ACCOMPLISHMENTS
The section will highlight projects that illustrate the quality of the science and the extent of the collaborative interactions, rather than catalog every funded project by each investigator (nearly all of whom are funded).
1) Biology of reactive oxygen species: Cysteine sulfenic acid residues in proteins as redox sensors and as markers of oxidative damage
Investigators in the DDCD Program have assumed a leadership role in an entirely new basic science area of cellular biochemistry that has major implications for regulatory control of both intracellular oxidant signaling and cellular sensitivity to oxidative stress. A novel discovery by Dr. Leslie Poole and her collaborators that peroxideoxins (Prxs) contain a cysteine sulfenic acid center led to her hypothesis that these peroxidase-like enzymes may act as a redox-sensitive “floodgates” which scavenge low levels of peroxides but self-inactivate to permit oxidant signaling at higher rates of oxidant generation. In a paper published in collaboration with Dr. Andy Karplus in Science, she showed that eukaryotic Prxs are sensitive to over-oxidation by hydrogen peroxide due to conserved structural motifs that confer rigidity. This allows the sulfenic acid center to act as a peroxide-sensitive switch, keeping peroxide levels low to prevent adventitious signaling by H2O2 until a “burst” of hydrogen peroxide inactivates the peroxidase and enables a pulse of peroxide to act as a signal transducer. Thus, the expression levels and evolved sensitivity of Prxs can be balanced against the size of the H2O2 burst to achieve optimal redox signaling in a given cell. Consistent with this idea, Prxs are over expressed in many tumors (dampening signals that would otherwise lead to apoptosis), and the overexpression of Prxs in cells interferes with some known peroxide signaling pathways. Dr. Poole and her colleague Dr. Jacqueline Fetrow of the WFU Physics Dept. are collaborating to develop a mathematical framework to define this model in kinetic terms (supported by RO1 GM075304). Dr. Poole has also collaborated with Dr. Bruce King (Chemistry Dept.) to develop novel probe reagents that are being used to pinpoint proteins that contain cysteine sulfenic acid residues, and to profile and compare these proteins in different cell types and under different redox conditions (supported by R21 CA112145). This will allow identification of proteins that have altered redox states, e.g. during transformation, as tumors have increased expression of peroxiredoxins, a feature that may suppress apoptosis.
Additional collaborations with Dr. Todd Lowther (Biochemistry, Center for Structural Biology) are aimed at determining the structural basis for the functions of the peroxiredoxins and other key proteins discovered to be sensitive to oxidation of protein sulfhydryl groups to cysteine sulfenic acids using the new probes developed by Dr. King. This novel approach to protein redox chemistry has generated a great deal of excitement, discussion, and also some controversy, and promises to continue to challenge the field of oxidative biology and redox regulation in the future.
In a related collaboration with Dr. Poole, Dr. Lowther has examined the in vivo oxidation state of peroxiredoxins within human cells during oxidative stress, and shown that Prxs are repaired via reduction at different rates (supported by R01 GM072866). These observations suggest that sulforedoxin (Srx) exhibits selectivity for substrates and employs novel sulfurchemistry during the reaction mechanism. He has determined the crystal structure of human Srx and found the structure clearly indicates how Srx interacts with the decameric Prx complex. The collaborations between Drs. Poole, Fetrow, King, and Lowther arose through the events organized by Dr. Townsend as DDCD Program functions.
2) DNA damage by alkylating agents: mechanisms of alkyl-guanine adduct mutagenesis
A novel class of N2-alkylguanine adducts are formed from byproducts of the metabolism of ethanol or nitrosamines. Research in the Akman and Perrino labs is devoted to understanding whether and how DNA damage in the form of alkyl guanine adducts that are caused by chemical carcinogens may result in mutations, and whether cellular defensive mechanisms exist that may repair or bypass these lesions. Dr. Steve Akman (DDCD) has developed a site-specific mutagenesis assay in his lab in collaboration with Dr. Jim Fishbein (previously at WFU, now at U. Md.) that utilizes a synthetic plasmid mini gene reporter construct containing a single alkylguanine adduct at a unique site in a bacterial supF gene. This assay measures the frequency of mutations generated during replication of the plasmid in mammalian cells, and is also used to measure repair of the adducted DNA over time. This assay was used to show that N2-ethyl– or N2-isopropylguanine is a mutagenic base modification that causes roughly equal numbers of transversions, transitions, and single base deletions, in contrast to O6-ethyl guanine, which causes mainly transitions (supported by RO1CA052881).
