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Environmental Factor, November 2015

Intramural papers of the month

By Robert Brown, Greg Buchold, Deacqunita Diggs, Gabriel Knudsen, and Qing Xu

NTP finds molecular differences between hepatoblastomas and hepatocellular carcinomas

National Toxicology Program (NTP) researchers demonstrated that mouse hepatoblastomas (HB) are molecularly distinct from hepatocellular carcinomas (HCC). They also determined that, at the genomic level, mouse HB is similar to human HB. The work suggests that mouse HB is a suitable model for evaluating human cancer.

HB, a poorly differentiated embryonal neoplasm that typically occurs late in life in the mouse, is commonly associated with HCC. Historically, scientists believed HB was a malignant variant of hepatocellular tumors despite morphological differences. To address the question of origin and whether mouse HB is relevant to human HB development following chemical exposures, HB, HCC, and normal liver samples from B6C3F1 mice were used to determine relationships in global gene expression and mutation spectra of Ctnnb1 and Hras, two common hepatic cancer genes.

The researchers found mouse and human HB share similarities at the pathway level, including embryonic development pathway dysregulation, stem cell pluripotency pathways, and genomic imprinting. In contrast, HB and HCC had strikingly different global gene expression and mutation spectra, although both may arise from a common hepatic progenitor cell. The authors suggest that exome sequencing at high read depths and cell lineage tracing experiments may further define the cellular origin of HB. (GK)

CitationBhusari S, Pandiri AR, Nagai H, Wang Y, Foley J, Hong HL, Ton TV, DeVito M, Shockley KR, Peddada SD, Gerrish KE, Malarkey DE, Hooth MJ, Sills RC, Hoenerhoff MJ. 2015. Genomic profiling reveals unique molecular alterations in hepatoblastomas and adjacent hepatocellular carcinomas in B6C3F1 mice. Toxicol Pathol; doi:10.1177/0192623315599853 [Online 18 August 2015].

Mitochondrial POLG2 disease variants impair cellular energy production

According to NIEHS scientists, a disease variant of the human mitochondrial DNA (mtDNA) polymerase gamma p55 subunit, encoded by the POLG2 gene, is unable to properly bind to the p140 catalytic subunit encoded by POLG. As a result, the energy reserve in each cell is greatly diminished, and the patient develops POLG2-related disease. Understanding the mechanisms that lead to the development of mitochondrial disease will help scientists design therapies to treat these debilitating conditions.

The p55 homodimer binds to the catalytic subunit forming the polymerase gamma trimer. Polymerase gamma is responsible for replication and repair of mtDNA. Because all currently identified POLG2 patients harbor heterozygous mutations, and because monomers within the p55 homodimer do not readily dissociate, the authors theorized that patients harbor a mixture of p55 molecules: 25 percent wild-type homodimers, 25 percent variant homodimers, and 50 percent heterodimers.

Using a tandem affinity strategy to study p55 variant heterodimers, the researchers found that one variant was dominant negative when associated with wild type and disturbed the mtDNA replication machinery. When they tagged p55 disease variants with green fluorescent protein and transfected them into human cells, two additional disease variants were found to misfold and were unable to localize to mtDNA. Both phenomena led to decreased respiratory capacity. This study aids in our understanding of how these complex disorders develop and provides valuable cell line models of mitochondrial disease. (RB)

CitationYoung MJ, Humble MM, DeBalsi KL, Sun KY, Copeland WC. 2015. POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity. Hum Mol Genet 24(18):5184-5197.

Transient metal ions mediate the direction of DNA synthesis

NIEHS researchers have revealed that transient metal ions are essential for DNA polymerase beta (pol beta) to carry out its reverse reaction, or pyrophosphorolysis. The findings help scientists understand the mechanisms of DNA replication and may aid in the development of pharmaceutical treatments for cancer.

Pol beta catalyzes DNA synthesis, which is its forward reaction, and pyrophosphorolysis. While the enzyme adds one nucleotide to the DNA primer during DNA synthesis, it excises the primer terminal nucleotide during pyrophosphorolysis, making the DNA one base shorter. Since the removal of chain-terminating nucleotides during HIV/AIDS therapy and cancer chemotherapy may cause drug resistance, understanding the events involved in this reaction with all DNA polymerases is vital to effective drug design.

