High UDG and BRCA1 expression is associated with adverse outcome in patients with pemetrexed treated non-small cell lung Cancer.

OBJECTIVE: The antifolate chemotherapy agent pemetrexed has been widely used to treat non-small-cell-lung-cancer (NSCLC), but there is no clinically validated biomarker to select patients likely to respond. The aim of this study was to assess two proteins involved in DNA repair mechanisms, uracil DNA glycosylase (UDG) and BRCA1 as potential prognostic biomarkers in NSCLC patients treated with pemetrexed-based chemotherapy. MATERIAL AND METHODS: Formalin-fixed-paraffin-embedded tumor specimens from 119 patients with advanced NSCLC treated with pemetrexed between 2004 and 2011 were retrospectively analyzed. Expression of UDG, BRCA1, and known prognostic factors ALK, TTF-1, thymidylate synthase and folylpolyglutamate synthase was assessed by immunohistochemistry using H-SCORE (product of percent stained cells and intensity of expression). Progression-free (PFS) and overall survival (OS) served as reference endpoint. RESULTS: Most NSCLC tumor samples had UDG positivity in at least 5% of tumor cells and 34% samples had more than 50% positive tumor cells. Using the median expression value as threshold, high UDG expression (H-SCORE≥75) was significantly associated with shorter median PFS (3-year PFS 7% vs. 37%, p = 0.045) and a trend for shorter OS (3-year OS 15% vs 42%, p = 0.066) compared to patients with low UDG. In multivariable Cox analysis, the association between high UDG and shorter PFS was close to statistically significant (p = 0.08) at a significance level of 0.05 after controlling for age, gender, ALK- and TTF1-status with hazard ratio of 2.1. Grouping patients according to combined UDG and BRCA1 expression, patients with a profile of UDGhigh/BRCA1high had the shortest PFS and OS compared to all other patient groups (p = 0.007 and 0.02, respectively). CONCLUSION: Our results demonstrate an important prognostic role for high UDG expression in pemetrexed-treated NSCLC patients, in addition to its previously reported role in pemetrexed cytotoxicity. High UDG expression was predictive of shorter PFS and OS, and patients with a combined profile of UDGhigh/BRCA1high had the poorest outcome following pemetrexed treatment.

Expression and recruitment of uracil-DNA glycosylase are regulated by E2A during antibody diversification.

B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID), which is positively regulated by the transcription factor E2A, is the key enzyme that initiates these processes by deaminating cytosine to uracil in Ig genes. Nuclear uracil-DNA glycosylase (UNG2) is subsequently required for uracil processing in the generation of high affinity antibodies of different isotypes. Here we show that the transcription factor E2A binds to the UNG2 promoter and represses UNG2 expression. Inhibition of E2A by binding of Ca(2+)-activated calmodulin alleviates this repression. Furthermore, we demonstrate that UNG2 preferentially accumulates in regions of the Ig heavy chain (IgH) gene containing AID hotspots. Calmodulin inhibition of E2A strongly enhances this UNG2 accumulation, indicating that it is negatively regulated by E2A as well. We show also that over-expression of E2A can suppress class switch recombination. The results suggest that E2A is a key factor in regulating the balance between AID and UNG2, both at expression and Ig targeting levels, to stimulate Ig diversification and suppress normal DNA repair processes.

Uracil-DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed.

Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthase. The base excision repair (BER) enzyme uracil-DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG(-/-) human colon cancer cells. Here, we examined the relationship between UNG expression and pemetrexed sensitivity in human lung cancer. We observed a spectrum of UNG expression in human lung cancer cells. Higher levels of UNG are associated with pemetrexed resistance and are present in cell lines derived from pemetrexed-resistant histologic subtypes (small cell and squamous cell carcinoma). Acute pemetrexed exposure induces UNG protein and mRNA, consistent with upregulation of uracil-DNA repair machinery. Chronic exposure of H1299 adenocarcinoma cells to increasing pemetrexed concentrations established drug-resistant sublines. Significant induction of UNG protein confirmed upregulation of BER as a feature of acquired pemetrexed resistance. Cotreatment with the BER inhibitor methoxyamine overrides pemetrexed resistance in chronically exposed cells, underscoring the use of BER-directed therapeutics to offset acquired drug resistance. Expression of UNG-directed siRNA and shRNA enhanced sensitivity in A549 and H1975 cells, and in drug-resistant sublines, confirming that UNG upregulation is protective. In human lung cancer, UNG deficiency is associated with pemetrexed-induced retention of uracil in DNA that destabilizes DNA replication forks resulting in DNA double-strand breaks and cell death. Thus, in experimental models, UNG is a critical mediator of pemetrexed sensitivity that warrants evaluation to determine clinical value.

