Antymutagenne białko MTH1 - rola biologiczna, regulacja aktywności enzymatycznej oraz znaczenie w procesie nowotworzenia, 124 s.

Rozprawa habilitacyjna (2005) - Karol Białkowski; DSc dissertation (2005) - Karol Bialkowski; Kolokwium habilitacyjne 24.11.2006Rozprawa ta stanowi podsumowanie opublikowanych wcześniej prac badawczych poświęconych antymutagennemu białku MTH1 ssaków, jego biologicznej roli, regulacji aktywności enzymatycznej oraz znaczeniu w procesie nowotworzenia. Białko MTH1 jest enzymem z grupy pirofosfohydrolaz, degradującym 5'-trifosforany nukleozydów purynowych do 5'-monofosforanów nukleozydów oraz nieorganicznego pirofosforanu. Enzym ten wykazuje szczególnie wysokie powinowactwo do niektórych produktów oksydacyjnych uszkodzeń 2'-deoksyguanozyno-5'-trifosforanu (dGTP) oraz 2'-deoksyadenozyno-5'-trifosforanu (dATP), które są substratami w procesie syntezy DNA. Jednym z najlepiej opisanych mutagennych nukleotydów, które mogą powstawać w komórce pod wpływem reaktywnych form tlenu, jest 8-oksy-2'-deoksyguanozyno-5'-trifosforan (8-oxo-dGTP). Jest on substratem dla licznych polimeraz DNA, które inkorporują go w postaci monofosfonukleozydu do nowosyntetyzowanej nici DNA w trakcie procesu replikacji. Z uwagi na zdolność parowania 8-oksy-2'-deoksyguanozny z 2'-deoksyadenozyną i 2'-deoksycytydyną, inkorporacja taka powodować może powstawanie mutacji punktowych po kolejnych rundach replikacyjnych (głównie transwersji AT do CG). Obecnie zakłada się, że zasadnicza funkcja biologiczna białka MTH1 polega na zapobieganiu mutacjom punktowym poprzez oczyszczanie puli wolnych nukleotydów z powstającego w komórkach 8-oxo-dGTP, który jest szczególnie efektywnie degradowany przez MTH1. W ramach przedstawionej rozprawy opracowano metodykę oznaczeń specyficznej aktywności 8-oxo-dGTPazowej białka MTH1 w komórkach i tkankach ssaków. Praktyczne zastosowanie tej metody umożliwiło: a) przeprowadzenie kinetycznej charakterystyki degradacji 8-oxo-dGTP przez białko MTH1 człowieka i chomika, b) poszukiwanie zależności pomiędzy poziomem aktywności 8-oxo-dGTPazowej białka MTH1 a zawartością promutagennej 8-oksy-2'-deoksyguanozyny w DNA tkanek mysich, c) zbadanie wpływu karcynogennych jonów kadmu i niklu in vivo na aktywność 8-oxo-dGTPazową białka MTH1 i zawartość 8-oksy-2'-deoksyguanozny w DNA narządów szczura oraz zaproponowanie nowego mechanizmu karcynogenezy wywoływanej przez jony Cd2+ w jądrach gryzoni, d) identyfikację całej klasy inhibitorów aktywności enzymatycznej białka MTH1 (5'-difosforanów nukleozydów), kinetyczną charakterystykę ich właściwości oraz analizę ich potencjalnego wpływu na efektywność hydrolizy 8-oxo-dGTP in vivo, e) poszukiwanie wyjaśnienia dla zróżnicowanego poziomu ekspresji białka MTH1 w różnych typach komórek fizjologicznych oraz jego nadekspresji w komórkach nowotworowych; w ramach tych poszukiwań określono wpływ różnych stanów