Quantitative Analysis Of Histone Post-translational Modification And Its Application In The Experimental Study Of Leukemia Cell Multi-drug Resistance Mechanisms

PurposeLeukemia is a devastating desease with the higher mortality. Now, therapeutic approachesfor leukemia may include chemotherapy, radiation therapy, bone marrow transplantation orimmunotherapy, even the traditional chinese medicine. However, chemotherapy, as a front-line therapy, has significant limitations with respect to the development of drug resistance.Mechanisms leading to primary resistance remain to be clarified.Resistance to chemotherapydrugs, such as doxorubicin, results from a variety of factors including over-expression ofmultiple genes during tumor progression, especially the amplification of multi-drugresistance gene mdr1and the overexpression of its encoding protein P-glycoprotein (Pgp).While epigenetic mechanisms, consisting of DNA base modifications and histonemodification show more and more critical importance in chronic remodeling disorders suchas drug resistance. At present, immunoprecipitation, immunoblotting andimmunofluorescence still dominant the research methods of histone Post-translationalmodifications. These methods mostly depend on highly specific antibodies targeting to theknowned modification sites, however, because of the big variety of post-translationalmodifications, it has been becoming more and more difficult to develop such antibodies.Furthermore, there are multiple non-specific interference in immunoassays, and Semi-quantitative can only be done by Western blot. Thus it is particularly important to developnew methods with more accuracy and ease of use.Here we have established the Ultra Performance Liquid Chromatography Electro SprayIonization Quadrupole Time of Flight mass spectrometry quantitative (UPLC-ESI-QTof MS)method to develope a new technique for the accurate quantification of histoneposttranslational modification in doxorubicin-resistant leukemia cell lines, and assessed theadvantages and disadvantages of the different sample processing propionylated stages. Wehave also compared the histone post-translational modification between leukemia cells andits drug-resistant cells, found out a number of potential sites whitch may affect doxorubicinresistance. These experiments might provide some approach for cancer Multi-DrugResistance Mechanisms investigation. MethodsThe cytotoxicity of doxorubicin toward HL60, HL60/ADR,K562and K562/ADRcell lines was measured using Cell Counting Kit-8assay.Synchronous cell cycles were induced by serum starvation to make the histonemodification comparable and to reduce experimental error.The histones were acid extracted and resolved by16%Tricine-SDS–PAGE, thenanalyzed at the intact protein level by LC/MS.Histones which were separated by Reverse-Phase High-Performance LiquidChromatography were identified by LC/MS at intact protein and peptide mappinglevel.The histone H3and H4of Leukemia cells HL60、K562and drug-resistantLeukemia cells HL60/ADR、K562/ADR were quantified by methylation stableisotope labeling of carboxylic acid groups.The histone H3and H4of Leukemia cells HL60、K562and drug-resistantLeukemia cells HL60/ADR、 K562/ADR were quantified by using propionicanhydride and methylation stable isotope to label the specific groups separatelyafter trypsin digestion.The propionylated histone H3and H4of Leukemia cells HL60、K562and drug-resistant Leukemia cells HL60/ADR、 K562/ADR were quantified by usingpropionic anhydride and methylation stable isotope to label the specific groupsafter typsin digestion.ResultsTheIC50ofdoxorubicininleukemiacellsHL60、K562wasroughly776timesand703times lower than HL60/ADR、K562/ADR cells respectively, which wasconfirmed in HL60/ADR, K562/ADR cells.The cell cycle is well synchronised by Serum starvation, and it was confirmedthat the histone extracted by acid with high purity and theoretical molecular weightby Tricine-SDS-PAGE electrophoresis analysis.It was comfirmed that the post-translational modification of histone changeddramatically both in leukemia HL60, K562and its resistant cells HL60/ADR byLC/MS at intact protein level. The histones were successfully separated and the histones including H2B, H4,H2A.Z, H2A1, H2A2, H2A.X, H3.1, H3.2, H3.3were identified by LC/MS/MS.Appraisal method for evaluation:1. Standard peptide was used to identify thehigh specific modification sites in lysine propionylation and methyl esterificationreaction, the modified ratio was up to95%;2. Propionylation was more suitablefor using RP-UPLC-ESI-QTof quantitative analysis of histone PTM,which canextend not only the retention time of small peptides, but also the peptideshomogenicity after trypsin digestion;3. The quantification of some peptides maybe affect by propionylation, such as histone H4peptide GKGGKGLGKGGAKR.It is confirmed that the acetylation level of histone H3and H4of HL60/ADRand K562/ADR cells and the H4K9and H4K20three methylation level wereincreased obviously by using the above three different quantitative methods. Theseresults show that LC/MS is expected to become an effective approach to quantifythe histone modification of leukemia cells, which may provide an effectiveapproach for screening treatment resistant cases and guiding clinical treatments.