W. Glenn McGregor

Inhibition of DNA Replication and Induction of S Phase Cell Cycle Arrest by G-rich Oligonucleotides

The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369–26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.

Participation of mouse DNA polymerase ι in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer

DNA polymerase ι (pol ι) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol ι performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol η, pol ι, or both. A deficiency in mouse DNA polymerase η (pol η) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol ι, indicating that pol ι participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was observed in wild-type cells that was diminished in pol η- and pol ι-deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asymmetric manner. To determine whether pol ι status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol η, pol ι, or both. Although pol ι deficiency alone had no effect, UV-induced skin tumors in pol η-deficient mice developed 4 weeks earlier in mice concomitantly deficient in pol ι. Collectively, these data reveal functions for pol ι in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer.

REV1 is implicated in the development of carcinogen-induced lung cancer

The somatic mutation hypothesis of cancer predicts that reducing the frequency of mutations induced by carcinogens will reduce the incidence of cancer. To examine this, we developed an antimutator strategy based on the manipulation of the level of a protein required for mutagenic bypass of DNA damage induced by the ubiquitous carcinogen benzo[a]pyrene. The expression of this protein, REV1, was reduced in mouse cells using a vector encoding a gene-specific targeting ribozyme. In the latter cells, mutagenesis induced by the activated form of benzo[a]pyrene was reduced by >90%. To examine if REV1 transcripts could be lowered in vivo, the plasmid was complexed with polyethyleneimine, a nonviral cationic polymer, and delivered to the lung via aerosol. The endogenous REV1 transcript in the bronchial epithelium as determined by quantitative real-time PCR in laser capture microdissected cells was reduced by 60%. There was a significant decrease in the multiplicity of carcinogen-induced lung tumors from 6.4 to 3.7 tumors per mouse. Additionally, REV1 inhibition completely abolished tumor formation in 27% of the carcinogen-exposed mice. These data support the central role of the translesion synthesis pathway in the development of lung cancer. Further, the selective modulation of members of this pathway presents novel potential targets for cancer prevention. The somatic mutation hypothesis of cancer predicts that the frequency of cancers will also be reduced. (Mol Cancer Res 2009;7(2):247–54)

Altered Ig Hypermutation Pattern and Frequency in Complementary Mouse Models of DNA Polymerase ζ Activity

To test the hypothesis that DNA polymerase ζ participates in Ig hypermutation, we generated two mouse models of Pol ζ function: a B cell-specific conditional knockout and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B cells had a reduction in mutation frequency at Ig loci in the spleen and in Peyer’s patches, whereas knock-in mice with a mutagenic Pol ζ displayed a marked increase in mutation frequency in Peyer’s patches, revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in Ig hypermutation.

2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine-induced DNA adducts and genotoxicity in chinese hamster ovary (CHO) cells expressing human CYP1A2 and rapid or slow acetylator N-acetyltransferase 2

Heterocyclic amine carcinogens such as 2‐amino‐1‐methyl‐6‐phenylimidazo [4,5‐b] pyridine (PhIP) are present in diet and cigarette smoke. Bioactivation in humans includes N‐hydroxylation catalyzed by cytochrome P4501A2 possibly followed by O‐acetylation catalyzed by N‐acetyltransferase 2 (NAT2). Nucleotide excision repair‐deficient Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A2 and NAT2 catalytic activities were undetectable in untransfected CHO cell lines. CYP1A2 catalytic activity levels did not differ significantly (P > 0.05) among the CYP1A2‐transfected cell lines. Cells transfected with NAT2*4 had significantly higher levels of N‐acetyltransferase (P = 0.0001) and N‐hydroxy‐PhIP O‐acetyltransferase (P = 0.0170) catalytic activity than cells transfected with NAT2*5B. PhIP caused dose‐dependent decreases in cell survival and significant (P < 0.001) increases in mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in all the CYP1A2‐transfected cell lines. Transfection with NAT2*4 or NAT2*5B did not further increase hprt mutagenesis. PhIP‐induced hprt mutant cDNAs were sequenced, and 80% of the mutations were single base substitutions at G:C base pairs. dG‐C8‐PhIP DNA adduct levels were dose‐dependent in the order: untransfected < transfected with CYP1A2 < transfected with CYP1A2 and NAT2*5B < transfected with CYP1A2 and NAT2*4. Following incubation with 1.2 µM PhIP, DNA adduct levels were significantly (P < 0.05) higher in CHO cells transfected with CYP1A2/NAT2*4 versus CYP1A2/NAT2*5B. These results strongly support an activation role for CYP1A2 in PhIP‐induced mutagenesis and DNA damage and suggest a modest effect of human NAT2 and its genetic polymorphism on PhIP DNA adduct levels.

Micro- and nanotechnology approaches for capturing circulating tumor cells.

Circulating tumor cells (CTC) are cells that have detached from primary tumors and circulate in the bloodstream where they are carried to other organs, leading to seeding of new tumors and metastases. CTC have been known to exist in the bloodstream for more than a century. With recent progress in the area of micro- and nanotechnology, it has been possible to adopt new approaches in CTC research. Microscale and nanoscale studies can throw some light on the time course of CTC appearance in blood and CTC overexpression profiles for cancer-related markers and galvanize the development of drugs to block metastases. CTC counts could serve as endpoint biomarkers and as prognostic markers for patients with a metastatic disease. This paper reviews some of the recent researches on using micro- and nanotechnology to capture and profile CTC.