fizjologicznych komórek w populacji na poziom aktywności 8-oxo-dGTPazy, w tym: faz cyklu komórkowego, fizycznych oddziaływań międzykomórkowych oraz tempa proliferacji."Antimutagenic MTH1 protein – biological role, regulation of its enzymatic activity and involvement in carcinogenesis". This D.Sc. dissertation summarizes the results of the research devoted to antimutagenic MTH1 protein, its biological role, regulation of enzymatic activity, and involvement in carcinogenesis. The MTH1 protein is a pyrophosphohydrolase that degrades purine nucleoside 5'-triphosphates to the corresponding nucleoside 5'-monophosphate and inorganic pyrophosphate. The enzyme hydrolyzes most specifically some products of oxidative damage to 2'-deoxyguanosine 5'-triphosphate (dGTP) and 2'-deoxyadenosine 5'-triphosphate which are substrates for DNA synthesis. One of the best characterized mutagenic nucleotides is 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) which likely forms in the cells under attack of reactive oxygen species. It is an erroneous substrate for many DNA polymerases that incorporate its monophosphate residue to a nascent DNA strand during DNA replication. Such an incorporation causes point mutations during subsequent replication rounds due to mispairing properties of 8-oxo-2'-deoxyguanosine that may form stable pairs with deoxycytidine as well as with deoxyadenosine. As MTH1 protein very effectively hydrolyzes 8-oxo-dGTP, its widely accepted function is assigned to preventing point mutations by sanitization of this promutagenic nucleotide out of cellular free nucleotide pool. Within the framework of this dissertation an assay of specific 8-oxo-dGTPase activity of MTH1 protein in biological material was developed and validated. The elaborated method allowed subsequently for the following research to be carried out: a) a kinetic characteristics of 8-oxo-dGTP hydrolysis by murine and human MTH1 proteins, b) investigations on the possible impact of 8-oxo-dGTPase activity on 8-oxo-2'-deoxyguanosine (8-oxo-dG) content in DNA of mouse organs, c) in vivo investigations on the effects of cadmium(II) and nickel(II) ions on the 8-oxo-dGTPase activity and 8-oxo-dG level in DNA of rat organs that resulted in a proposal of a new mechanism of cadmium testicular carcinogenesis in rodents, d) an identification and kinetic description of a whole class of mammalian 8-oxo-dGTPase activity inhibitors (nucleoside 5'-diphosphates) and the analysis of their potential influence on the effectiveness of 8-oxo-dGTP decomposition in vivo, e) a search for explanation to diverse MTH1 expression levels in different physiological cell types and MTH1 overexpression in cancer cells; in order to partially elucidate those phenomena different experimental approaches were used to relate cellular 8-oxo-dGTPase activity of MTH1 protein to cell cycling, proliferation rate and cell-to-cell contact

