The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.

DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

PCR microfluidic devices for DNA amplification

It has been demonstrated that in humans certain factors such as early menarche, late pregnancy, and nulliparity are associated with a higher risk of developing breast cancer, while early pregnancy acts as a protective factor. Induction of mammary cancer in rats by administration of the chemical carcinogen 7, 12-dimethylbenz(a)anthracene reveals that the same factors influencing human breast cancer risk also affect the susceptibility of the rat mammary gland to the chemical carcinogen. Nulliparous rats and rats undergoing pregnancy interruption are more susceptible to developing carcinomas. This fact has been attributed to the incomplete differentiation of the gland at the time of carcinogen administration. Parous rats are resistant to the carcinogenic effect of DMBA, which is explained by the complete development of the gland attained during pregnancy and lactation. This development is manifested by the differentiation of terminal end buds into secretory units, which have a smaller proliferative compartment; the epithelial cells of these secretory units have a longer cell cycle, less avidity for binding DMBA, and possess a more efficient DNA excision repair capacity.

Differentiation of the mammary gland and susceptibility to carcinogenesis

MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents.

The DNA base excision repair (BER) pathway is responsible for the repair of cellular alkylation and oxidative DNA damage. A crucial and the second step in the BER pathway involves the cleavage of baseless sites in DNA by an AP endonuclease. The major AP endonuclease in mammalian cells is Ape1/ref-1. Ape1/ref-1 is a multifunctional protein that is not only responsible for repair of AP sites, but also functions as a reduction-oxidation (redox) factor maintaining transcription factors in an active reduced state. Ape1/ref-1 has been shown to stimulate the DNA binding activity of numerous transcription factors that are involved in cancer promotion and progression such as Fos, Jun, NF(B, PAX, HIF-1(, HLF and p53. Ape1/ref-1 has also been implicated in the activation of bioreductive drugs which require reduction in order to be active and has been shown to interact with a subunit of the Ku antigen to act as a negative regulator of the parathyroid hormone promoter, as well as part of the HREBP transcription factor complex. Ape1/ref-1 levels have been found to be elevated in a number of cancers such as ovarian, cervical, prostate, rhabdomyosarcomas and germ cell tumors and correlated with the radiosensitivity of cervical cancers. In this review, we have attempted to try and assimilated as much data concerning Ape1/ref-1 and incorporate the rapidly growing information on Ape1/ref-1 in a wide variety of functions and systems.

Going APE over ref-1.

The antineoplastic agent arabinofuranosyl cytosine (ara-C) produces an inhibition of the pyrimidine dimer excision system of human DNA repair. Alkaline sucrose gradient analysis of DNA from normal human skin fibroblasts exposed to 20 J/m/sup 2/ of ultraviolet radiation (254 nm) shows an accumulation of DNA single-strand breaks when DNA repair is attempted in the presence of 10 ..mu..M ara-C. Cells from complementaion groups of xeroderma pigmentosum that are defective in early steps of excision repair show reduced numbers of DNA single strand breaks/10/sup 8/ daltons when compared with normal cells. Cesium chloride gradient analysis of radioactive precurser uptake during repair replication indicates that ara-C causes a 6 to 56% reduction in the number of nucleotide bases inserted into the DNA at concentrations of 1 and 10 ..mu..M, respectively. These concentrations result in the substitution for deoxycytidine (dCyd) by ara-C of 40 and 100%, respectively, in repaired regions. Repair inhibition is reversed by 50% upon removal of ara-C and by > 95% with the addition of 100 ..mu..M dCyd. Chromatography of digested DNA shows that incorporated ara-C is not removed during dCyd reversal, suggesting that ara-C incorporation per se does not play a significant role in the inhibition of repair synthesis.more » The repair inhibition observed here is dependent on 2 mM hydroxyurea, presumably due to reduction in the intracellular pool of dCyd. The overall results suggest the possibility that ara-C is a weak competitive inhibitor of DNA polymerases associated with ultraviolet-induced excision repair.« less

Inhibition of DNA excision repair in human cells by arabinofuranosyl cytosine: effect on normal and xeroderma pigmentosum cells.

Detailed comparative map of human chromosome 19q and related regions of the mouse genome

N-Alkylpurines induced in DNA by simple monofunctional alkylating agents are known to be cytotoxic and possibly indirectly mutagenic. These adducts are removed by the ubiquitous N-methylpurine-DNA glycosylase (MPG) in a multistep repair pathway. Chinese hamster ovary (CHO) cell clones expressing 2- to 16-fold enhanced levels of MPG activity were isolated from cells stably transfected with human MPG cDNA expression plasmids. The in vivo removal of 3-methyladenine and 7-methylguanine from some of these lines was analyzed and was observed to reflect their MPG levels. These cell lines did not develop increased resistance, as compared to the control, in regards to cytotoxic, mutagenic and sister chromatid exchange inducing effects of the alkylating agents that induce 3-alkyladenine and 7-alkylguanine as the major alkyl adducts in DNA. These results suggest that the MPG activity is not limiting in the multi-step repair pathway of N-alkylpurines in CHO cells.

Overexpression of human DNA repair protein N-methylpurine-DNA glycosylase results in the increased removal of N-methylpurines in DNA without a concomitant increase in resistance to alkylating agents in Chinese hamster ovary cells.

William C. Dunn

Papers

Inhibition of DNA excision repair in human cells by arabinofuranosyl cytosine: effect on normal and xeroderma pigmentosum cells.

Detailed comparative map of human chromosome 19q and related regions of the mouse genome

Overexpression of human DNA repair protein N-methylpurine-DNA glycosylase results in the increased removal of N-methylpurines in DNA without a concomitant increase in resistance to alkylating agents in Chinese hamster ovary cells.

Age-dependent modulation of tissue-specific repair activity for 3-methyladenine and O6-methylguanine in DNA in inbred mice.

PCR amplification and analysis of simple sequence length polymorphisms in mouse DNA using a single microchip device.