Thomas A. Rosenquist

TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.

Aristolochic acid and the etiology of endemic (Balkan) nephropathy

Endemic (Balkan) nephropathy (EN), a devastating renal disease affecting men and women living in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia, is characterized by its insidious onset, invariable progression to chronic renal failure and a strong association with transitional cell (urothelial) carcinoma of the upper urinary tract. Significant epidemiologic features of EN include its focal occurrence in certain villages and a familial, but not inherited, pattern of disease. Our experiments test the hypothesis that chronic dietary poisoning by aristolochic acid is responsible for EN and its associated urothelial cancer. Using 32P-postlabeling/PAGE and authentic standards, we identified dA-aristolactam (AL) and dG-AL DNA adducts in the renal cortex of patients with EN but not in patients with other chronic renal diseases. In addition, urothelial cancer tissue was obtained from residents of endemic villages with upper urinary tract malignancies. The AmpliChip p53 microarray was then used to sequence exons 2–11 of the p53 gene where we identified 19 base substitutions. Mutations at A:T pairs accounted for 89% of all p53 mutations, with 78% of these being A:T → T:A transversions. Our experimental results, namely, that (i) DNA adducts derived from aristolochic acid (AA) are present in renal tissues of patients with documented EN, (ii) these adducts can be detected in transitional cell cancers, and (iii) A:T → T:A transversions dominate the p53 mutational spectrum in the upper urinary tract malignancies found in this population lead to the conclusion that dietary exposure to AA is a significant risk factor for EN and its attendant transitional cell cancer.

Mesd Encodes an LRP5/6 Chaperone Essential for Specification of Mouse Embryonic Polarity

Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. Here, we demonstrate that mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3(-)(/)(-) embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling.

The novel DNA glycosylase, NEIL1, protects mammalian cells from radiation-mediated cell death

DNA damage mediated by reactive oxygen species generates miscoding and blocking lesions that may lead to mutations or cell death. Base excision repair (BER) constitutes a universal mechanism for removing oxidatively damaged bases and restoring the integrity of genomic DNA. In Escherichia coli, the DNA glycosylases Nei, Fpg, and Nth initiate BER of oxidative lesions; OGG1 and NTH1 proteins fulfill a similar function in mammalian cells. Three human genes, designated NEIL1, NEIL2 and NEIL3, encode proteins that contain sequence homologies to Nei and Fpg. We have cloned the corresponding mouse genes and have overexpressed and purified mNeil1, a DNA glycosylase that efficiently removes a wide spectrum of mutagenic and cytotoxic DNA lesions. These lesions include the two cis-thymineglycol(Tg) stereoisomers, guanine- and adenine-derived formamidopyrimidines, and 5,6-dihydrouracil. Two of these lesions, fapyA and 5S,6R thymine glycol, are not excised by mOgg1 or mNth1. We have also used RNA interference technology to establish embryonic stem cell lines deficient in Neil1 protein and showed them to be sensitive to low levels of gamma-irradiation. The results of these studies suggest that Neil1 is an essential component of base excision repair in mammalian cells; its presence may contribute to the redundant repair capacity observed in Ogg1 -/- and Nth1 -/- mice.

Mutational Signature of Aristolochic Acid Exposure as Revealed by Whole-Exome Sequencing