Hepatitis B Virus X Protein Increases 8-Oxo-7,8-Dihydro-2ʹ-Deoxyguanosine (8-Oxodg) Level via Repressing MTH1/ MTH2 Expression in Hepatocytes

Background/Aims: Chronic hepatitis B virus (HBV) infection markedly increases the risk of development of hepatocellular carcinoma (HCC). Among the seven viral proteins that HBV encodes, HBV X protein (HBx) appears to have the most oncogenic potential. The mitochondria-associated HBx can induce oxidative stress in hepatocytes, leading to the production of abundant reactive oxygen species (ROS). High levels of ROS usually induce oxidative DNA damage and 8-hydroxy-2-deoxyguanosine (8-OHdG), also known as 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is one of the major products of DNA oxidation and an important biomarker for oxidative stress and carcinogenesis. Cells have evolved a mechanism to prevent oxidized nucleotides from their incorporation into DNA through nucleotide pool sanitization enzymes of MTH1 (NUDT1), MTH2 (NUDT15), MTH3 (NUDT18) and NUDT5. However, little is known as to whether HBx can regulate the expression of those enzymes and modulate the formation and accumulation of 8-oxodG in hepatocytes. Methods: The level of 8-oxodG was assessed by ELISA in stable HBV-producing hepatoma cell lines, an HBV infectious mouse model, HBV and HBx transgenic mice and HBV-infected patients versus their respective controls. Expression of MTH1, MTH2, MTH3 and NUDT5 was determined by a real-time quantitative PCR and western blot analysis. Transcriptional regulation of MTH1 and MTH2 expression by HBx and the effect of HBx on MTH1 and MTH2 promoter hypermethylation were examined using a luciferase reporter assay and bisulfite sequencing analysis. Results: In comparison with controls, significantly higher levels of 8-oxodG were detected in the genome and culture supernatant of stable HBV-producing HepG2.2.15 cells, in the sera and liver tissues of HBV infectious mice and HBV or HBx transgenic mice, and in the sera of HBV-infected patients. Expression of HBx in hepatocytes significantly increased 8-oxodG level and reduced the expression of MTH1 and MTH2 at both mRNA and protein levels. It was also demonstrated that HBx markedly attenuated the MTH1 or MTH2 promoter activities through hypermethylation. Furthermore, enhancement of 8-oxodG production by HBx was reversible by overexpression of MTH1 and MTH2. Conclusion: Our data show that HBx expression results in the accumulation of 8-oxodG in hepatocytes through inhibiting the expression of MTH1 and MTH2. This may implicate that HBx may act as a tumor promoter through facilitating the mutational potential of 8-oxodG thus connecting a possible link between HBV infection and liver carcinogenesis

Deoxyguanosine triphosphate, a possible target for reactive oxygen species-induced mutagenesis

Intracellular dNTP pool sizes are highly asymmetric, with dGTP usually comprising 5 to 10% of the sum of the dNTP pools. The work presented in this dissertation addresses the question of whether the underrepresentation of dGTP is related to its potential to be oxidized by reactive oxygen species. 8-oxo-guanine is important in oxidative mutagenesis, and current evidence indicates that this lesion arises in DNA partly through oxidation of dGTP, followed by incorporation of 8-oxo-dGTP into DNA. The bacterial MutT protein and its mammalian homolog catalyze the hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP in vitro. It is a widely accepted premise that the primary function of these enzymes is to remove 8-oxo-dGTP from the nucleotide pool of cells so that it cannot be used as a substrate for DNA synthesis. However, this model has been called into question by observations that some mutT strains of E. coli display a mutator phenotype when grown anaerobically, and by kinetic studies that showed 8-oxo-dGTP to be a poor DNA polymerase substrate. In this study, the dNTP pools of mammalian cells cultured in varying oxygen conditions were measured, with the expectation that the dGTP pool would expand under low oxygen conditions if it were a target for damage by reactive oxygen species. HeLa cells cultured in 2% 0₂ showed no change in the dGTP pool when compared to cells cultured in 20% 0₂; however, in V79 cells, the dGTP pool did expand in 2% 0₂. This result was not specific to the dGTP pool, as pools of dATP and dTTP also increased when V79 cells were cultured at 2% 0₂. These results suggest that there may be increased turnover of the dGTP pool when cells are cultured in high oxygen, but these experiments did not address the reason for this oxygen-dependent change. In order to determine whether 8-oxo-dGTP accumulates to levels that are sufficient to cause mutagenesis in cells, an analytical method for the measurement of 8-oxo-dGTP from cell extracts was developed. By use of this method, which involves reversed-phase high performance liquid chromatography coupled with electrochemical detection, no 8-oxo-dGTP was detected in mutT E. coli cells, even when they were cultured in the presence of H₂0₂. The estimated upper limit of 8-oxo-dGTP in these cells is about 240 molecules per cell, which corresponds to an intracellular concentration of approximately 0.34 μM. When 8-oxo-dGTP was added at this concentration to an in vitro DNA replication system in which replication errors could be scored as mutations, along with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect on the frequency of mutation. Therefore, the presence of 8-oxo-dGTP at physiologically relevant concentrations does not appear to be significantly mutagenic. The results presented in this dissertation suggest that the mechanism by which the MutT enzyme counteracts mutagenesis should be reevaluated