Effect of Rapid Human N-acetyltransferase 2 Haplotype on DNA Damage and Mutagenesis Induced by 2-Amino-3-methylimidazo [4,5-f] quinoline (IQ) and 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx)

Heterocyclic amines such as 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) are dietary carcinogens generated when meats are cooked well-done. Bioactivation includes N-hydroxylation catalyzed by cytochrome P4501A2 (CYP1A2) followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles were treated with IQ or MeIQx to examine the effect of NAT2 genetic polymorphism on IQ- or MeIQx-induced DNA adducts and mutagenesis. MeIQx and IQ both induced decreases in cell survival and significantly (p<0.001) greater number of endogenous hypoxanthine phosphoribosyl transferase (hprt) mutants in the CYP1A2/NAT2*4 than the CYP1A2/NAT2*5B cell line. IQ- and MeIQx-induced hprt mutant cDNAs were sequenced and over 85% of the mutations were single-base substitutions with the remainder exon deletions likely caused by splice-site mutations. For the single-base substitutions, over 85% were at G:C base pairs. Deoxyguanosine (dG)-C8-IQ and dG-C8-MeIQx adducts were significantly (p<0.001) greater in the CYP1A2/NAT2*4 than the CYP1A2/NAT2*5B cell line. DNA adduct levels correlated very highly with hprt mutants for both IQ and MeIQx. These results suggest substantially increased risk for IQ- and MeIQx-induced DNA damage and mutagenesis in rapid NAT2 acetylators.

RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage

Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or poleta only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm.

RAD18 Signals DNA Polymerase IOTA to Stalled Replication Forks in Cells Entering S-phase with DNA Damage

Endogenously generated reactive oxygen species and genotoxic carcinogens can covalently modify bases in cellular DNA. If not recognized and removed prior to S-phase of the cell cycle, such modifications can block DNA replication fork progression. If blocked forks are not are not resolved, they result in double strand breaks and cell death. Recent data indicate that the process of translesion DNA synthesis (TLS) is a highly conserved mechanism for bypassing lesions in template DNA. Although not fully understood, in yeast a ubiquitin ligase (RAD18) signals error-prone Y family polymerases to the blocked fork to bypass the damage with potentially mutagenic consequences. Homologs of the yeast proteins are found in higher eukaryotic cells, including human. We are examining the hypothesis that RAD18 acts as a proximal signal to Y-family polymerases to bypass damage, in a manner analogous to yeast but with additional layers of complexity. Here we report that RAD18 accumulates in nuclear foci after UV irradiation only in cells entering S-phase with DNA damage. These foci co-localize with proliferating cell nuclear antigen (PCNA). In addition, a newly described DNA polymerase, pol iota, also forms nuclear foci in a damage- and S-phase dependent manner. These data support our overall hypothesis that RAD18 accumulates at blocked forks and initiates the signal to recruit TLS polymerases.

Abstract 3662: Absence of leukotriene B4 receptor-1 (BLT1) protects against lung cancer

Chemokines direct leukocyte trafficking, which plays a major role in immune and inflammatory responses, thus contributing to (or) modulating a number of disease conditions. During carcinogenesis leukocytes infiltrate the site of the tumor microenvironment and could exert a major influence on promotion or suppression of cancer depending on their activation status. Leukotriene B4, a pro-inflammatory lipid chemokine, mediates many of its activities through BLT1, a high affinity G-protein coupled receptor. BLT1 is expressed on an array of immune cells and has been implicated to play a critical role in many inflammatory diseases. We investigated whether BLT1 also influences lung carcinogenesis, the leading cause of cancer-related deaths worldwide. In this study, we used BLT1-deficient mice (BLT1-/-) to determine their susceptibility to lung cancer development in a transgenic model expressing an oncogenic form of K-ras (K-ras LA1) as well as in carcinogen (3-Methylcholanthrene; 3 MCA)-induced and inflammation (butylated hydroxy toluene; BHT) – promoted murine lung cancer model. We found that in the activated K-ras transgenic background the BLT1-/- mice showed fewer and smaller lung tumors as compared to the BLT1+/+ mice. In the MCA-BHT induced lung cancer model also the BLT1-/- mice showed fewer and smaller lung tumors than the BLT1+/+ mice. Lung lesions in BLT1-/- mice were more of adenomatous hyperplasia type and fewer adenomas when compared to those in BLT1+/+ mice. Moreover, the frequency of induced mutations in cells derived from the lungs of BLT1-/- mice was greatly reduced compared to BLT1+/+ mice. Pulmonary inflammation was assessed in bronchoalveolar lavage (BAL) fluids following one, three or six weekly injections of BHT. At one and three week time points macrophages, and at six weeks lymphocytes constituted the predominant leukocyte subtypes in the BAL fluids of BLT1+/+ mice. In BLT1-/- mice both of these responses were significantly attenuated leading to an overall reduction in pulmonary inflammation. Based on these results, we conclude that BLT1 mediated pulmonary inflammation has a promoting role in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3662. doi:10.1158/1538-7445.AM2011-3662