Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′- and 5′- ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetics studies showed that the 5′-activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC50 = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.

DNA topoisomerase IIα (170 kDa, TOP2α/170) is essential in proliferating cells since it resolves DNA topologic entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated TOP2α/90 isoform, detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide (J Pharmacol Exp Ther 2017;360:152-63). TOP2α/90 (786 amino acids) is the translation product of a TOP2α mRNA that retains a processed intron 19. TOP2α/90 lacks the active-site tyrosine-805 (Tyr805) required to generate double-strand DNA breaks as well as the nuclear localization signals present in the TOP2α/170 isoform (1531 amino acids). The function of TOP2α/90 is unknown. Here, we found that TOP2α/90, like TOP2α/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Importantly, co-immunoprecipitation of endogenous TOP2α/90 and TOP2α/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2α/90 in K562 cells suppressed, while siRNA-mediated knockdown of TOP2α/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated K562 or K/VP.5 cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2α/90 in K562 cells inhibited etoposide cytotoxicity assessed by soft agar colony formation assays. qPCR and immunoassays demonstrated expression of TOP2α/90 mRNA and protein in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2α/90 expression decreases drug-induced TOP2α-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2α/170. Alternative processing of TOP2α pre-mRNA, and subsequent synthesis of TOP2α/90, may be an important mechanism regulating the formation and/or stability of TOP2α/170-DNA covalent complexes in response to TOP2α-targeting agents. Citation Format: Ragu Kanagasabai, Soumendra Karmahapatra, Yang Yu, Victor A. Hernandez, Corey A. Kientz, Evan E. Kania, Terry S. Elton, Jack C. Yalowich. The novel C-terminal truncated 90-kDa isoform of topoisomerase IIα, TOP2α/90, is a determinant of etoposide resistance in K562 leukemia cells via heterodimerization with the TOP2α/170 isoform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 904.

Background: hMPG initiates repair of mutagenic and tumorigenic modified purine bases. Results: Two germ line hMPG variants showed reduced activity due to low affinity for DNA and facilitated lesion-induced mutations. Conclusion: Changes in amino acid sequence alter the function of hMPG, leading to genomic instability. Significance: Individuals possessing these hMPG variants may be at higher risk for genomic instability-related diseases.

BackgroundTopo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details.ResultsIn this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg2+/Mn2+, and that neither Zn2+ or nor Ca2+ can activate hTDP2. Mg2+ also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg2+ controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg2+ concentration above which it is inhibitory for hTDP2 activity.ConclusionThese results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.

Interest in the mechanisms of DNA repair pathways, including the base excision repair (BER) pathway specifically, has heightened since these pathways have been shown to modulate important aspects of human disease. Modulation of the expression or activity of a particular BER enzyme, N-methylpurine DNA glycosylase (MPG), has been demonstrated to play a role in carcinogenesis and resistance to chemotherapy as well as neurodegenerative diseases, which has intensified the focus on studying MPG-related mechanisms of repair. A specific small molecule inhibitor for MPG activity would be a valuable biochemical tool for understanding these repair mechanisms. By screening several small molecule chemical libraries, we identified a natural polyphenolic compound, morin hydrate, which inhibits MPG activity specifically (IC50 = 2.6 µM). Detailed mechanism analysis showed that morin hydrate inhibited substrate DNA binding of MPG, and eventually the enzymatic activity of MPG. Computational docking studies with an x-ray derived MPG structure as well as comparison studies with other structurally-related flavanoids offer a rationale for the inhibitory activity of morin hydrate observed. The results of this study suggest that the morin hydrate could be an effective tool for studying MPG function and it is possible that morin hydrate and its derivatives could be utilized in future studies focused on the role of MPG in human disease.

Mammalian apurinic/apyrimidinic endonuclease (APE1) initiates the repair of abasic sites (AP-sites), which are highly toxic, mutagenic, and implicated in carcinogenesis. Also, reducing the activity of APE1 protein in cancer cells and tumors sensitizes mammalian tumor cells to a variety of laboratory and clinical chemotherapeutic agents. In general, mouse models are used in studies of basic mechanisms of carcinogenesis, as well as pre-clinical studies before transitioning into humans. Human APE1 (hAPE1) has previously been cloned, expressed, and extensively characterized. However, the knowledge regarding the characterization of mouse APE1 (mAPE1) is very limited. Here we have expressed and purified full-length hAPE1 and mAPE1 in and from E. coli to near homogeneity. mAPE1 showed comparable fast reaction kinetics to its human counterpart. Steady-state enzyme kinetics showed an apparent Km of 91 nM and kcat of 4.2 s−1 of mAPE1 for the THF cleavage reaction. For hAPE1 apparent Km and kcat were 82 nM and 3.2 s−1, respectively, under similar reaction conditions. However, kcat/Km were in similar range for both APE1s. The optimum pH was in the range of 7.5–8 for both APE1s and had an optimal activity at 50–100 mM KCl, and they showed Mg2+ dependence and abrogation of activity at high salt. Circular dichroism spectroscopy revealed that increasing the Mg2+ concentration altered the ratio of “turns” to “β-strands” for both proteins, and this change may be associated with the conformational changes required to achieve an active state. Overall, compared to hAPE1, mAPE1 has higher Km and kcat values. However, overall results from this study suggest that human and mouse APE1s have mostly similar biochemical and biophysical properties. Thus, the conclusions of mouse studies to elucidate APE1 biology and its role in carcinogenesis may be extrapolated to apply to human biology. This includes the development and validation of effective APE1 inhibitors as chemosensitizers in clinical studies.