Role of Operon aaoSo-mutT in Antioxidant Defense in Streptococcus oligofermentans

Previously, we have found that an insertional inactivation of aaoSo, a gene encoding L-amino acid oxidase (LAAO), causes marked repression of the growth of Streptococcus oligofermentans. Here, we found that aaoSo and mutT, a homolog of pyrophosphohydrolase gene of Escherichia coli, constituted an operon. Deletion of either gene did not impair the growth of S. oligofermentans, but double deletion of both aaoSo and mutT was lethal. Quantitative PCR showed that the transcript abundance of mutT was reduced for 13-fold in the aaoSo insertional mutant, indicating that gene polarity derived from the inactivation of aaoSo attenuated the expression of mutT. Enzymatic assays were conducted to determine the biochemical functions of LAAO and MutT of S. oligofermentans. The results indicated that LAAO functioned as an aminoacetone oxidase [47.75 nmol H2O2 (min·mg protein)–1]; and MutT showed the pyrophosphohydrolase activity, which removed mutagens such as 8-oxo-dGTP. Like paraquat, aaoSo mutations increased the expression of SOD, and addition of aminoacetone (final concentration, 5 mM) decreased the mutant’s growth by 11%, indicating that the aaoSo mutants are under ROS stress. HPLC did reveal elevated levels of cytoplasmic aminoacetone in both the deletion and insertional gene mutants of aaoSo. Electron spin resonance spectroscopy showed increased hydroxyl radicals in both types of aaoSo mutant. This demonstrated that inactivation of aaoSo caused the elevation of the prooxidant aminoacetone, resulting the cellular ROS stress. Our study indicates that the presence of both LAAO and MutT can prevent endogenous metabolites-generated ROS and mutagens. In this way, we were able to determine the role of the aaoSo-mutT operon in antioxidant defense in S. oligofermentans

Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells

7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [14C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 106 nucleotides, 5–6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis

Effect of sequence and metal ions on UVB-induced anti cyclobutane pyrimidine dimer formation in human telomeric DNA sequence

Irradiation of G-quadruplex forming human telomeric DNA with ultraviolet B (UVB) light results in the formation of anti cyclobutane pyrimidine dimers (CPDs) between loop 1 and loop 3 in the presence of potassium ions but not sodium ions. This was unexpected because the sequences involved favor the nonphotoreactive hybrid conformations in K+ solution, whereas a potentially photoreactive basket conformation is favored in Na+ solution. To account for these contradictory results, it was proposed that the loops are too far apart in the basket conformation in Na+ solution but close enough in a two G-tetrad basket-like form 3 conformation that can form in K+ solution. In the current study, Na+ was still found to inhibit anti CPD formation in sequences designed to stabilize the form 3 conformation. Furthermore, anti CPD formation in K+ solution was slower for the sequence previously shown to exist primarily in the proposed photoreactive form 3 conformation than the sequence shown to exist primarily in a nonphotoreactive hybrid conformation. These results suggest that the form 3 conformation is not the principal photoreactive conformation, and that G-quadruplexes in K+ solution are dynamic and able to access photoreactive conformations more easily than in Na+ solution

Estrés oxidativo y expresión de proteínas relacionadas en enfermedades de alto riesgo cardiovascular: estudio especial de la hipertensión arterial

