DNA Adducts, DNA Damage, and DNA Repair
DNA damage and its repair, or lack thereof, are thought to be major mechanisms underlying cancer development. Chemicals can damage the DNA directly and/or indirectly due to inflammation and oxidative stress; this damage needs to be repaired. DNA damage can cause mutations or loss of expression of DNA repair proteins. The latter would lead to more mutations and genetic instability, which are hallmarks of tumor cells. This Program is conducting extensive studies of DNA adducts and DNA damage by chemical carcinogens. There are also investigations of how these lesions are repaired and of the effects of chemical carcinogens on DNA repair. Examples include:
- Research which found that excess Rad51 sensitizes srs2Δ cells to the DNA-damaging agent MMS and the replication-stalling agent hydroxyurea (HU). We proposed that inappropriate use of the recombination-mediated pathway results in induction of DNA damage that cannot be repaired. These results highlight the sensitive balance between the different pathways used for replication restart.
- Studies showing that nucleotide excision repair (NER) proteins excises bulky catechol 4-hydroxyequilenin (4-OHEN) adducts in vitro with efficiencies that depend on the stereochemical properties of the adducts and the nature of the modified DNA base. Mechanisms by which human DNA repair enzymes recognize and excise bulky DNA adducts, such as those derived from B[a]P diol epoxides, as well as the molecular/structural factors and mechanisms involved in translesion synthesis catalyzed by representative bypass polymerases (Dpo4, pol ê , and pol ç ) in vitro, have been extensively studied. Modern computational and modeling techniques based on NMR structural studies have been employed to derive insights into adduct conformational properties and specific DNA distortions in the vicinity of the adducts that serve as signals of recognition and removal of the lesions by nucleotide excision repair proteins.
- Investigations finding that multiple low-dose exposures to 4-hydroxyestradiol, a common metabolite of estrogen, are mutagenic in the cII assay in BB rat2 cells. Furthermore, the mutational spectrum of 4-hydroxyestradiol contains a considerable proportion of mutations at A:T base pairs, consistent with the known ability of E(2)-3,4-quinone to form a significant fraction of DNA adducts at adenines.
- Research which reported that tankyrases are poly(ADP-ribose) polymerases that bind to the telomere repeat binding protein TRF1 and regulate telomere length and sister telomere cohesion.
- A study showing that TIN2/TPP1/POT1, which binds to both telomere repeat binding proteins (TRF1 and TRF2), controls telomere length and chromosome end protection. Telomere dysfunction is recognized by the cell as DNA damage and, thus, increased understanding of how the cell responds to telomere dysfunction will provide insight into the mechanisms of DNA repair.
- Perlmutter Cancer Center investigators showed that TRF2 is also lost from human telomeres, and that the TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1, as well as several factors involved in the DNA damage response.
- Our scientists have mapped the Cr(III)- DNA distribution in exons 5, 7, and 8 of the p53 gene. They found that the sequence specificities of Cr(III)- DNA and Cr(III)-histidine-DNA adducts in the p53 gene sequence are identical and that both types of adducts are preferentially formed at -NGG- sequences, including codons 245, 248, and 249-the mutational hotspots in human lung cancer.
Carcinogenesis and Animal Models
The development and use of appropriate informative animal models for chemical carcinogenesis will help us understand which chemicals cause cancer in humans and the mechanisms involved. This program makes extensive use of animal models to study chemical carcinogenesis.
- Perlmutter Cancer Center researchers have been studying the function of cell cycle checkpoint components, including the Polo family of protein kinases (Plks), Bub1, BubR1, and Sgo1. These studies have improved our understanding of the molecular mechanisms by which deregulated DNA-damage checkpoint and spindle checkpoint result in aneuploidy, chromosomal instability, and malignant transformation.
- Our investigators have exposed mice to mainstream cigarette smoke and identified inbred mouse strains that are susceptible or resistant to the development of lung cancer. To identify genetic host factors that predispose mice to cigarette smoke-induced lung tumors, they bred F2 intercross mice from the susceptible and resistant strains and exposed them to cigarette smoke. In addition, they intend to develop serum biomarkers that are associated with cigarette smoke-induced lung cancer in collaboration.
- Scientists exposed groups of hairless mice to arsenite (As) alone, solar ultraviolet (UV) radiation alone, or arsenite plus solar UV. They found that untreated mice and mice treated with arsenite alone did not develop tumors. Mice treated with UV alone developed a small number of locally invasive squamous cell carcinomas. Mice exposed to arsenite plus UV exhibited a dose-related increase in skin cancers that was significantly higher compared with mice exposed to UV alone. This is the first animal model for As-induced cancers.
There are a number of chemical agents that can prevent the development of tumors or their recurrence. These agents are being studied in vitro, in animal models, and in human epidemiological studies.
- When cells are concomitantly treated with arsenite (As) and 0.5 μM caffeic acid phenethyl ester (CAPE), cell transformation does not occur. Gene arrays and Western blots showed that, in comparison to parental HOS cells, As causes cell transformation and many changes in gene expression, including down-regulation of inflammatory cytokine mRNAs, p53, and antioxidant enzyme mRNAs, whereas CAPE alters a number of these changes at the same time as it suppresses As-induced cell transformation. Further, a CAPE-containing diet inhibits growth of MCF-7 (breast cancer cell)-induced tumors in a nude mouse xenograft model.
