Acid-specific formaldehyde donor is a potential, dual targeting cancer chemotherapeutic/chemo preventive drug for FANC/BRCA-mutant cancer.

BACKGROUND: Development of chemotherapeutic/preventive drugs that selectively kill cancer - the Holy Grail of cancer research - is a major challenge. A particular difficulty arises when chemotherapeutics and radiation are found to be rather ineffective against quiescent cancer cells in solid tumors. In the limited oxygen condition within a solid tumor, glycolysis induces an acidic environment. In such an environment the compound hexamethylenetetramine (HMTA) will act as a formaldehyde donor. HMTA has been characterized a non-carcinogen in experimental animals and causes no major adverse side-effects in humans. We previously reported that both a chicken B-lymphocyte cell line transformed with an avian leucosis virus and human colon cancer cells deficient in the FANC/BRCA pathway are hypersensitive to formaldehyde. Thus, we assessed the potential usage of HMTA as a chemotherapeutic agent. RESULTS: The differential cytotoxicity of HMTA was tested using chicken DT40 cells deficient in DNA repair under neutral and acidic conditions. While HMTA is not efficiently hydrolyzed under neutral conditions, all HR-deficient DT40 cells tested were hypersensitive to HMTA at pH 7.3. In contrast, HMTA clearly increased cell toxicity in FANCD2-, BRCA1- and BRCA2- deficient cells under acidic conditions. CONCLUSION: Here we show that in vitro experiments showed that at low pH HMTA causes drastic cytotoxicity specifically in cells deficient in the FANC/BRCA pathway. These results strongly suggest that HMTA may be an attractive, dual-targeting chemotherapeutic/preventive drug for the selective delivery of formaldehyde to solid tumors and causes cell death in FANC/BRCA-deficient cells without major adverse effects.

Homologous Recombination and Translesion DNA Synthesis Play Critical Roles on Tolerating DNA Damage Caused by Trace Levels of Hexavalent Chromium.

Contamination of potentially carcinogenic hexavalent chromium (Cr(VI)) in the drinking water is a major public health concern worldwide. However, little information is available regarding the biological effects of a nanomoler amount of Cr(VI). Here, we investigated the genotoxic effects of Cr(VI) at nanomoler levels and their repair pathways. We found that DNA damage response analyzed based on differential toxicity of isogenic cells deficient in various DNA repair proteins is observed after a three-day incubation with K2CrO4 in REV1-deficient DT40 cells at 19.2 µg/L or higher as well as in TK6 cells deficient in polymerase delta subunit 3 (POLD3) at 9.8 µg/L or higher. The genotoxicity of Cr(VI) decreased ~3000 times when the incubation time was reduced from three days to ten minutes. TK mutation rate also significantly decreased from 6 day to 1 day exposure to Cr(VI). The DNA damage response analysis suggest that DNA repair pathways, including the homologous recombination and REV1- and POLD3-mediated error-prone translesion synthesis pathways, are critical for the cells to tolerate to DNA damage caused by trace amount of Cr(VI).

SWI/SNF complexes are required for full activation of the DNA-damage response.

SWI/SNF complexes utilize BRG1 (also known as SMARCA4) or BRM (also known as SMARCA2) as alternative catalytic subunits with ATPase activity to remodel chromatin. These chromatin-remodeling complexes are required for mammalian development and are mutated in ~20% of all human primary tumors. Yet our knowledge of their tumor-suppressor mechanism is limited. To investigate the role of SWI/SNF complexes in the DNA-damage response (DDR), we used shRNAs to deplete BRG1 and BRM and then exposed these cells to a panel of 6 genotoxic agents. Compared to controls, the shRNA knockdown cells were hypersensitive to certain genotoxic agents that cause double-strand breaks (DSBs) associated with stalled/collapsed replication forks but not to ionizing radiation-induced DSBs that arise independently of DNA replication. These findings were supported by our analysis of DDR kinases, which demonstrated a more prominent role for SWI/SNF in the activation of the ATR-Chk1 pathway than the ATM-Chk2 pathway. Surprisingly, γH2AX induction was attenuated in shRNA knockdown cells exposed to a topoisomerase II inhibitor (etoposide) but not to other genotoxic agents including IR. However, this finding is compatible with recent studies linking SWI/SNF with TOP2A and TOP2BP1. Depletion of BRG1 and BRM did not result in genomic instability in a tumor-derived cell line but did result in nucleoplasmic bridges in normal human fibroblasts. Taken together, these results suggest that SWI/SNF tumor-suppressor activity involves a role in the DDR to attenuate replicative stress and genomic instability. These results may also help to inform the selection of chemotherapeutics for tumors deficient for SWI/SNF function.

Convenient, multi-well plate-based DNA damage response analysis using DT40 mutants is applicable to a high-throughput genotoxicity assay with characterization of modes of action.

Chemists continually synthesize myriad new chemicals (∼2,000/year), some of which make their way into the environment or otherwise pose possible threats to humans who potentially become exposed to the compounds. Regulators must determine whether these, along with the glut (∼80,000) of existing, chemicals are toxic and at what exposure levels. An important component of this determination is to ascertain the mode of action (MOA) of each compound as it relates to the pathway the compound uses to induce genotoxicity. Several assays have traditionally been used to reveal these effects to the genome: the Ames test, tests with yeast and mammalian cell lines, and animal studies. Previously, we described a new multi-well plate-based method which makes use of the DT40 isogenic cell line and its dozens of available mutants knocked out in DNA repair and cell cycle pathways and we now provide a detailed protocol of the further improvement of the assay. Although the DT40 line has existed for some time and has been used in numerous studies of DNA repair pathways, little use has been made of this valuable resource for toxicological investigations. Our method introduces the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide dye scheme determination of cell survival in a manner that greatly increases throughput and reduces cost while maintaining reasonable sensitivity. Although this new genotoxicity assay requires validation with many more mutagens before becoming an established, regulatory decision-making analysis tool, we believe that this method will be very advantageous if eventually added to the repertoire of those investigating MOAs of potentially genotoxic substances.

Cells deficient in the FANC/BRCA pathway are hypersensitive to plasma levels of formaldehyde.

Formaldehyde is an aliphatic monoaldehyde and is a highly reactive environmental human carcinogen. Whereas humans are continuously exposed to exogenous formaldehyde, this reactive aldehyde is a naturally occurring biological compound that is present in human plasma at concentrations ranging from 13 to 97 micromol/L. It has been well documented that DNA-protein crosslinks (DPC) likely play an important role with regard to the genotoxicity and carcinogenicity of formaldehyde. However, little is known about which DNA damage response pathways are essential for cells to counteract formaldehyde. In the present study, we first assessed the DNA damage response to plasma levels of formaldehyde using chicken DT40 cells with targeted mutations in various DNA repair genes. Here, we show that the hypersensitivity to formaldehyde is detected in DT40 mutants deficient in the BRCA/FANC pathway, homologous recombination, or translesion DNA synthesis. In addition, FANCD2-deficient DT40 cells are hypersensitive to acetaldehyde, but not to acrolein, crotonaldehyde, glyoxal, and methylglyoxal. Human cells deficient in FANCC and FANCG are also hypersensitive to plasma levels of formaldehyde. These results indicate that the BRCA/FANC pathway is essential to counteract DPCs caused by aliphatic monoaldehydes. Based on the results obtained in the present study, we are currently proposing that endogenous formaldehyde might have an effect on highly proliferating cells, such as bone marrow cells, as well as an etiology of cancer in Fanconi anemia patients.