The mutational signature of aristolochic acid exemplifies how genome-wide sequencing can be used to identify environmental exposures leading to cancer. Carcinogen AAlert Aristolochic acid (AA) is a natural compound derived from plants in the Aristolochia genus. For centuries, Aristolochia has been used throughout Asia to treat a variety of ailments as a component of traditional Chinese medicine. In recent years, however, a more sinister side of this herb has come to light when it was linked to kidney damage and cancers of the urinary tract. Now, two studies by Poon et al. and Hoang et al. present a “molecular signature” of AA-induced DNA damage, which helps to explain the mutagenic effects of AA and may also be useful as a way to detect unsuspected AA exposure as a cause of cancer. The molecular signature seen in AA-associated tumors is characterized by a predominance of A:T-to-T:A transversions, a relatively unusual type of mutation that is infrequently seen in other types of cancer, including those caused by other carcinogens. These mutations concentrate at splice sites, causing the inappropriate inclusion or exclusion of entire exons in the resulting mRNA. The overall mutation rate is another notable feature of AA-associated cancers, because it is several times higher than the rate of mutations caused by other carcinogens such as tobacco and ultraviolet light. In both studies, the authors also used the molecular signature to discover that AA was a likely cause of tumors previously attributed to other carcinogens. In one case, a urinary tract cancer that had been attributed to smoking and, in the other case, a liver cancer previously attributed to a chronic hepatitis infection were both identified as having the telltale signature of AA mutagenesis. The identification of a specific molecular signature for AA has both clinical and public health implications. For individual patients, the molecular signature could help physicians identify which tumors were caused by AA. Although this information cannot yet be used to optimize the treatment of individual patients, those who are diagnosed with AA-associated cancers could be monitored more closely for the appearance of additional tumors. Meanwhile, a better understanding of the mutagenic effects of AA should also help to strengthen public health efforts to decrease exposure to this carcinogenic herb. In humans, exposure to aristolochic acid (AA) is associated with urothelial carcinoma of the upper urinary tract (UTUC). Exome sequencing of UTUCs from 19 individuals with documented exposure to AA revealed a remarkably large number of somatic mutations and an unusual mutational signature attributable to AA. Most of the mutations (72%) in these tumors were A:T-to-T:A transversions, located predominantly on the nontranscribed strand, with a strong preference for deoxyadenosine in a consensus sequence (T/CAG). This trinucleotide motif overlaps the canonical splice acceptor site, possibly accounting for the excess of splice site mutations observed in these tumors. The AA mutational fingerprint was found frequently in oncogenes and tumor suppressor genes in AA-associated UTUC. The AA mutational signature was observed in one patient’s tumor from a UTUC cohort without previous indication of AA exposure. Together, these results directly link an established environmental mutagen to cancer through genome-wide sequencing and highlight its power to reveal individual exposure to carcinogens.

Loss of Nectin-2 at Sertoli-Spermatid Junctions Leads to Male Infertility and Correlates with Severe Spermatozoan Head and Midpiece Malformation, Impaired Binding to the Zona Pellucida, and Oocyte Penetration

Abstract The members of the nectin/CD155 gene family represent a growing class of novel cell adhesion molecules of the immunoglobulin superfamily. In the present study, we describe the generation of a mouse line lacking a functional nectin-2 gene (nectin-2LacZ/LacZ) and analyze the resulting male-specific infertility phenotype. Although nectin-2LacZ/LacZ males produced normal amounts of motile spermatozoa, scanning electron microscopy revealed severe malformations of the spermatozoan head and midpiece. Besides a 4-fold reduction in migration of nectin-2LacZ/LacZ spermatozoa to the oviducts, in vitro binding to zona-intact mouse oocytes was reduced 6-fold. On the other hand, nectin-2LacZ/LacZ spermatozoa bound to zona-free hamster oocytes at near-wild type levels but, remarkably, failed to penetrate. In addition to the previously reported expression of nectin-2 and nectin-3 at Sertoli-spermatid junctions and of nectin-2 at inter-Sertoli cell junctions, we also found nectin-2 to localize at apical cell-cell junctions of the epididymal epithelium. Expression analysis of a LacZ knockin gene into the defunct nectin-2 gene in nectin-2LacZ/LacZ mice provided additional support for our earlier conjecture that in normal testis, nectin-2 is produced exclusively by Sertoli cells. Finally, we found Sertoli-spermatid junctions in nectin-2LacZ/LacZ mice to be virtually devoid of the actin-bundling protein espin, suggesting that ectoplasmic specializations fail to form in the absence of nectin-2. Our functional analyses indicate that the infertility phenotype of nectin-2-deficient male mice is caused by a combination of reduced migration to the oviduct, spermatozoa-zona binding, and sperm-oocyte fusion. We corroborate our previous description of a heterotypic adhesion complex between Sertoli cells and elongated spermatids that is maintained by nectin-2 and nectin-3, respectively.

