SaOs 2 and U2OS cells necessary greater doses. Right here, a clear lower from the relative number of viable cells was only reached past ten mM. The LD50 for U2OS was concerning ten and thirty mM and for SaOs two among 30 and 60 mM. In all scenarios, we observed a slightly greater sensitivity of p53 replete cells than of p53 deficient cells, notably at lower doses. Whenever we treated U2OS and HCT116 cells with all the p53 inhibitor pifithrin a, we also observed a slightly lowered sensitivity, signifying a contribution of p53 to LiCl mediated cell death. Having said that, whenever we knocked p53 down by siRNA, we found at most effective a slightly greater survival in the presence of p53, supporting the notion that p53 isn’t an impor tant mediator of LiCl induced cell death.
When we investigated the colony forming capability of HCT116 cells, we observed a somewhat increased sensitivity for LiCl than found with the MTT assay. Here, prolif eration of p53 replete cells was previously somewhat reduced at 1 mM LiCl, whereas p53 deficient cells demanded chk2 inhibitor at the very least three mM LiCl for inhibition of colony forming capability. To investigate if the reduction in proliferation in response to LiCl is likely to be as a consequence of inhibition of GSK three, we repeated these experiments with one other inhibitor of GSK three, alsterpaullone. Right here, proliferation of HCT116 was by now reduced at a dose of 0. three uM alsterpaullone when the other cell lines expected not less than one uM from the drug for development suppression. LD50s had been at about 3 uM for MEFs, concerning 0. six and 1 uM for HCT116 and concerning one and 3 uM for the two osteo sarcoma cell lines.
We upcoming examined cell proliferation just after treating the cells for various days with LiCl or alsterpaullone at about the LD50 dose. For MEFs and HCT116 cells, we saw a continuous raise in cell number with time. This enhance was, though, considerably weaker during the presence of LiCl or alsterpaullone. For U2OS cells, we observed a slightly numerous image. Right here we found that after additional resources an preliminary improve in cell num ber, even inside the presence of LiCl or alsterpaullone, the amount of cells remained a lot more or much less frequent, or maybe declined. For SaOs 2, we observed a strong reduction in proliferation at first time points, but at later on time points inhibition of proliferation ceased. LiCl induces cell death in p53 constructive and p53 negative cells We subsequent investigated no matter if the lessen in prolifera tion soon after treatment of tumour cells with LiCl was due to the induction of cell death.
By performing FACS ana lysis, we observed both in p53 good and in p53 nega tive HCT116 cell lines a clear grow from the sub G1 peak starting up at 16h soon after LiCl addition and increasing thereafter. This maximize in the sub G1 peak was more prominent within the p53 proficent cell line. At the identical time, we observed a significant lower in G1 and S phase cells and an increase in G2 cells, which was transient within the case of p53 replete cells and persistent inside the case of p53 deficient cells.