1985 |
Cordeiro-Stone, Marila |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Effects of Bpde On Dna Replication in Human Fibroblasts @ University of North Carolina Chapel Hill
The objective of this project is to characterize the mechanisms of inhibition of DNA replication by the carcinogen 7Beta, 8Alpha-dihydroxy-9Alpha, 10Alpha-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE-I) in cultured human fibroblasts. Effects on initiation of replicons, elongation and joining of nascent DNA strands to high molecular weight, and RNA synthesis will be evaluated in BPDE-I treated cells. By using mutant cell strains exhibiting deficiencies in excision repair or replicative by-pass the modifying effects of these processes will also be examined. Velocity sedimentation analyses will be used to describe the size distribution and quantity of pulse-labeled nascent DNA after BPDE-I treatment. In this technique, an inhibition of replicon initiation is manifested by an initial and selective inhibition of synthesis of nascent DNA with the same length as an average replicon (20 Mum); with time this inhibition spreads to include even larger molecules. Inhibition of DNA chain elongation is manifested by a uniform and dose-dependent reduction in synthesis of DNA in replicons that were already in operation at the time of carcinogen treatment. The relationship between this inhibition and the concurrent synthesis of abnormally small nascent molecules will be investigated using S1 nuclease as a probe for single-stranded regions opposite gaps in daughter DNA. These S1 nuclease-sensitive regions will be detected by the formation of small duplex DNA molecules which contain nascent strands synthesized after BPDE-I damage of the template DNA. Using aphidicolin to synchronize cells at the beginning of the S phase, the growth rates of nascent DNA will be determined in control and BPDE-I-treated cells. The synchronization should produce a less complex distribution of nascent DNA in the alkaline sucrose gradients facilitating analysis of its transition to higher molecular weights. A possible indirect effect of BPDE-I on replication resulting from inhibition of RNA transcription will be investigated by comparing the effects of known inhibitors of RNA synthesis on replicon initiation and DNA chain elongation. These studies should further clarify whether BPDE-I exerts its critical effects in a manner similar to ultraviolet radiation and help to establish the generality of a specific pattern of effects of carcinogens on DNA replication.
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1 |
1986 — 1988 |
Cordeiro-Stone, Marila |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Timing of Oncogene Replication and Carcinogenesis @ University of North Carolina Chapel Hill
The objective is to test the hypothesis that early S phase specificity for malignant transformation of cells by chemical carcinogens is due to the alteration of cellular oncogenes, at the time of their replication in the early S phase of the cell cycle. This hypothesis is supported by the 0ollowing observations: a) proliferating cells are very sensitive to chemical carcinogens and a higher frequency of transformation is observed when cells are treated at the beginning of the S phase; b) genes are replicated in S phase according to a specific temporal order that is maintained from one generation to the next; c) replicating DNA is more susceptible to chemical modifications by methylating carcinogens than bulk DNA; this characteristic, coupled to a smaller probability of repair before replication, should favor the fixation of genetic alterations; d) a recognized mechanism of activation of cellular oncogenes is through site-specific mutations. The above hypothesis will be tested by determining the time of replication of various cellular oncogenes, in several normal cells and their transformed counterparts, obtained in our laboratories according to defined protocols. The oncogenes to be studied in detail are the cellular homologs of v-erb, v-Ha-ras, v-Ki-ras, v-fos, v-sis and v-myc. To accomplish this task we will first develop a simple method for mapping the time of replication of specific genes. This method will be based on the enrichment of replicating sequences upon binding to nitrocellulose filters of DNA isolated from synchronized cells at different time points in the S phase. Subsequently, these filters will be blot-hybridized with specific gene-probes. The validity of this method will be checked by using DNA probes complementary to sequences that are known to be early- or late-replicating in fibroblasts. Furthermore, it will be used to verify whether transcription competence affects the time of replication of tissue-specific genes in fibroblasts, as compared to epithelial cells. We also consider important to compare the time of replication of oncogenes with their time of maximum transcription. It is conceivable that genes are also more susceptible to mutation by chemical carcinogens during a time when they are highly transcribed. Therefore, the results of these studies should help to elucidate the possible relationship between replication (or transcription) of cellular oncogenes and the higher frequency of transformation by chemical carcinogens in early S phase of the cell cycle.
