Xu Tian

Detection of PIGO-Deficient Cells Using Proaerolysin: A Valuable Tool to Investigate Mechanisms of Mutagenesis in the DT40 Cell System

While isogenic DT40 cell lines deficient in DNA repair pathways are a great tool to understand the DNA damage response to genotoxic agents by a comparison of cell toxicity in mutants and parental DT40 cells, no convenient mutation assay for mutagens currently exists for this reverse-genetic system. Here we establish a proaerolysin (PA) selection-based mutation assay in DT40 cells to identify glycosylphosphatidylinositol (GPI)-anchor deficient cells. Using PA, we detected an increase in the number of PA-resistant DT40 cells exposed to MMS for 24 hours followed by a 5-day period of phenotype expression. GPI anchor synthesis is catalyzed by a series of phosphatidylinositol glycan complementation groups (PIGs). The PIG-O gene is on the sex chromosome (Chromosome Z) in chicken cells and is critical for GPI anchor synthesis at the intermediate step. Among all the mutations detected in the sequence levels observed in DT40 cells exposed to MMS at 100 µM, we identified that ∼55% of the mutations are located at A:T sites with a high frequency of A to T transversion mutations. In contrast, we observed no transition mutations out of 18 mutations. This novel assay for DT40 cells provides a valuable tool to investigate the mode of action of mutations caused by reactive agents using a series of isogenic mutant DT40 cells.

A purified MAA-based ELISA is a useful tool for determining anti-MAA antibody titer with high sensitivity

Atherosclerosis is widely accepted to be a chronic inflammatory disease, and the immunological response to the accumulation of LDL is believed to play a critical role in the development of this disease. 1,4-Dihydropyridine-type MAA-adducted LDL has been implicated in atherosclerosis. Here, we have demonstrated that pure MAA-modified residues can be chemically conjugated to large proteins without by-product contamination. Using this pure antigen, we established a purified MAA-ELISA, with which a marked increase in anti-MAA antibody titer was determined at a very early stage of atherosclerosis in 3-month ApoE-/- mice fed with a normal diet. Our methods of Nε-MAA-L-lysine purification and purified antigen-based ELISA will be easily applicable for biomarker-based detection of early stage atherosclerosis in patients, as well as for the development of an adduct-specific Liquid Chromatography/Mass Spectrometry-based quantification of physiological and pathological levels of MAA.

Evidence that endogenous formaldehyde produces immunogenic and atherogenic adduct epitopes

Endogenous formaldehyde is abundantly present in our bodies, at around 100 µM under normal conditions. While such high steady state levels of formaldehyde may be derived by enzymatic reactions including oxidative demethylation/deamination and myeloperoxidation, it is unclear whether endogenous formaldehyde can initiate and/or promote diseases in humans. Here, we show that fluorescent malondialdehyde-formaldehyde (M2FA)-lysine adducts are immunogenic without adjuvants in mice. Natural antibody titers against M2FA are elevated in atherosclerosis-prone mice. Staining with an antibody against M2FA demonstrated that M2FA is present in plaque found on the aortic valve of ApoE−/− mice. To mimic inflammation during atherogenesis, human myeloperoxidase was incubated with glycine, H2O2, malondialdehyde, and a lysine analog in PBS at a physiological temperature, which resulted in M2FA generation. These results strongly suggest that the 1,4-dihydropyridine-type of lysine adducts observed in atherosclerosis lesions are likely produced by endogenous formaldehyde and malondialdehyde with lysine. These highly fluorescent M2FA adducts may play important roles in human inflammatory and degenerative diseases.

Homologous recombination and translesion DNA synthesis play critical roles on tolerating DNA damage caused by trace levels of hexavalent chromium

Contamination of potentially carcinogenic hexavalent chromium (Cr(VI)) in the drinking water is a major public health concern worldwide. However, little information is available regarding the biological effects of a nanomoler amount of Cr(VI). Here, we investigated the genotoxic effects of Cr(VI) at nanomoler levels and their repair pathways. We found that DNA damage response analyzed based on differential toxicity of isogenic cells deficient in various DNA repair proteins is observed after a three-day incubation with K2CrO4 in REV1-deficient DT40 cells at 19.2 μg/L or higher as well as in TK6 cells deficient in polymerase delta subunit 3 (POLD3) at 9.8 μg/L or higher. The genotoxicity of Cr(VI) decreased ~3000 times when the incubation time was reduced from three days to ten minutes. TK mutation rate also significantly decreased from 6 day to 1 day exposure to Cr(VI). The DNA damage response analysis suggest that DNA repair pathways, including the homologous recombination and REV1- and POLD3-mediated error-prone translesion synthesis pathways, are critical for the cells to tolerate to DNA damage caused by trace amount of Cr(VI).

