From Basic Research in Toxicology to a Career in Regulatory Toxicology: An Industry Perspective.

Regulatory toxicology helps define the balance between health risk and societal benefit of chemicals by applying a science-based approach, thus representing a potential career opportunity for scientists involved in biomedical research.

O6-methylguanine-induced transcriptional mutagenesis reduces p53 tumor-suppressor function.

Altered protein function due to mutagenesis plays an important role in disease development. This is perhaps most evident in tumorigenesis and the associated loss or gain of function of tumor-suppressor genes and oncogenes. The extent to which lesion-induced transcriptional mutagenesis (TM) influences protein function and its contribution to the development of disease is not well understood. In this study, the impact of O6-methylguanine on the transcription fidelity of p53 and the subsequent effects on the protein's function as a regulator of cell death and cell-cycle arrest were examined in human cells. Levels of TM were determined by RNA-sequencing. In cells with active DNA repair, misincorporation of uridine opposite the lesion occurred in 0.14% of the transcripts and increased to 14.7% when repair by alkylguanine-DNA alkyltransferase was compromised. Expression of the dominant-negative p53 R248W mutant due to TM significantly reduced the transactivation of several established p53 target genes that mediate the tumor-suppressor function, including CDKN1A (p21) and BBC3 (PUMA). This resulted in deregulated signaling through the retinoblastoma protein and loss of G1/S cell-cycle checkpoint function. In addition, we observed impaired activation of apoptosis coupled to the reduction of the tumor-suppressor functions of p53. Taking these findings together, this work provides evidence that TM can induce phenotypic changes in mammalian cells that have important implications for the role of TM in tumorigenesis.

Mechanism of RNA polymerase II stalling by DNA alkylation.

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

Minor Groove 3-Deaza-Adenosine Analogues: Synthesis and Bypass in Translesion DNA Synthesis.

Anticancer drugs that alkylate DNA in the minor groove may give rise to 3-alkyl-adenosine adducts that interfere with replication, inducing apoptosis in rapidly dividing cancer cells. However, translesion DNA synthesis (TLS) by polymerase enzymes (Pols) with the capacity to bypass DNA adducts may contribute to damage tolerance and drug resistance. 3-Alkyl-adenosine adducts are unstable and depurinate, which is a barrier to addressing chemical and enzymatic aspects of how they impact the progress of DNA Pols. To characterize structure-based relationships of 3-adenine alkylation relevant to cancer drugs on duplex stability and DNA Pol-catalyzed DNA synthesis, we synthesized stable 3-deaza-3-alkyl-adenosine analogues, including 3-deaza-3-phenethyl-adenosine and 3-deaza-3-methoxynaphthylethyl-adenosine, and incorporated them into oligonucleotides. A moderate reduction of duplex stability was observed on the basis of thermal denaturation data. Replication studies using purified Y-family human DNA Pols hPol η, κ, and ι indicated that these enzymes can perform TLS over the modified bases. hPol η had higher misincorporation rates when synthesizing opposite the modified bases compared with adenine, whereas hPol κ and ι maintained high fidelity. These results provide insight into how alterations in chemical structure reduce bypass of minor-groove adducts, and provide novel chemical probes for evaluating minor-groove DNA alkylation.