- Dietary supplement of celecoxib at doses of 400 ppm, 600 ppm and 1000 ppm are very effective against mouse prostatic intraepithelial neoplasia (mPIN) and adenocarcinoma of the prostate. Tumor growth inhibition by celecoxib was associated with increased rate of apoptosis. Dietary intake of exisulind in combination with celecoxib for 12 months reduced PIN lesions at doses of 500 and 1000 ppm. Histological examination of the dorsolateral prostate indicated a reduction of PIN lesions from 82 percent to 15 percent and adenocarcinomas from 68 percent to 15 percent. TUNEL assay showed a higher rate of apoptosis (60 percent) induced by these agents when used in combination.
- In a collaboration with urology clinicians and pathologists at NYU, a clinical trial with prostate cancer patients is continuing to determine the preventive efficacy of dietary soy protein isolate, which contains putative anticancer isoflavones such as genistein. This trial is designed to determine whether a two-year dietary intervention with soy protein isolate reduces biochemical recurrence, i.e., elevation of prostate-specific antigen (PSA), in men who are at high risk for cancer recurrence after surgery (radical prostatectomy).
- Lycopene, soy-derived genistein, and other dietary chemopreventives (such as vanillin and cinnamaldehyde) act as antimutagens in the human HNPCC hMLH1 mismatch repair-deficient cell line HCT116. Within 24 hours, both lycopene (2 μM) and genistein (3.125 μM) reduced the elevated spontaneous mutant frequency at the HPRT gene in MCF-7, MDA-MB-468 and BT-474 breast tumor cells by 35 to 50 percent, as well as the spontaneous levels of micronuclei in all three cell lines. TUNEL assays and flow cytometry suggest that an underlying mechanism for these effects does not involve apoptosis at the low physiological concentrations used.
Cell Signaling and Epigenetic Mechanisms of Carcinogenesis
DNA damage and mutations are the classical mechanisms by which chemical carcinogens induce tumors in humans. However, in recent years, the effects of chemical carcinogens on inheritable epigenetic parameters as well as cell signaling have received considerably more attention. This program includes a number of investigators who study these alternative mechanisms of carcinogenesis.
- A new class of enzymes in demethylating histone H3 lysine 9 (H3K9) were discovered. These enzymes require iron, oxoglutarate, oxygen, and ascorbic acid to oxidatively demethylate mono- and di-methylated H3K9me 1 H3K9me 2 yielding formaldehyde and, in the process, decarboxylated oxoglutarate releases carbon dioxide. Nickel ions are potent inhibitors of these enzymes and cause an increase in the mono- and di-methylation of H3K9. The methylation of H3K9 in the nucleosomes of promoter regions causes DNA methylation and gene silencing.
- Since suppression of apoptosis is thought to contribute to carcinogenesis, an ongoing investigation focuses on the mechanisms implicated in nickel-induced anti-apoptotic effects via COX-2 induction. Perlmutter Cancer Center researchers found that nickel ions induce COX-2, which is the primary inhibitor of apoptosis via JNK1 but not JNK2.
- We found that BRCA1 promoter activity could be suppressed by benzo[a]pyrene, a carcinogen found in cigarette smoke. Results from this study imply the strong deleterious effects of cigarette smoking in BRCA1 mutation carriers, as the expression of the remaining functional wild type BRCA1 could be turned off by benzo[a]pyrene, thereby promoting or accelerating the carcinogenic process.
- Although asbestos induces the secretion of TNF-alpha and the expression of TNF-alpha receptor I in human mesothelial cells, a treatment of mesothelial cells with TNF-alpha significantly reduces asbestos cytotoxicity. Moreover, TNF-alpha activates NF-kappaB, which leads to mesothelial cell survival and resistance to the cytotoxic effects of asbestos. Hence, there seems to be a critical role for TNF-alpha and NFkappaB signaling in mediating cellular responses to asbestos.
There are substantial efforts under way in this program to develop markers for the early detection of lung, breast, cervical, and ovarian cancers.
- We found that anti-HMdU (5-hydroxymethyl-2'-deoxyuridine, an oxidized thymidine) autoantibody (aAb) levels are higher in sera of women with transient HPV infection than in non-infected women, and are further elevated in the sera of persistently infected subjects.
- A study showed that LPA increases EOC metastatic potential by enhancing MT1-MMP-mediated proMMP-2 activation and increasing urokinase-type plasminogen activator (uPA). LPA also elevates levels of proangiogenic factors, including VEGF and IL-8, to facilitate EOC cell invasion and migration in vitro. We also reported that LPA stimulates membrane-bound vesicle formation. Membrane vesicles derived from women with malignant or nonmalignant ovarian ascites contain activated MMP-2, MMP-9, and uPA, suggesting that vesicle-stimulated proteinase activation leads to increased extracellular matrix degradation, which may facilitate tumor cell invasion and metastasis.
- DNA adducts, DNA methylation, protein markers, fluorescence, and ultra-thin bronchoscopies to obtain biopsies of preneoplastic lesions are biomarkers studied in individuals with preneoplastic lung lesions. The main goal of this research is to evaluate potential biomarkers in patients with early lung cancer, high-risk smokers with solid nodules or GGOs, and controls (either smokers with normal CT scans or never smokers).
- Osteopontin (OPN) could be an early marker for mesothelioma. OPN is overexpressed in many types of cancer, including lung. It mediates cell-matrix interactions and is regulated by proteins in cell-signaling pathways that have been associated with asbestos-mediated causes of cancer.