Abstract 5363: Genotoxicity of trace level of hexavalent chromium existing in city water

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tens of millions of Americans consume trace amounts of potentially toxic hexavalent chromium (Cr(VI)) through drinking city water every day. According to the studies conducted by the Environmental Working Group, 89% of the water samples from U.S. cities are contaminated with Cr(VI) at levels ranging from 0.03 to 12.9 μg/L. While trivalent chromium (Cr(III)) is among the essential trace elements for normal carbohydrate, lipid, and protein metabolism in humans, Cr(VI) has been extensively studied as a toxic heavy metal ion. Cr(VI) is a well-known human carcinogen by inhalation; in contrast, it is a rodent carcinogen by ingestion of extremely high doses (57,300 μg/L or higher) in drinking water. The United States Environmental Protection Agency (USEPA) and other regulatory agencies in the world have set the maximum contaminant levels (MCL) for total Cr or Cr(VI) in human drinking water at 50 or 100 μg/L, although the USEPA is currently re-evaluating these regulations. The mechanism by which Cr(VI) causes genotoxicity is well-characterized at concentrations equivalent to current regulatory levels or higher. The genotoxic mode of action of Cr(VI) appears to be due to the intracellular reduction of Cr(VI) to Cr(III) leading to the formation of a ternary complex between DNA, Cr(III), and intracellular reducing molecules. However, little information is available regarding the biological effects of Cr(VI) at doses lower than 100 μg/L. In this study, we investigated the effects of Cr(VI) at nanomolar ranges (1 μg/L or higher) on the DNA damage response in isogenic cultured cells deficient in various DNA repair genes. Here we show that the DNA damage response is observed after a three-day incubation with Cr(VI) at 2-5 μg/L (ppb), which is lower than the Cr(VI) concentrations detected in some of city water in the US. Strikingly, the genotoxicity of Cr(VI) decreased ∼3000 times depending on its incubation time, ranging from three days to ten minutes. Our dose- and time-dependent Cr(VI) genotoxicity results, combined with the estimated transit time of Cr(VI) in the digestive tracts of humans, strongly suggest that trace amounts of Cr(VI) in drinking water at levels 12.9 μg/L or lower are unlikely to cause a significant impact on inducing carcinogenicity in the oral cavity and small intestine. Citation Format: Xu Tian, Keyur Patel, John R. Ridpath, James A. Swenberg, Jun Nakamura. Genotoxicity of trace level of hexavalent chromium existing in city water. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5363. doi:10.1158/1538-7445.AM2014-5363

Abstract 1281: POLD3 is required for DNA damage response to endogenous and exogenous DNA damage in human cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Methyl methanesulfonate (MMS) induces mutations in a nonlinear (hockey-stick-shaped) dose-response curve in DT40 cells, about 55% of which are located at A:T sites with a high frequency of A to T mutations. We hypothesized that these transversion mutations are due to base excision repair (BER) intermediates, such as abasic sites and single strand breaks derived from N3-methyladenine. One candidate polymerase to bypass abasic sites is polymerase delta (POLD). POLD is responsible for the lagging strand during DNA replication. POLD is referred to as a high fidelity DNA polymerase because of its ability to discriminate deoxynucleotides and its 3’->5’ exo-nuclease activity. One of the POLD subunits, POLD3, is important to recruit PCNA binding to the POLD core enzyme and is implied to have critical roles for translesion DNA synthesis. In yeast, cells deficient in POLD3 homologous gene POL32 are sensitive to DNA damaging agents and resistant to mutations caused by mutagens, such as MMS and UV. Here we generated POLD3 knock-down (KD) cells via lentivirus based shRNA in three different human cell lines. All POLD3 KD cells grow slower than the control cells, suggesting POLD3 function tolerates endogenous DNA lesions. Furthermore, we found that POLD3 KD cells were sensitive to various genotoxic agents including MMS and potassium chromate. To further investigate the role of POLD3 in the DNA damage response, we introduced a series of deletion mutations of POLD3 in POLD3 deficient cells. We found that both the POLD2 binding domain and the middle linker domain are critical for the activity of POLD3 while the C-terminal PCNA binding domain is dispensable. While there is little homology between human POLD3 and yeast POL32, function of these two proteins appears to be quite similar in structure biology analysis. Our results also suggest that POLD3 plays important roles to bypass DNA lesions caused by endogenous and various exogenous genotoxic agents. Citation Format: Xu Tian, James Swenberg, Jun Nakamura. POLD3 is required for DNA damage response to endogenous and exogenous DNA damage in human cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1281. doi:10.1158/1538-7445.AM2013-1281

Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells

Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS.

