Research Program of the Essigmann Lab

Cellular Responses to DNA Damage

The research objective of the Essigmann laboratory is to understand the relationship between the structures of lesions formed in the genome by DNA damaging agents and the specific biological endpoints of mutation, cancer, and cell death. In the area of carcinogenesis, we probe the molecular etiology of human cancer. Our parallel studies on genotoxic drugs focus upon uncovering the mechanism of action of existing drugs. Based upon that understanding, we design novel compounds that could be useful for the treatment of cancer, polycystic kidney disease, and viral diseases.


Background. The biological effects of ionizing radiation, most chemical carcinogens, and many antitumor drugs are dependent upon the ability of these agents to act as electrophiles in the vicinity of DNA (inside broken lines). Chemical carcinogens, for example, are either inherently electrophilic or they are activated to reactive species by cellular enzymes. Electrophiles are also produced intracellularly by normal metabolic processes.  Similarly, reactive compounds are produced by inflammatory cells of the immune system.

The reactive intermediates modify DNA, RNA and protein, forming covalent adducts in which the chemical residue is joined to nucleophilic atoms of the constituent nucleotides or amino acids.  Adducts within DNA have special significance in view of their potential to force replication or repair errors and thus lead to heritable genetic alterations. The resulting mutations may constitute an important step in the pathway leading to neoplastic transformation.

The work of this laboratory addresses the biochemical mechanisms by which cells respond to specific forms of DNA damage. The rationale of our work stems from the possibility that the DNA adducts caused by DNA damaging agents will be either mutagenic or cytotoxic, or both


Mutagenic Properties of DNA Adducts

Exposure of cells to DNA carcinogenic damaging agents usually results in the formation of a vast population of structurally heterogeneous chemical-DNA adducts. It is widely believed that misreplication of the adducts initiates cells along the pathway to malignancy. A large body of evidence indicates, however, that only a subset of the adduct population is likely to contribute to mutagenicity and carcinogenicity. When we began our work, a central problem in the field of carcinogenesis was the lack of an experimental system to identify which DNA lesion gave rise to which types of mutations. Our laboratory established such a system.


As shown in this figure, the genome of a virus or plasmid is processed by using recombinant DNA techniques to situate a small gap at a specific site. An oligonucleotide containing a single DNA adduct is then synthesized and ligated into the gap. The site specifically modified genome is introduced into a bacterial or mammalian cell, allowed to replicate intra- or extra-chromosomally and, finally, progeny are isolated. Reduction in the yield of progeny is an indication of the genotoxicity of the adduct. We also determine the type, amount, and genetic requirements for mutagenesis induced by the adduct. This technology enables one to rank the mutagenic and genotoxic potentials of the various adducts that form in the genomes of cells treated with DNA damaging agents. Using this system we have defined the genetic effects of the DNA lesions induced by oxidants and ionizing radiation, simple alkylating agents, aflatoxin B1, cisplatin, 4-aminobiphenyl, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), vinyl chloride, and a host of other agents.  By studying mutagenesis and toxicity of single DNA lesions in cells with defects in replication or repair, we have sometimes been able to define the roles of specific repair proteins in countering the effects of DNA adduct or of specific replication proteins as the mediators of mutagenesis.  Occasionally we follow up those genetic studies by defining the biochemical mechaism of a new DNA repair enzyme (e.g., our studies on the AlkB family proteins). 

Our current studies are in four areas: (i) understanding how aflatoxin B1 (AFB1) causes mutations that give rise to tumors, (ii) understanding how oxidized DNA bases give rise to mutations, (iii) trying to apply knowledge of mutagenesis toward the development of drugs that use mutagenesis as their mechanism of action (lethal mutagenesis of viruses or mutagenic expansion of neoantigen arrays on tumor cells) and (iv) probing how patterns of mutagenesis in intact tissues of animals helps explain the process of malignant transformation.  In this latter area, we have focused on aflatoxin in the past and are funded to study nitrosamines and alcohol in future work.  Aflatoxin, N-nitrosodimethylamine and ethyl alcohol are a major causes of concern worldwide owing to their ability to induce liver cancer. In earlier studies we showed that the exo-epoxide of AFB1 gives rise to its DNA adducts, and recently we showed that the N7 guanine adduct formed by the epoxide has a mutational signature that matches (in terms of genetic requirements) that of the toxin in cellular systems. Human tumors from Asia and Africa, where exposure to AFB1 is a major problem, show the same mutation as seen in our studies.  We have probed the biology of AFB1-treated liver in mice and rats by using transcriptional arrays and high-resolution mutational spectrometry, with a specific emphasis on understanding why young animals are more sensitive to cancer than older ones (figure below).  Prenatal exposure to the toxin was recently shown to enhance the mutagenic threat of aflatoxin by up to 20-fold, and current studies are aimed at understanding the molecular basis for this observation.