RESUMEN Entre las múltiples y variadas implicaciones fisiopatológicas del Estrés Oxidativo, los trastornos cardiovasculares son, sin duda, uno de los objetivos prioritarios y motivo de interés de un gran número de investigadores biomédicos. Uno de los principales factores de riesgo es el desarrollo de una serie de enfermedades frecuentes en nuestra población como son la hipertensión arterial y las dislipemias. Éstos trastornos, tienen una vinculación más o menos estrecha, directa o indirecta con el fenómeno que conocemos como estrés oxidativo y los 3 tipos de patologías poseen aumentados los niveles de EO de manera que se potencia el riesgo de aparición de eventos cardiovasculares. Objetivo de este estudio es comparar los niveles de EO en las diferentes patologías cardiovasculares seleccionadas (HTA con y sin Síndrome Metabólico asociado, HF y HFC) midiendo la respuesta de las células mononucleares frente al EO crónico a través de la medida de los niveles de ARNm de las actividades antioxidantes en cada una de ellas. Dicho estudio comparativo se va a realizar mediante el análisis de los siguientes parámetros oxidativos en orina, plasma y en células circulantes de los grupos seleccionados y el grupo control: 1.- Estudio de los productos de oxidación, marcadores de EO y metabolitos relacionados (GSH, GSSG, 8-oxo-dG nuclear, mitocondrial y urinaria, MDA, proteínas carboniladas y niveles de NO3/NO2 . 2.- Determinación de la actividad de los principales sistemas antioxidantes y prooxidante (SOD Cu-Zn y Mn, Cat, GPx, GSR y XO) . 3.- Cuantificación de los niveles de ARNm por RT-PCR cuantitativo a tiempo real, de los genes que codifican dichas actividades enzimáticas, además de OGG1 y G6PDH. CONCLUSIONES 1) En las tres patologías, HTA, HF y HFC, los resultados obtenidos sugieren que el EO representa un mecanismo común y subyacente en la patogenicidad de dichos procesos y posiblemente contribuya al desarrollo de complicaciones cardiovasculares, si bien, el mecanismo bioquímico-molecular responsable del proceso oxidativo presenta características diferenciales entre ellas, así como la intensidad de respuesta al EO. 2) Los niveles elevados de EO en estas patologías no dependen exclusivamente de un aumento en su producción ya que existe una menor actividad antioxidante. La disminución de los mecanismos antioxidantes se debe principalmente a: - Actividad reducida de los genes antioxidantes y del sistema GSH. - Menores niveles de ARNm, disminuyendo así su capacidad de producir enzimas y sistemas protectores frente a EO. 3) Los pacientes de HTA sin tratamiento se encuentran peor protegidos frente a EO que los controles, a pesar de la activación de algunos sistemas antioxidantes como el sistema Tiorredoxina y la enzima Manganeso Superóxido Dismutasa. 4) Los pacientes de HTA con y sin síndrome metabólico asociado no presentan diferencias significativas entre sí, lo que nos confirma que es la HTA el factor asociado al estrés oxidativo, siendo indiferentes las otras características que determinan el diagnóstico de síndrome metabólico. 6) La hipertensión arterial progresa con una marcada oxidación del material genético que se verifica por el aumento en la producción de la base modificada y mutagénica 8-oxo-dG como resultado del estrés oxidativo y de la disminución de enzima reparadora OGG1 en las células mononucleares de los pacientes hipertensos. 7) De los marcadores analizados, la liberación de 8-oxo-dG en orina es el parámetro más representativo del estrés oxidativo en la hipertensión arterial. 8) Los tres modelos de EO crónico analizados poseen un aumento en los niveles de EO pero distinta intensidad en su respuesta. Los pacientes se encuentran desprotegidos frente al EO. __________________________________________________________________________________________________The main aim of this study is to compare the EO levels in some different cardiovascular diseases, previously selected (control, HTA with and without an associate metabolic syndrome, HF and HFC), by measuring the response of mononuclear cells against the chronicle EO through the RNAm level in the antioxidant activity of each one. This comparative study is carried out in different groups by the analyse of different oxidative parameters in urine, plasma and circulated cells. 1.- Study of oxidation products , EO markers and related metabolites. 2.- Main antioxidant and prooxidant systems activity determination. 3.- RNAm level quantification by a real time quantitative RT-PCR. CONCLUSIONS: 1.- The results of the three pathologies (HTA, HF and HFC) propose that EO represents a common and underlying mechanism in their pathologies process and probably it contributes to develop cardiovascular complications, although the responsible biochemistry-molecular mechanism of the oxidative process shows many different features between them. 2.- The high levels of EO in these pathologies do not exclusively depends on an increase of its production because the oxidant activity is lower. 3.- HTA patients without a treatment are worse protected against EO that the controled ones, despite of the activation of some antioxidant systems like Tiorredoxine System and SOD Mn enzyme. 4.- HTA patients with and without the associated metabolic syndrome do not show great differences between them, so it confirms that HTA is the associated factor to the oxidative stress. 5.- The High blood pressure progresses with a high oxidation of the genetic material, that it is verified by the increase of the production of modify base and mutagenig 8- oxo-dG as a result of oxidative stress and the decrease of repairing enzyme OGG1 in the mononuclear cells. 6.- In analysed markers, the 8-oxo-dG liberation in urine is the most representative parameter of oxidative stress in high blood pressure. 7.- The three EO chronicle models analysed have an increase on EO level but different intensity in their response so patients are unprotected against EO

