Supplementary Materialsijms-20-01230-s001

Supplementary Materialsijms-20-01230-s001. proven. 2.4. An Additional TP53 Mutation Emerged Asunaprevir (BMS-650032) in MV4-11-R The wild-type p53 protein functions as a tumor suppressor to promote cell senescence and trigger apoptosis; however, we observed higher amounts of p53 protein in MV4-11-R. Mutations in the gene were shown to correlate with the growth-inhibitory potency of chemotherapeutic drugs in a number of malignancy cell lines, including leukemia cell lines [20,21]. We analyzed the gene sequence in MV4-11-P, showing that it is mutated at codon 248 from CGG (arginine) to UGG (tryptophan), designated as the R248W mutation. Asunaprevir (BMS-650032) In MV4-11-R, we detected another point mutation at codon 281 from GAC (aspartic acid) to GGC (glycine), designated as the D281G mutation (Physique 5A), in addition to the R248W mutation. Pyrosequencing analysis revealed that the percentage of D281G mutant alleles increased from 1% to 41% during the transition of MV4-11-P to MV4-11-R, while the percentage of R248W mutant alleles only slightly shifted from 54% to 65% (Physique 5B). Further cloning analysis verified that most D281G alleles were from wildtype R248 alleles, resulting in only 13.3% wild-type alleles remained in MV4-11-R cells against 43.5% wild-type alleles in MV4-11-P cells. This suggests that a cell populace harboring the D281G mutation emerged in the MV4-11-R collection, and the reduction in wild-type p53 resulted in a growth benefit in comparison to MV4-11-P cells. Open up in another home window Body 5 Sequencing analyses from the introduction end up being uncovered with the gene of a fresh mutation, D281G, in MV4-11-R. (A) The R248W (CGG TGG, crimson body) mutation was discovered in MV4-11-P, while both R248W and D281G (GAC GGC, crimson body) mutations had been seen in MV4-11-R using Sanger sequencing evaluation. (B) The percentage of mutant antisense-alleles for D281G and R248W mutations in MV4-11-P and MV4-11-R was dependant on pyrosequencing. To reply whether mutations keep company with cytarabine level of resistance, we compared position among cell lines in the National Asunaprevir (BMS-650032) Cancers institute-60 (NCI-60) -panel and their IC50 data for cytarabine from online data source CancerDR [22,23]. It demonstrated that cell lines bearing mutations generally have higher IC50 of cytarabine (Supplementary Body S3, Supplementary Desk S2). Using data from Genomics of Medication Sensitivity in Cancers [24], a feasible link was noticed Asunaprevir (BMS-650032) between mutations and elevated cytarabine level of resistance from data of 876 cancers cell lines (= 0.0321), though it is not thought as a significant relationship because of high false discovery rate (FDR%) (Supplementary Table S3). These data further support that this emergence of a mutation in MV4-11-R may contribute to cytarabine resistance. 2.5. Examination of the Cytarabine Metabolic Pathway and Multidrug Resistance Genes in MV4-11-R We assessed whether transporters and enzymes in the cytarabine metabolic pathway are involved in cytarabine resistance in MV4-11-R. Our qPCR results showed that there are no significant differences in the mRNA expression of between MV4-11-P and MV4-11-R. We also examined the expression of ATP-binding cassette transporters such as multidrug resistance 1 (and between MV4-11-P and MV4-11-R. 2.6. Cabozantinib Effectively Inhibits Tumorigenic Features of MV4-11-P and MV4-11-R Both In Vitro and In Vivo We further tested the responses of MV4-11-P and MV4-11-R to a number of anti-cancer drugs. MV4-11-P and MV4-11-R cells showed similar sensitivity to cabozantinib (a multi-kinase inhibitor), sorafenib (a multi-kinase inhibitor), and MK2206 (an Akt inhibitor) (Physique 6ACC). On the other hand, MV4-11-R was less sensitive than MV4-11-P to CI-1040 (a MEK inhibitor) or idarubicin; the IC50 values ENG for both drugs in MV4-11-R were approximately five-fold higher than those in MV4-11-P (Physique 6D,E). Further examination of ERK and Akt.