While the translesion bypass of the large aromatic adducts during DNA replication has been widely studied, little is known about the polymerization across small adducts that are the result of alkylation of the N2-guanineand N6-adenine in DNA. Using the Akman model and in vitro polymerase fidelity assays, Dr. Fred Perrino (DDCD) has examined the role of translesion synthesis by the human Y-family polymerases past these adducts as a mechanism of mutagenicity. In collaboration with Dr. Akman, Dr. Perrino demonstrated that theN2-ethylGua and N2-isopropylGua lesions strongly block replication by the replicative DNA polymerases alpha, delta, and epsilon, and are mutagenic and genotoxic in cells. In contrast, in the presence of the Y family DNA polymerases eta, iota, or kappa, efficient translesion bypass of these DNA adducts occurred, resulting in errorprone replication. A second collaboration between Dr. Perrino and Dr. Tom Hollis (DDCD) of the WFU Center for Structural Biology investigates the structural features of the Y-family polymerases involved in adduct bypass. These intraprogrammatic collaborations exemplify interactions that arose from recognition of complementary goals among Program members.
3) DNA maintenance and repair: Role in prevention of carcinogenesis A new exonuclease that likely plays a role in DNA repair was first purified, characterized, cloned, and sequenced by Dr. Fred Perrino. Over the past decade, he has published most of the papers on this novel class of enzymes, many in the leading journals in the field. Recently, in collaboration with Dr. Perrino, Dr. Tom Hollis has determined the crystal structure of the human 3' – 5’ DNA exonuclease, TREX2, a dimeric enzyme that accounts for the majority of 3' exonclease activity in human cells and is probably involved in the maintenance of DNA 3' termini during repair or replication processes. Structural studies have provided the first pictures of adimeric 3' deoxyribonuclease and serve as the foundation for further structural and biochemical experiments to fully understand the role of dimerization in catalysis.
In other DNA repair studies, Dr. Karin Drotschmann has found that mismatch repair proteins are required for the induction of cell death in response to certain mismatch errors. She has observed that overexpression of one of the key MMR genes results in the induction of cell death. She has also identified functional requirements in key MMR proteins that distinguish damage response from repair. These results are the foundation for her project to discover the role of MMR proteins in the cellular decision between cell cycle arrest and DNA repair, versus commitment to apoptosis (supported by R01 CA101829). In related translational studies, she has found altered expression levels of key MMR genes associated with prostatic cancer. A recent collaboration with Dr. Fred Salsbury of the WFU Physics Dept. and Dr. Tom Hollis of the WFUSM Biochemistry Dept. has led to identification of the specific structures of the MutS protein – DNA interactions for damaged DNA, as opposed to mismatched DNA.
A major achievement, conceived by Dr. Steve Akman (DDCD) was the discovery of a human enzyme that resolves parallel tetramolecular (quadruplex) DNA. His group, in collaboration with Dr. Jim Vaughn (CGS) is the first to identify, purify, characterize, and express recombinant protein for this “G4 resolvase”, which exhibits a 300-fold preference for G4 quadruplex DNA over duplex DNA. He is now beginning to explore the biologic roles of this enzyme using transgenic overexpression and dominant negative mutants, with the hypothesis that it may have a role during rapid DNA replication, e.g. as occurs in cancer. This enzyme may also have a role in maintenance of genomic stability.
4) Cellular defenses against DNA damage by electrophiles
The cancer demographics of the next few decades portend a significant increase in cancer diagnoses due to an aging population and lifestyle issues. Thus, unless major new therapeutic breakthroughs are made, the best hope for reversal of this trend is likely to be the development of effective strategies to prevent, or at least delay, the progression of abnormal (i.e. damaged) cells to frank malignancy. Fully half of the members of the DDCD Program are devoted to finding answers to this problem. A prime example is the work by Drs. Townsend and Morrow to elucidate mechanisms whereby enzymes involved in xenobiotic metabolism either exacerbate the reactivity and toxicity of procarcinogens (e.g. via oxidation by P450) or intercept and detoxifythe reactive products of P450 activation via conjugation and export of carcinogens (e.g. via GST and MRP).
The dynamic competition between phase I activation and phase II detoxification has been examined via stable transfection and heterologous expression in the same cell for the first time in novel studies by Dr. Alan Townsend, utilizing cell lines stably transfected with P450 expression vectors, or P450 together with glutathione S-transferase (GST) vectors (supported by RO1ES10175). These experiments showed that the degree of protection varied substantially for different endpoints such as cytotoxicity, DNA adduct formation, and mutagenicity. The mechanistic basis for these differences remains under investigation, and likely involves preferential conjugation and possibly also efflux of specific metabolites.