N-Cadherin Expression And EMT Progression In Arsenic- And Cadmium-Transformed Urotsa

Environmental agents are common causes of bladder cancer. Specifically, arsenic (As3+) and cadmium (Cd2+) are known carcinogens implicated in the development of bladder cancer. Previous studies from our laboratory have shown that As3+ and Cd2+ can cause malignant transformation of normal immortalized bladder urothelial cells, which can form tumors when injected subcutaneous or intraperitoneal into nude mice. Microarray analysis of repeated metal transformation in parallel revealed that N-cadherin was the most upregulated gene in As3+ transformants, and a top induced gene in Cd2+-transformed cells. The switch from E-cadherin to N-cadherin is a well-known indicator of the epithelial-to-mesenchymal transition occurring in bladder cancer. Further, N-cadherin upregulation is correlated with tumor stage, increased recurrence, and decreased survival in patients. While the factors mediating the decrease in E-cadherin expression are well-established, little is known of the factors regulating the increase in N-cadherin expression. The goal of the present study was to determine how As3+ and Cd2+ regulate N-cadherin expression, whether this expression is maintained in heterotransplant models, and if N-cadherin is promoting the epithelial-to-mesenchymal transition in As3+- and Cd2+-transformed UROtsa cells in vitro. This work has demonstrated that N-cadherin is induced in As3+- and Cd2+-transformed UROtsa cell lines, and that the expression is maintained in intraperitoneal, but not subcutaneous, tumor xenografts. Further, tumor-initiating cells derived from transformed UROtsa cells did not express N-cadherin. This suggests that tumor microenvironment and heterogeneity of cell populations are important factors for the use of animal models in cancer research. The As3+ and Cd2+ UROtsa cell lines represent the initial phases of EMT in bladder cancer, and may demonstrate a unique EMT pathway specific to heavy metal carcinogens. Transcriptional regulation of N-cadherin, which is mostly unknown, may be elucidated by the investigation of the transcription factor Twist and epigenetic regulation, particularly histone acetylation

Prenatal cadmium exposure dysregulates sonic hedgehog and Wnt/beta-catenin signaling in the thymus resulting in immunomodulatory effects

