MTH1 suppression enhances the stemness of MCF7 through upregulation of STAT3.

MTH1 protein can sanitize the damaged (d)NTP pool and MTH1 inhibitors have been developed to impede the growth of rapidly proliferating tumor cells; however, the effect of MTH1 inhibition on breast cancer stemness has not been reported yet. Here, we constructed breast cancer cell lines with the stable depletion of MTH1. MTH1 suppression clearly increased the ratio of CD44+CD24-/low subpopulations and promoted the formation of tumorspheres in MCF7 and T47D cells. RNA expression profiling, RT-qPCR and Western blotting showed the upregulation of master stem cell transcription factors Sox2, Oct4 and Nanog in MTH1 knockdown cells. GSEA suggested and Western blotting verified that MTH1 knockdown increased the expression of phosphorylated STAT3 (Tyr705). Furthermore, we indirectly demonstrated that the increased concentration of 8-oxo-dGTP and 8-oxo-GTP in MTH1-knockdown cells and exogenous 8-oxoGTP, rather than 8-oxo-dGTP, could significantly increase the phosphorylation of STAT3. In conclusion, this work indicates that MTH1 inhibition increased the proportion of breast cancer stem cells (BCSCs) and promoted stemness properties in MCF7 cells.

The high expression of MTH1 and NUDT5 promotes tumor metastasis and indicates a poor prognosis in patients with non-small-cell lung cancer.

MutT Homolog 1 (MTH1) is a mammalian 8-oxodGTPase for sanitizing oxidative damage to the nucleotide pool. Nudix type 5 (NUDT5) also sanitizes 8-oxodGDP in the nucleotide pool. The role of MTH1 and NUDT5 in non-small-cell lung cancer (NSCLC) progression and metastasis remains unclear. In the present study, we reported that MTH1 and NUDT5 were upregulated in NSCLC cell lines and tissues, and higher levels of MTH1 or NUDT5 were associated with tumor metastasis and a poor prognosis in patients with NSCLC. Their suppression also restrained tumor growth and lung metastasis in vivo and significantly inhibited NSCLC cell migration, invasion, cell proliferation and cell cycle progression while promoting apoptosis in vitro. The opposite effects were observed in vitro following MTH1 or NUDT5 rescue. In addition, the upregulation of MTH1 or NUDT5 enhanced the MAPK pathway and PI3K/AKT activity. Furthermore, MTH1 and NUDT5 induce epithelial-mesenchymal transition both in vitro and in vivo. These results highlight the essential role of MTH1 and NUDT5 in NSCLC tumor tumorigenesis and metastasis as well as their functions as valuable markers of the NSCLC prognosis and potential therapeutic targets.

An identification and functional evaluation of a novel CYP2C9 variant CYP2C9*62.

Warfarin is the most commonly used anticoagulant in the clinical treatment of thromboembolic diseases. The dose of warfarin varies significantly within populations, and the dose is closely related to the genetic polymorphisms of the CYP2C9 and VKORC1 genes. In this study, a new CYP2C9 nonsynonymous mutation (8576C > T) was detected after the genetic screening of 162 patients took warfarin. This mutation, named as the new allele CYP2C9*62, can result in an arginine to cysteine amino acid substitution at position 125 of the CYP2C9 protein (R125C). When expressed in insect cells, the protein expression of CYP2C9.62 was significantly lower than that of the wild-type, and its metabolic activity was also significantly decreased after the addition of three typical CYP2C9 probe drugs, suggesting that the new mutant can dramatically affect the metabolism of CYP2C9 drugs in vitro.

The Pol ß variant containing exon α is deficient in DNA polymerase but has full dRP lyase activity.

DNA polymerase (Pol) ß is a key enzyme in base excision repair (BER), an important repair system for maintaining genomic integrity. We previously reported the presence of a Pol ß transcript containing exon α (105-nucleotide) in normal and colon cancer cell lines. The transcript carried an insertion between exons VI and VII and was predicted to encode a ~42 kDa variant of the wild-type 39 kDa enzyme. However, little is known about the biochemical properties of the exon α-containing Pol ß (exon α Pol ß) variant. Here, we first obtained evidence indicating expression of the 42 kDa exon α Pol ß variant in mouse embryonic fibroblasts. The exon α Pol ß variant was then overexpressed in E. coli, purified, and characterized for its biochemical properties. Kinetic studies of exon α Pol ß revealed that it is deficient in DNA binding to gapped DNA, has strongly reduced polymerase activity and higher Km for dNTP during gap-filling. On the other hand, the 5'-dRP lyase activity of the exon α Pol ß variant is similar to that of wild-type Pol ß. These results indicate the exon α Pol ß variant is base excision repair deficient, but does conduct 5'-trimming of a dRP group at the gap margin. Understanding the biological implications of this Pol ß variant warrants further investigation.

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program.

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.

Effect of CYP2C9 Genetic Polymorphism in a Chinese Population on the Metabolism of Mestranol in vitro.

BACKGROUND: Mestranol is a widely used estrogen, which is converted into its active metabolite ethinyl estradiol by cytochrome P450 (CYP) 2C9. To comprehensively examine the enzymatic activity of reported CYP2C9 variants in Chinese individuals in response to mestranol, wild-type CYP2C9*1 and 35 allelic variants were highly expressed in Sf21 insect cell microsomes and used for the detection of their enzymatic values in vitro. These results showed that the majority of tested variants exhibited decreased clearance values compared to wild type, except for CYP2C9*40 and *36. METHOD: Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.25-8 µmol/l mestranol for 30 min at 37°C. Then, the production of the metabolite of mestranol, ethinyl estradiol, was analyzed using high-performance liquid chromatography. RESULTS: Most CYP-catalyzed reactions were sufficiently described by classical Michaelis-Menten kinetic parameters (e.g., Km and Vmax), while 9 variants exhibited atypical or non-Michaelis-Menten kinetic values, which were largely due to the self-inhibitory effect in response to mestranol. CONCLUSION: This is the first report of these rare alleles for mestranol metabolism, which provides fundamental data for further clinical studies on CYP2C9 alleles for mestranol metabolism.

Lowered nudix type 5 (NUDT5) expression leads to cell cycle retardation in HeLa cells.

The molecule 8-oxo-7,8-dihydroguanine (8-oxoGua), an oxidized form of guanine, can pair with adenine or cytosine during nucleic acid synthesis. Moreover, RNA containing 8-oxoGua causes translational errors, thus leading to the production of abnormal proteins. Human NUDT5, a MutT-related protein, catalyzes the hydrolysis of 8-oxoGDP to 8-oxoGMP, thereby preventing misincorporation of 8-oxoGua into RNA. To investigate the biological roles of NUDT5 in mammalian cells, we established cell lines with decreased level of NUDT5 expression. In NUDT5 inhibited cells, the RNA oxidation was not significantly higher than that of normal cells. However, the cell cycle G1 phase was significantly delayed, and cell numbers in both S and G2/M phases were reduced, indicating that cell proliferation was hampered by NUDT5 suppression. Key proteins for preventing the G1-S transition, including p53, p16, and Rb were increased, while the Rb phosphorylation was decreased. These results suggested that the NUDT5 protein may play significant roles in regulating the G1-S transition in mammalian cells.