Abstract 2410: Crucial role of the non-BRCT automodification domain of PARP1 in either DNA binding and PARP inhibitor-mediated cell toxicity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Poly(ADP-ribose)polymerase (PARP) inhibitor (PARPi)-mediated synthetic lethality is an attractive new paradigm for cancer therapy. While it has been recently proposed some of PARPi may cause DNA single strand break (SSB)-PARP1 or -PARP2 complexes, leading to cell toxicity, the mode of action of PARPi-mediated synthetic lethality is not well understood. We analyzed DNA damage response to PARPi using an isogenic set of DT40 cells. Both PARP-1 ko mutants and PARP-1 ko mutants expressing DNA bindin domain-defficient PARP1 showed markedly more resistance to olaparib than parental DT40 cells, indicating that PARP1 binding to DNA strand break through DNA binding domain is critical in olaparib-mediated cell toxicity. While non-BRCT automodomain domain deletion mutant showed a resistant phenotype, olaparib causes similar toxicity in BRCT domain deletion mutant and parental DT40 cells. Furthermore, PARP1 localized in chromatin binding fraction in BRCT1 deletion mutant, but not in non-BRCT automodomain domain deletion mutant. These results indicate that non-BRCT automodification domain plays crucial role for the formation of stable DNA SSB-PARP1 complex and olaparib-mediated synthetic lethality. Citation Format: Jun Nakamura, Xu Tian. Crucial role of the non-BRCT automodification domain of PARP1 in either DNA binding and PARP inhibitor-mediated cell toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2410. doi:10.1158/1538-7445.AM2014-2410

Abstract LB-151: REV1 has critical roles in base excision repair intermediate-mediated transversion mutations but not for O6-methylguanine-initiated mutations in vertebrate cells.

Isogenic chicken DT40 cells deficient in various DNA repair pathways are a great tool for understanding the DNA damage response and mutagenesis caused by genotoxic agents by a comparison of mutants and parental DT40 cells. We recently found that ~55% of PIGO mutations are located at A:T sites with a high frequency of A to T transversion mutations in DT40 cells exposed to methyl methanesulfonate (MMS). These results suggest that A to T transversion mutations found in MMS exposure-derived PIGO mutant cells are due to base excision repair intermediates, such as abasic sites and single strand breaks originated from N3-methyladenine. It has been reported that DNA repair protein REV1 acts as not only as a deoxycytidine transferase across abasic sites but also as a scaffold protein to coordinate translesion DNA synthesis (TLS) polymerase switching. To address effects of REV1 on MMS-induced PIGO mutations in DT40 cells, we exposed REV1 knockout DT40 cells to low-dose MMS. REV1 knockout DT40 cells showed hyper-sensitivity in cell survival after MMS treatment with no increase in PIG-O mutations. These results indicate that PIG-O mutations in DT40 cells exposed to MMS are caused through REV1-dependent, but REV1 deoxycytidyl transferase-independent, TLS. In contrast to MMS, REV1 and parental DT40 cells showed similar toxicity and mutagenicity caused by a combination of low-dose N-methyl-N-nitrosourea and O6-benzylguanine. These results suggest that base excision repair intermediates appear to initiate REV1-dependent TLS, whereas, O6-methylguanine could be by-passed by replicative polymerases, such as polymerase delta complexes, with an associated increase in G to A transition mutations. Citation Format: Jun Nakamura, Xu Tian, James Swenberg. REV1 has critical roles in base excision repair intermediate-mediated transversion mutations but not for O6-methylguanine-initiated mutations in vertebrate cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2013-LB-151

Abstract 2552: Detection of PIGO-deficient cells using proaerolysin: a valuable tool to investigate mechanisms of mutagenesis in the DT40 cell system

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL A DNA damage response analysis makes use of the chicken DT40 isogenic cell line and its dozens of available mutants knocked out in various DNA repair and cell cycle pathways. While this DT40 cell system is valuable for understanding the DNA damage response to genotoxic agents, no convenient mutation assay for mutagens currently exists for this reverse-genetic system. Here we establish a proaerolysin (PA) selection-based mutation assay in DT40 cells to identify glycosylphosphatidylinositol (GPI)-anchor deficient cells. Using PA, we detected an increase in the number of PA-resistant DT40 cells exposed to MMS for 24 hours followed by a 5-day period of phenotype expression. GPI anchor synthesis is catalyzed by a series of phosphatidylinositol glycan complementation groups (PIGs). The PIG-O gene is on the sex chromosome (Chromosome Z) in chicken cells and is critical for GPI anchor synthesis at the intermediate step. We identified that all PA-resistant DT40 cells analyzed have mutations in the PIG-O gene. This novel assay for DT40 cells provides a valuable tool to investigate the mode of action of mutations caused by reactive agents using a series of isogenic mutant DT40 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2552. doi:1538-7445.AM2012-2552