Experiments by Dr. Charles Morrow were the first to demonstrate a potent synergy between glutathione S transferases (GSTs) and the multidrug resistance proteins (MRP-1 and MRP-2) that enhances detoxification of both carcinogens and anticancer drugs, including the drug chlorambucil. This finding has resulted in a paradigm shift in the field of GST functions in detoxification, as it has shown that while GST expression alone often does not confer protection, the presence of MRP synergistically potentiates GST detoxification functions. The mechanism appears to be via removal of inhibitory GSH conjugates that would otherwise block GST activity, and in some cases via removal of the conjugates, which may have some residual toxicity when they accumulate to high levels in the cell.
Ferritin is an iron storage protein composed of 24 heteromeric H and L subunits which previously was thought to function solely in the storage of excess iron. Studies by the Torti group have shown that cells react to TNF treatment by up regulating production of the ferritin heavy chain, and that these cells are then protected against inflammation. These results indicated, for the first time, that ferritin drives the balance of reactive iron in cells. TNF-dependent induction of ferritin is mediated by NF-kappa B, and, as demonstrated in recent studies published in Cell by the Torti group and their collaborators, is the central player in the cyto protective response mediated by NF-kappa B. Ferritin has subsequently been shown by this lab to bind to kininogen, a process that inhibits the release of inflammatory mediators in serum. Further work by the Torti group has detailed the genetic elements in the ferritin H promoter which respond to oxidant stresses such as hydrogen peroxide or to phase II chemical inducers. These studies demonstrated a role for ferritin in the antioxidant response elicited by synthetic chemopreventive inducers such as oltipraz, as well as natural compounds such as sulforaphane that are found in cruciferous vegetables. To extend these observations to an in vivo setting, a transgenic ferritin mouse strain has been developed by Dr. John Wilkinson, working with the Torti lab. In this model, expression of ferritin H in specific tissues is controlled through treatment of mice with the antibiotic doxycycline. Currently, the impact of ferritin on iron metabolism in the kidney has been assessed, and the in vivo findings support results in tissue culture that changes in ferritin control tissue-reactive iron availability in vivo. The ability of increased ferritin expression to protect against oxidant stress, and to intervene in carcinogenesis, is currently being explored in both kidney- and lung- specific models.
5) Chemistry–biology interface at CCCWFU: synthesis and testing of novel chemotherapeutic and chemopreventive agents
Several DDCD investigators are engaged in research projects to develop novel chemotherapeutic agents or new approaches to the application of existing drugs. In a search for novel hybrid inorganic-organic molecules for applications in cancer chemotherapy and transcription regulation, a new series of platinum analogs conjugated to intercalating an moiety based on a 9-aminoacridine derivative has been synthesized by Dr. Uli Bierbach (DDCD; Chemistry Dept.). In collaborative studies with Dr. Greg Kucera (DDCD Program; Hem/Onc), interesting and unique properties were demonstrated for some of these new agents, including intercalation in the minor groove of DNA. This dual-acting metalating-intercalating agent showed promising activity in vitro in several solid tumors, including ovarian and lung cancer cell lines, and potent activity against cells selected for resistance to cis-platinum. The targeting of adenine residues at 5'-TA sites in the minor groove of DNA by the prototype conjugate Pt-bis(ACRAMTU) is unprecedented and complementary to the guanine-targeted classical drug cis-platinum. Recent results demonstrated that this binding mode is observed in TATA sequences, known to play a crucial role in eukaryotic transcription initiation. This approach may thus be applicable to the design of sequence-specific inhibitors of transcription. This project is an example of a fruitful collaboration representing the chemistry-biology interface at CCCWFU (supported by 1R01 CA 101880-01), as well as intraprogrammatic strengths within the CCCWFU.
Ionizing radiation induces DNA single-strand and double-strand breaks through a mechanism involving the radiolytic scission of water to form extremely reactive hydroxyl radicals, which then immediately attack DNA or other macromolecules such as proteins or lipids. This process, though mutagenic and detrimental under normal circumstances, is used to advantage in radiation therapy to preferentially destroy rapidly dividing cancer cells. Ionizing radiation remains a useful tool for treatment of solid tumors. However, like chemotherapy, it also often lacks sufficient selectivity for tumor versus normal tissue toxicity, and hence there is an ongoing need and search for agents that selectively enhance the sensitivity of tumor cells to radiation more than that of normal tissues. As a result of pilot studies with agents already under investigation for their chemotherapeutic or chemopreventive activity, several DDCD investigators have discovered radiosensitizing properties of the therapeutic drugs or chemopreventive neutriceuticals they were studying. The novel synthetic oligo-FdUMP polymer of 5-fluorodeoxyuridine, and iron chelator tachypyridine, each being developed as potential chemotherapeutics under the aegis of RAID grants to Dr. Suzy Torti and Dr. Bill Gmeiner and a RAND grant to Dr. Torti, were both found to significantly enhance the radio sensitivity of tumor cells in vitro. Similarly, Dr. Costas Koumenis found that caffeic acid phenethyl ester (CAPE), a chemopreventive nutriceutical, also had potent radiosensitizing activity both in vitro and in vivo using a xenograft model.