Cadmium (Cd) is both an environmental pollutant as well as a component of cigarette smoke. Recent data suggests increased cancer risks and increased mortality in environmentally exposed populations. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports in the literature of immunomodulatory effects of prenatal exposure to Cd. The sonic hedgehog (Shh) and Wnt/ss-catenin pathways are required for thymocyte maturation. Several studies have demonstrated that Cd exposure affects these pathways in different organ systems. Our experiments were designed to investigate the effect of prenatal Cd exposure on thymocyte development, and to determine if these effects were linked to dysregulation of Shh and Wnt/ss-catenin pathways. In addition, longer term effects of prenatal Cd on the immune system were investigated. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose (10 ppm) of Cd during pregnancy and effects on the thymus of the offspring were assessed on post-natal day 0 (PND0), while effects on the thymus and spleen were assessed on PND14 and 49. On PND0, thymocyte phenotype was determined by flow cytometry. A Gli:luciferase reporter cell line was used to measure Shh signaling. Transcription of target genes and translation of key components of both signaling pathways was assessed using real-time RT-PCR and western blot, respectively. On PND14 and 49, thymocyte and splenocyte phenotypes were analyzed, and cytokine production of splenic T cells was determined by ELISA. On PND0, prenatal Cd exposure increased the number of CD4+ cells and a subpopulation of double-negative cells (DN; CD4-CD8-). Shh and Wnt/ss-catenin signaling were both decreased in the thymus; however, this was not due to altered Shh and Wnt protein levels. Target genes of Shh and Wnt/ss-catenin were affected differentially among thymocyte subpopulations. On PND14 and 49, prenatal Cd exposure increased the number of DN thymocytes. In the spleen, prenatal Cd exposure had a cell type-, sex-specific effect on splenocyte phenotype and cytokine production. Collectively, these findings suggest that even very low exposure to Cd during gestation dysregulates two signaling pathways in the thymus resulting in altered thymocyte development, and this dysregulation can result in long term detrimental effects on the immune system of the offspring

Effect of DNA Sequence Context, DNA structure, and Excitation Method on Cyclobutane Pyrimidine Dimer Formation

The cis-syn cyclobutane pyrimdine dimer (CPD) is the major photoproduct resulting from UV irradiation of duplex DNA that results in C to T mutations found in human skin. CPDs with the anti stereochemistry were recently discovered to be formed in human telomeric DNA that adopts a quadruplex structure in vitro and may also play a role in the effects of sunlight in vivo. In this thesis, the effect of telomeric DNA structure on the formation of the anti-CPDs is investigated, as well as the effect of sequence context on cis-syn CPD formation which could help explain the origin of DNA mutation hot spots and cold spots. Originally it was hypothesized that anti CPDs formed from G-quadruplex structures, but I now show that anti CPDs can also form in human telomeric DNA sequences when complexed with lithium ions that are known to disfavor G-quadruplex formation. I also show that anti CPDs can also form in the presence of potassium ion when selected guanine bases are changed to inosine to interfere with G-quartet formation. Most significantly weI show that anti-CPDs form in sequences containing A’s in place of G’s that cannot form Hoogsteen hairpins, but can form reverse Hoogsteen hairpins. These results suggest that reverse Hoogsteen hairpins may play a hitherto unrecognized role in the biology and photoreactivity of DNA in telomeres, and possibly in other purine-rich sequences found in regulatory regions. To study sequence context effects, we designed 129-mer sequences containing all 64 possible NPyPyN tetrads (where PyPy is CC, CT, TC, or TT, and N is A, C, G or T), and used a T4 endonuclease gel electrophoresis assay to determine the relative yields of photoproduct formation at the different sites. The results show that CPD yields for different tetrads varied by as much as an order of magnitude. The yield of CPDs under UVC irradiation at a given site decrease in the order TT \u3e TC \u3e CT \u3e CC, whereas the yield of CPDs under UVB irradiation decreased in the order TT \u3e TC \u3e CC \u3e CT. The yield of CPD formation was lowest with a 5’-G and highest with a 5’-T, whereas the yield was lowest with either a 3’-C, G or T, and highest with a 3’-flanking A. We also studied the sequence context effect on photosensitized CPD formation in the presence of acetone and norfloxacin. The results not only show that the efficiency of photosensitized CPD formation depends on the flanking bases, but also indicates that the efficiency depends on the structure and properties of the photosensitizer. These results suggest that the photosensitizers may result in unique mutation spectra that can be used to identify endogenous photosensitizers such as those implicated in chemi-excitation pathways in melanoma induction