Previous data had shown that DNA polymerases use two metal ions, known as catalytic and nucleotide-binding metals, for the DNA synthesis reaction. The NIEHS team, however, identified a new metal ion-binding site in the enzyme. In this study, the researchers modeled the role of metal ions in pyrophosphorolysis by using computational calculation and biochemical assays. They found that the new metal ion is required to initiate the reaction of pyrophosphorolysis, but must be removed later for the reaction to proceed. Similar to a game of tug of war, the transient metal ions contribute to shifting the reaction in the reverse. (QX)

CitationPerera L, Freudenthal BD, Beard WA, Shock DD, Pedersen LG, Wilson SH. 2015. Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse. Proc Natl Acad Sci U S A 112(38):E5228-E5236.

The AF-2 domain of estrogen receptor alpha suppresses AF1

The C terminus ligand binding domain (AF-2) of estrogen receptor alpha (ERalpha) can inhibit the ligand-independent activation function in the N terminus (AF-1) of ERalpha, according to a team led by NIEHS researchers. The finding explains how the binding of estrogen activates its receptor by disrupting the inhibitory AF-2/AF-1 interaction. Since many estrogen-dependent tumors are treated with selective estrogen receptor modulators (SERMs) that inhibit ERalpha activity, discovery of this mechanism may help to determine the exposure risk from SERMs and other xenoestrogens. 

The scientists assigned various estrogenic compounds to distinct categories, using a cell culture- based assay with different mutant forms of ERalpha lacking the AF-1 domain, or mutations in single nucleotides of distinct structural regions within the AF-2 domain. They propose a model in which the inhibitory interaction of one subregion of AF-2 with AF-1 is suppressing other activating interactions, including another copy of the receptor which allows dimerization or interaction with activating cofactors (coregulator proteins). How estrogenic compounds produce distinct effects in different tissues is under study. (GB)

CitationArao Y, Coons LA, Zuercher WJ, Korach KS. 2015. Transactivation function-2 of estrogen receptor alpha contains transactivation function-1-regulating element. J Biol Chem 290(28):17611-17627.

Improved detection methods for environmental chemicals

Researchers at NIEHS recently proposed a novel covariate-adjusted standardization method that effectively stabilizes the variability in urinary measures by adjusting for diluteness using excreted creatinine. Measuring environmental chemicals in urine or blood can be error-prone, due to variations in urinary diluteness caused by recent water intake and in blood concentrations of lipophilic chemicals caused by recent fat consumption. A similar standardization method for blood measurements adjusts for serum lipid levels.

To measure chemicals in urine, seven approaches were compared by means of data simulated to mimic a variety of realistic causal scenarios. Of those approaches, the proposed new method, which divides by the ratio between the observed and the covariate-predicted creatinine level, performed best. Depending on the causal scenario judged to capture the research setting, one might simultaneously need to separately adjust for creatinine as a covariate in the model.

In scenarios where lipophilic chemicals are measured in serum, a separate set of simulations revealed that the method that calls for dividing by serum lipids out-performed an alternative method that has recently been promulgated as superior. Therefore, the more classical approach for lipid adjustment is evidently better suited for epidemiological studies. (DD)

CitationO’Brien KM, Upson K, Cook NR, Weinberg CR. 2015. Environmental chemicals in urine and blood: Improving methods for creatinine and lipid adjustment. Environ Health Perspect; doi:10.1289/ehp.1509693 [Online 24 July 2015].

(Robert Brown, Ph.D., is an Intramural Research and Training Award (IRTA) fellow in the NIEHS Cell Biology Group. Greg Buchold, Ph.D., is a former NIEHS postdoctoral fellow in the NIEHS Reproductive and Developmental Biology Laboratory. Deacqunita Diggs, Ph.D., is a National Health and Environmental Effects Laboratory fellow in the U.S. Environmental Protection Agency Developmental Toxicity Branch. Gabriel Knudsen, Ph.D., is a research fellow in the National Cancer Institute, Center for Cancer Research, Laboratory of Toxicology and Toxicokinetics. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)



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