Glutathione determines chronic myeloid leukemia vulnerability to an inhibitor of CMPK and TMPK.

Bcr-Abl transformation leads to chronic myeloid leukemia (CML). The acquirement of T315I mutation causes tyrosine kinase inhibitors (TKI) resistance. This study develops a compound, JMF4073, inhibiting thymidylate (TMP) and cytidylate (CMP) kinases, aiming for a new therapy against TKI-resistant CML. In vitro and in vivo treatment of JMF4073 eliminates WT-Bcr-Abl-32D CML cells. However, T315I-Bcr-Abl-32D cells are less vulnerable to JMF4073. Evidence is presented that ATF4-mediated upregulation of GSH causes T315I-Bcr-Abl-32D cells to be less sensitive to JMF4073. Reducing GSH biosynthesis generates replication stress in T315I-Bcr-Abl-32D cells that require dTTP/dCTP synthesis for survival, thus enabling JMF4073 susceptibility. It further shows that the levels of ATF4 and GSH in several human CML blast-crisis cell lines are inversely correlated with JMF4073 sensitivity, and the combinatory treatment of JMF4073 with GSH reducing agent leads to synthetic lethality in these CML blast-crisis lines. Altogether, the investigation indicates an alternative option in CML therapy.

A Convenient and Sensitive Method for Deoxynucleoside Triphosphate Quantification by the Combination of Rolling Circle Amplification and Quantitative Polymerase Chain Reaction.

Measurement of four dNTP pools is important for investigating metabolism, genome stability, and drug action. In this report, we developed a two-step method for quantitating dNTPs by the combination of rolling circle amplification (RCA) and quantitative polymerase chain reaction (qPCR). We used CircLigase to generate a single-strand DNA in circular monomeric configuration, which was then used for the first step of RCA reaction that contained three dNTPs in excess for quantification of one dNTP at limiting levels. The second step is the amplification of RCA products by qPCR, in which one primer was designed to be completely annealed with the polymeric ssDNA product but not the monomeric template DNA. Using 1 amol of the template in the assay, each dNTP from 0.02 to 2.5 pmol gave a linearity with r2 > 0.99, and the quantification was not affected by the presence of rNTPs. We further found that the preparation of biological samples for the RCA reaction required methanol and chloroform extraction. The method was so sensitive that 1 × 104 cells were sufficient for dNTP quantification with the results similar to those determined by a radio-isotope method using 2 × 105 cells. Thus, the RCA/qPCR method is convenient, cost-effective, and highly sensitive for dNTP quantification.

Regulation of adult human mesenchymal stem cells into osteogenic and chondrogenic lineages by different bioreactor systems.

The aim of this study was to examine the feasibility of expanding and regulating mesenchymal stem cells (MSCs) from isolated adult human bone marrow mononuclear cells, seeded on gelatin-hyaluronic acid biomatrices, and then to quantitatively compare the gene expression in three different culture systems. Individual and interactive effects of model system parameters on construct structure, function, and molecular properties were evaluated. The results showed that these adult human MSCs even at old age not only expressed primitive mesenchymal cell markers but also maintained a high level of colony-forming efficiency and were capable of differentiating into osteoblasts, chondrocytes, and adipocytes upon appropriate inductions. After 21 days of culture, we found that the osteoblastic and chondrocytic lineage gene expression were earlier and higher expressed in spinner flask bioreactor culture group when compared with the static culture and rotating wall vessel reactor culture. The osteogenic lineage proteins type I collagen, alkaline phosphatase, and osteocalcin were strongly stained in histological sections of spinner flask bioreactor culture, whereas these were less detected in the other two groups, especially in rotating wall vessel reactor culture. As for the markers associated with the chondrogenic lineage differentiation proteins, type II collagen was apparently expressed in spinner flask culture group, while the expression of proteoglycans (aggreacan, decorin) in three culture conditions took the lead of each other. We conclude that the spinner flask bioreactor with appropriate induction medium reported in this study may be used to rapidly expand adult MSCs and is likely to possess better induction results toward osteoblastic and chondrocytic lineages.