The Long-Evans Cinnamon (LEC) rat is an animal model for Wilson’s disease (WD). This animal is genetically predisposed to copper accumulation in the liver, increased oxidative stress, accumulation of DNA damage, and the spontaneous development of hepatocellular carcinoma (HCC). Thus, this animal model is useful for studying the relationship of endogenous DNA damage to spontaneous carcinogenesis. In this study, we have investigated the apurinic/apyrimidinic endonuclease 1 (APE1)-mediated excision repair of endogenous DNA damage, apurinic/apyrimidinic (AP)-sites, which is highly mutagenic and implicated in human cancer. We found that the activity was reduced in the liver extracts from the acute hepatitis period of LEC rats as compared with extracts from the age-matched Long Evans Agouti (LEA) rats. The acute hepatitis period had also a heightened oxidative stress condition as assessed by an increase in oxidized glutathione level and loss of enzyme activity of glyceraldehyde 3- phosphate dehydrogenase, a key redox-sensitive protein in cells. Interestingly, the activity reduction was not due to changes in protein expression but apparently by reversible protein oxidation as the addition of reducing agents to extracts of the liver from acute hepatitis period reactivated APE1 activity and thus, confirmed the oxidation-mediated loss of APE1 activity under increased oxidative stress. These findings show for the first time in an animal model that the repair mechanism of AP-sites is impaired by increased oxidative stress in acute hepatitis via redox regulation which contributed to the increased accumulation of mutagenic AP-sites in liver DNA.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and chronic inflammation seems to be the most important risk factor. Since inflammation induces oxidative stress and DNA damage, base excision repair (BER) of oxidative damage in prevention of mutations and subsequent tumorigenesis in an inflammatory environment seems to be important, but a direct link between deficient DNA repair and carcinogenesis in the highly damaging environment of the inflamed liver has not been shown. Furthermore, the longstanding question about how and in which cells cancer initiation occurs and the roles that DNA damage and repair play in the process are larger problems that remain unanswered. In our study we utilize the Long Evans Cinnamon (LEC) rat, which develops spontaneous HCC after consecutive phases of acute and chronic hepatitis induced by the accumulation of copper due to a mutation in the copper transporter gene ATP7B, mimicking Wilson's disease in humans. We have previously shown that the LEC rat exhibits deficient BER during acute hepatitis, particularly in the activity of 8-oxoguanine DNA glycosylase (Ogg1), and activity returns during chronic hepatitis as preneoplastic foci begin to form. In order to study the role of BER in cancer initiation, we have generated three new cell lines from the animal. We report here that we have isolated and grown apparently spontaneously immortalized LEC hepatocytes from the acute hepatitis liver (LEC-AH) and the chronic hepatitis liver (LEC-CH) in long term culture. We have also developed a carcinoma cell line from a LEC liver tumor (LEC-T). The newly generated cell lines express albumin, and preliminary karyotyping analysis shows that the LEC-AH and LEC-CH cells are cytogenetically normal (average 42 chromosomes). As observed in the acute and chronic hepatitis tissues, LEC-AH cells exhibit decreased activity (-2.5 fold) of Ogg1 compared to LEC-CH cells. Furthermore, at 12 days post-confluency, we have observed spontaneous foci formation in the LEC-AH cells (96 foci per 106 cells) in culture while LEC-CH cells do not form foci (<1 foci per 106 cells). These preliminary results indicate a correlation between foci formation in culture and Ogg1 activity, demonstrating a potential direct link between decreased BER and preneoplastic foci formation. Additionally, the foci may represent an enriched population of initiated cells, providing a unique opportunity to study the mechanism of spontaneous cancer initiation in vitro and identify initiation markers. Experiments elucidating the specific roles of Ogg1, other BER enzymes, and oxidative damage in foci formation of LEC-AH cells are in progress, as is further molecular analysis of the foci-forming cell populations as they may reflect the biochemical and molecular characteristics of preneoplastic foci in vivo. (Supported by a grant from NIH/NCI RO1 CA113447) 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 4430. doi:1538-7445.AM2012-4430