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1 |
1991 — 2004 |
Cordeiro-Stone, Marila |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Replication of Damaged Dna in Human Cells @ University of North Carolina Chapel Hill
The long-term goal of this project is to characterize at the molecular level a pathway of replication of UV-damaged DNA in human cells. Our hypothesis is that normal human cells contain a factor (or factors) that contributes to the replication of UV-induced pyrimidine dimers in a error-free manner and without detectable inhibition of DNA strand growth. Xeroderma pigmentosum (XP) variant cells are hypersensitive to mutagenesis and carcinogenesis by UV, even though they have normal levels of excision repair. Instead, XP variant cells have an increased sensitivity to inhibition of DNA strand growth by UV lesions present in chromosomal DNA. We have proposed to establish whether this phenomenon can be detected also during the replication of episomal DNA damaged by UV. We will determine the optimal conditions for replication in normal and XP variant cells of a plasmid carrying the SV40 genome (Specific Aim 1). We expect that both cell types will be equally proficient in replicating the undamaged plasmid. Then, we will measure the effects of a defined number of pyrimidine dimers or benzo[a]pyrene-diolepoxide-I (BPDE) adducts on the replication of the SV40 plasmid (Specific Aim 2). We expect the UV-irradiated plasmid to replicate better in normal than in XP variant cells. However, this differential response is not predicted for experiments in which the plasmid DNA will be adducted by BPDE-I. Origin-defective plasmids will be used as negative controls in our replication studies and to measure the incorporation of labeled precursors in plasmid DNA by excision repair. The data generated in this project will guide us in the development of in vitro assays that can detect differences in whole extracts of different human cells in their capabilities to carry on the replication of UV-damaged DNA templates (Specific Aim 3). Finally, we will initiate complementation studies to identify putative factors that participate in this process. These specific aims will extend our knowledge of molecular events operating when replication forks encounter damaged sites in DNA. They seek to demonstrate the existence of carcinogen-dependent pathways of replication of damaged DNA. This project will begin to dissect into its molecular components the UV-dependent pathway. Overall, this study will focus on a DNA metabolic pathway that contributes to the protection of normal human beings from the carcinogenic effects of UV light, thus reducing their susceptibility to skin cancers.
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1 |
2007 — 2011 |
Cordeiro-Stone, Marila |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cellular Responses to Environmental Insults and Risk For Melanoma Development @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): This project will test the hypothesis that defects in DNA repair are acquired during stages of melanoma development and produce a chromosomal-mutator phenotype in UV-irradiated cells. Focus will be on nucleotide excision repair (NER), post-replication repair (PRR), DNA double-strand break (dsb) repair and intra-S checkpoint responses to the component of sunlight (UV radiation) that is most efficient in inducing DNA damage. In the three specific aims, DNA repair will be quantified in strains of normal human melanocytes, in melanoma cell lines, and in melanocytes expressing mutated genes commonly found in melanoma. Specific Aim 1 will determine whether NER capacity is attenuated or lost during stages of melanomagenesis. PRR capacity (translesions synthesis and gap repair) and the intra-S checkpoint response of inhibition of replicon initiation will be measured in Specific Aim 2. Proposed studies will also investigate the hypothesis that UV-induced DNA damage triggers a signaling pathway that results in trans-inhibition of DNA chain elongation;this novel element of the intra-S checkpoint is postulated to reduce the rate of fork displacement in replicons that have not yet encountered a template lesion. A DNA fiber-spreading and immuno-staining assay will enable visualization of replication dynamics and measurement of individual replication tracks. Knockdown of the Timeless-interacting protein (Tipin), together with ectopic expression of RNAi-resistant Tipin, will test whether this protein regulates the rate of displacement of DNA replication forks through its binding to RPA. Induction of chromosomal aberrations and INK4a allelic deletions in UV-treated cells will be examined in Specific Aim 3. Aberrations and deletions are thought to be associated with DNA dsb generated at collapsed replication forks and other single-strand DNA regions formed during replication of the UV-damaged DNA. Phospho-histone H2AX/phospho-ATM/Mre11-positive nuclear foci will be quantified to monitor the formation and repair of UV-induced DNA dsb. Studies will determine whether genetic alterations associated with melanoma development enhance UV-clastogenesis in cultured melanocytes. The proposed studies will shed light on how an environmental carcinogen, sunlight, induces skin cancer by expanding our knowledge of DNA damage responses in normal human melanocytes and providing insight into the mechanisms of genetic instability in melanoma.
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1 |
2010 — 2011 |
Chastain, Paul D Cordeiro-Stone, Marila |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Sunlight and Melanomagenesis @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): While considerable attention has been given to mapping sites of solar radiation-induced mutations in a number of skin cancer-related genes, less is known about the overall distribution of the original DNA damage and how that damage influences replication and genomic integrity. The goal of this revised R21 project is to initially visualize and analyze the distribution of specific photoproducts (e.g. cyclobutane pyrimidine dimers (CPDs) and [6-4] pyrimidine-pyrimidone photoproducts (6-4PPs)) and relate sites of damage to selected genomic locations and areas undergoing replication. We will then determine whether specific chromosomal regions or genes associated with different types of skin cancer, such as p53 (basal cell carcinomas and squamous cell carcinomas), PTCH1 (basal cell carcinomas), CDKN2A (melanoma), chromosomal region 9p (basal cell carcinomas), are more susceptible to UV-induced DNA damage formation or replication stalling. These two factors increase risk for point mutations through error-prone translesion synthesis and for deletions and translocations originated during the recombinational repair of the stalled replication forks that have collapsed. PUBLIC HEALTH RELEVANCE: While considerable attention has been given to mapping sites of solar radiation-induced mutations in a number of skin cancer-related genes, less is known about the overall distribution of the original DNA damage and how that damage influences replication and the probability of mutation fixation. The goal of this R21 project is to initially visualize and analyze the distribution of specific photoproducts and relate the damage sites to selected genomic locations and areas undergoing replication. We will then determine whether genes associated with skin cancer formation are more susceptible to UV- induced DNA damage formation or replication stalling. These two factors increase risk for point mutations through error-prone translesion synthesis and for deletions and translocations originated during the repair of double-strand DNA breaks that are formed by the collapse of replication forks.
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0.988 |