A novel approach to fluoropyrimidine drug development by Dr. Bill Gmeiner (DDCD) is the synthesis of oligomeric 5-fluorodeoxyuridine monophosphate (FdUMP[10]), a new form of an old drug. Renal cancer cells and prostate cancer cells were highly sensitive to FdUMP[10] but were not sensitive to 5FU. This research was initially funded through a CCCWFU Chairman’s challenge grant and a “Push” grant from the CCCWFU, and is currently funded by an NIH RAID grant to Dr. Gmeiner and Dr. Pommier, his internal collaborator at NCI. Dr. Greg Kucera is a co-Investigator on Dr. Gmeiner’s UO1 award (CA 102532). One interesting and important insight from this work is that FdUMP[10], the prototype polymeric form, is cytotoxic as a result of misincorporation of FdUTP into DNA and trapping of Topoisomerase I cleavage complexes. As described above, a more recent finding in a collaboration with Dr. Costas Koumenis (CGS) is that the oligomeric FdUMP[10] exhibits radiosensitizing activity against tumor cells in vitro.
The divalent metal chelator tachypyridine is another novel chemotherapeutic drug candidate that has been studied by Dr. Suzy Torti under the aegis of an NIH RAID grant, and more recently, also by a RAND award. This drug, which chelates both iron and zinc, has been shown be a potent inducer of apoptosis in a p53-independent manner. More recently, in collaborative studies with Dr. Costas Koumenis, tachypyridine has shown excellent activity as a radio sensitizer, via cell cycle effects mediated by checkpoint kinases.
6) Development of novel strategies for chemoprevention of cancer
Elucidating mechanisms of chemoprevention of genotoxicity by either dietary or pharmacologic agents is a major emphasis of research in the DDCD Program. A collaboration that developed at the chemistry-biology interface has grown into a synergistic relationship for synthesis and testing of novel chemopreventive agents. Dr. Mark Welker has developed synthetic organic chemistry methods that he is now applying to preparation of a series of structural variants of dithiolthiones based on the structure of oltipraz, an effective chemopreventive agent currently in a clinical trial (supported by R15 CA109144). In collaboration with Drs. Suzy Torti and Townsend an in vitro cell culture screen with a responsive cell line is used to assay for increased expression ofNQO1, ferritin or glutathione transferase isozymes. Several of the novel synthetic compounds are effective inducers of the chemopreventive response in these cells.
Research by Dr. Costas Koumenis has shown that plant phenolic antioxidants related to 3, 4-dihydroxycinnamic acid, like caffeic acid phenethyl ester (CAPE) and curcumin, exhibit potent antiproliferative, antimitogenic and antiangiogenic properties (supported by RO1 CA104922). He has shown that CAPE and curcumin inhibit activation of the proto-oncogene kinase Akt/PKB that lies upstream of NF-kB activation. Akt is abnormally activated in a variety of tumors and promotes cell survival, cell proliferation and angiogenesis.
In a new line of investigation, Dr. Suzy Torti has found that the chemopreventive compound curcumin may function as an iron chelator, and that some of its potent antiproliferative activity may be a result of this affinity (supported by AICR). Discovery of this hitherto unrecognized property also suggests that iron chelation may represent a novel additional mechanism for the chemopreventive activity of curcumin. These studies have led to a funded AICR award to Dr. Suzy Torti, with Drs. Mark Miller (DDCD) and Mark Cline (CGS) as collaborators, and a grant proposal by Dr. Brigitte Miller (Clinical) with Drs. S. Torti (DDCD) and Koumenis for the conduct of a clinical trial of topical curcumin against cervical intraepithelial neoplasia.
The encouraging recent findings on curcumin chemopreventive activity have led Drs. Mark Miller and Townsend to include this agent in a chemoprevention pilot study originally designed to test the activity of sulindac and other COX2 inhibitors in prevention of lung tumors in the mutant Ras transgenic mouse model developed by Dr. Miller. This model, which has a mutant Ras transgene under the control of aninducible Tet-on promoter, exhibits lung adenomas within eight weeks of treatment with the inducing agent doxycycline. Preliminary results have also shown that both COX2 and iNOS are strongly induced in lung tissue within two weeks after Doxinduction of the mutant Ras gene expression, and that this response is completely blocked by treatment with sulindac sulfide. These studies will be continued with the addition of curcumin, along with the plannedCOX2 inhibitor celecoxib and also an iNOS inhibitor.