many vital cellular processes, including DNA repair, apoptosis, cell cycle arrest and metabolism5. Expression of p53 is tightly controlled through the formation of complexes with the E3 ligases MDM2 and MDM4 and consequent degradation in a ubiquitin-proteasome dependent manner6. Missense mutations in the TP53 gene lead to either loss of anti-tumor or gain of novel oncogenic activity, Y16 which is associated with both drug resistance and tumor exacerbation7, 8. Genetic analysis of p53 mutations revealed that the GC-? ?AT transition of CpG dinucleotides at codons 175, 248 and 2739 and deletion induced by hemizygous loss at the 17p chromosomal region are two frequent types of mutations. Thus, a tremendous effort has been put to restore the wild-type function of p53. The transcription factor p73 belongs to the p53 family of proteins and exists in at least 14 different isoforms, arising from two independent promoters on the TP73 gene and further alternative splicing of the transcripts10. The transactivation (TA) domain containing TAp73 and the amino-terminal domain-deleted Np73 represent two major isoforms. The overall biological outcome of the p73 protein seems to be highly dependent to the relative expression of these two isoforms with TAp73 being Rabbit polyclonal to AHCYL1 pro-apoptotic and Np73 being a potential oncogene that counteracts the tumor suppressor activity of both TAp73 and p5310C12. On the other hand, bortezomib, also known as Velcade or PS-341, is a bronic dipeptide proteasome inhibitor, and the first of its class to receive FDA approval for the treatment of multiple myeloma. The drug has also shown potent inhibition of tumor cell growth and progression at IC50 values down to the nanomolar range in a wide spectrum of malignancy models including breast, prostate, lung and liver cancer, as well as CRC13C16. Clinically, with regards to multiple myeloma, the drug demonstrated remarkable efficacy Y16 and relatively few side effects17, 18, however resistance emerges in the majority of patients receiving it17. The most well characterized mechanism of bortezomib-induced cell death is the inhibition of the proteolytic activity of the 26S proteasome, which comprises two outer 19S regulatory complexes and one inner 20S core particle13, 14. The role of p53 in proteasome inhibitor-mediated apoptosis is controversial. Studies have shown that p53 is required for inducing apoptosis in LNCaP18, KIM-219, TT20 and FRO cells20 in response to proteasome inhibition, but not in HeLa21, DHL22 and PC-3 cells23. Therefore, the precise molecular mechanism of bortezomib appears to be cancer type-dependent. Although previous results showed potent anti-proliferative effects of bortezomib in HCT116 cells, the impact of p53 on these effects is still controversial24C28. In our initial experiment, we carefully re-evaluated bortezomibs anti-proliferative activity in HCT116 wt (wild-type) and p53?/? cells under different experimental conditions. We observed transient resistance in p53?/? cells to bortezomib after 24 hrs of treatment, which was diminished upon long-term treatments. Studying the molecular mechanism revealed the essential role of TAp73, a transcriptionally active isoform of the p53-homologue, p73, in inducing apoptosis in p53-deficient cells, but not in wt. Knocking down p73 by a CRISPR/Cas9 plasmid in HCT116 p53?/? cells or a p73 siRNA in HT-29 carrying mutated p53 significantly enhanced the resistance to bortezomib, confirming the anti-tumorigenic role Y16 of TAp73 in cells lacking functional p53. Results Transient resistance to bortezomib in HCT116 p53?/? cells Previous reports have shown contradicting results regarding the resistance of HCT116 p53?/? cells to bortezomib24C28. To address this controversy, we re-evaluated the anti-proliferative effect of bortezomib in HCT116 wt and p53?/? cells at three seeding densities,.

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