Positive and negative regulation in the promoter of the ORF46 gene of Kaposi's sarcoma-associated herpesvirus.

The ORF46 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes uracil DNA glycosylase, an enzyme involved in DNA repair. In this study, we show that the transcriptional start site of the ORF46 gene is located at nucleotide 69,425 of the viral genome and ORF50 protein, a latent-lytic switch transactivator, activates the ORF46 promoter via RBP-Jκ protein. Three consensus RBP-Jκ-binding sites found in the ORF46 promoter are critical for the binding of RBP-Jκ protein and conferring the ORF50 responsiveness. In addition, a negative regulatory region has been determined in the ORF46 promoter, which mediates the suppression of the ORF50 responsiveness. The functional negative region of the ORF46 promoter is mainly composed of the Sp1-binding sites. Like the negative region of the ORF46 promoter, addition of Sp1-binding sequences alone in an ORF50-responsive promoter efficiently confers the suppression of the ORF50 responsiveness. Furthermore, sodium butyrate, a pleiotropic inducing agent for the KSHV lytic cycle, is able to relieve the negative regulation of the ORF46 promoter in the latently KSHV-infected cells. The identification of multiple positive and negative cis-acting regulatory elements in the viral promoters emphasizes the elaborate controls in the KSHV lytic cycle, which ensure the adequate expression of each viral lytic gene.

Folate deficiency increases mtDNA and D-1 mtDNA deletion in aged brain of mice lacking uracil-DNA glycosylase.

Strong epidemiological and experimental evidence links folate deficiency and resultant hyperhomocysteinemia with cognitive decline and neurodegeneration. Here, we tested the hypothesis that uracil misincorporation contributes to mitochondrial pathology in aged brain following folate deprivation. In a 2 × 2 design, 14-month-old mice lacking uracil DNA glycosylase (Ung-/-) versus wild-type controls were subjected to a folate-deficient versus a regular diet for six weeks. Folate-deficient feeding significantly enhanced mtDNA content and overall abundance of the D-1 mtDNA deletion in brain of Ung-/-, but not of wild-type mice. Independent of folate status, the frequency of the D-1 mtDNA deletion in mtDNA was significantly increased in Ung-/- mice. The rate of mitochondrial biogenesis as assessed at six weeks of the experimental diet by mRNA expression levels of transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and of mitochondrial transcription factor A (Tfam) was not affected by either Ung-/- genotype or short-term folate deficiency. Similarly, citrate synthase (CS) activity in the brain did not differ across experimental groups. By contrast, independent of genotype, lactate dehydrogenase (LDH) activity was significantly reduced in folate-deficient animals. Our results suggest that impaired uracil excision repair causes an increase in mitochondrial mutagenesis in aged brain along with a compensatory increase in mtDNA content in response to low folate status. Folate deficiency may contribute to neurodegeneration via mtDNA damage.

5-Fluorouracil incorporated into DNA is excised by the Smug1 DNA glycosylase to reduce drug cytotoxicity.

5-Fluorouracil (FU) has been widely used for more than four decades in the treatment of a range of common cancers. The fluorine-substituted uracil analogue is converted to several active metabolites but the mechanism of cytotoxicity has remained unclear. In a widely cited but unsubstantiated model, FU is thought to kill cells via the inhibition of thymidylate synthase and increased use of dUTP in place of TTP during DNA replication, with subsequent excision of high levels of uracil causing the fragmentation of newly synthesized DNA. Using gene-targeted cell lines defective in one or both of the two mammalian uracil-DNA glycosylase repair enzymes, we were able to test this model of FU cytotoxicity. Here, we show that incorporation of FU itself into DNA has been previously underestimated and is a predominant cause of cytotoxicity. FU readily becomes incorporated into the DNA of drug-treated cells, and accumulation of FU in the genome, rather than uracil excision, is correlated with FU cytotoxicity in mammalian cells. Furthermore, the Smug1, but not the Ung, uracil-DNA glycosylase excises FU from DNA and protects against cell killing. The data provides a clearer understanding of the action of FU, suggesting predictive biomarkers of drug response and a mechanism for acquired resistance in tumors.

Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor.

Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 A, beta = 109.7 degrees. X-ray data from the crystal have been collected for structure analysis.