Inactivation of mammalian 8-oxoguanine-DNA glycosylase by cadmium(II): implications for cadmium genotoxicity.

Cadmium(II) is a toxic, mutagenic and carcinogenic metal (IARC Class 1 human carcinogen). It causes damage to eukaryotic cells both in acute and chronic modes of exposure via multiple biochemical mechanisms. In particular, Cd diminishes the capacity of cells to repair oxidative DNA damage. Oxidative DNA lesions are important precursors to mutations and ultimately may lead to neoplastic transformation of human cells. We investigated interactions of Cd with murine Ogg1 (mOgg1), an enzyme that removes 8-oxoguanine (8-oxoG), an abundant oxidative lesion, from DNA. Cd(2+) and Zn(2+), but not other divalent cations tested, suppressed mOgg1-catalyzed reactions. The apparent inhibition by Cd consisted of at least two independent processes: irreversible, DNA-independent first-order inactivation of mOgg1 and DNA-dependent inhibition. Irreversibly inactivated mOgg1 has nearly normal affinity for damaged DNA and a normal catalytic rate constant but is defective in formation of the covalent reaction intermediate. When both modes of inhibition are in effect, the catalytic rate constant is dramatically lowered, while affinity to damaged DNA is decreased moderately. Potential sites for Cd binding in mOgg1 and mOgg1-DNA complex are identified. Inactivation of Ogg1 may play a role in the mutagenic and carcinogenic action of Cd.

Biomonitoring of Aristolactam-DNA Adducts in Human Tissues Using Ultra-Performance Liquid Chromatography/Ion-Trap Mass Spectrometry

Aristolochic acids (AAs) are a structurally related family of nephrotoxic and carcinogenic nitrophenanthrene compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs have been implicated in the etiology of so-called Chinese herbs nephropathy and of Balkan endemic nephropathy. Both of these disease syndromes are associated with carcinomas of the upper urinary tract (UUC). 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I) is a principal component of Aristolochia herbs. Following metabolic activation, AA-I reacts with DNA to form aristolactam (AL-I)-DNA adducts. We have developed a sensitive analytical method, using ultraperformance liquid chromatography-electrospray ionization/multistage mass spectrometry (UPLC-ESI/MS(n)) with a linear quadrupole ion-trap mass spectrometer, to measure 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I) and 7-(deoxyguanosin-N(2)-yl) aristolactam I (dG-AL-I) adducts. Using 10 μg of DNA for measurements, the lower limits of quantitation of dA-AL-I and dG-AL-I are, respectively, 0.3 and 1.0 adducts per 10(8) DNA bases. We have used UPLC-ESI/MS(n) to quantify AL-DNA adducts in tissues of rodents exposed to AA and in the renal cortex of patients with UUC who reside in Taiwan, where the incidence of this uncommon cancer is the highest reported for any country in the world. In human tissues, dA-AL-I was detected at levels ranging from 9 to 338 adducts per 10(8) DNA bases, whereas dG-AL-I was not found. We conclude that UPLC-ESI/MS(n) is a highly sensitive, specific and robust analytical method, positioned to supplant (32)P-postlabeling techniques currently used for biomonitoring of DNA adducts in human tissues. Importantly, UPLC-ESI/MS(n) could be used to document exposure to AA, the toxicant responsible for AA nephropathy and its associated UUC.

Cytochrome P450 1A2 Detoxicates Aristolochic Acid in the Mouse

Aristolochic acids (AAs) are plant-derived nephrotoxins and carcinogens responsible for chronic renal failure and associated urothelial cell cancers in several clinical syndromes known collectively as aristolochic acid nephropathy (AAN). Mice provide a useful model for study of AAN because the renal histopathology of AA-treated mice is strikingly similar to that of humans. AA is also a potent carcinogen in mice with a tissue spectrum somewhat different from that in humans. The toxic dose of AA in mice is higher than that in humans; this difference in susceptibility has been postulated to reflect differing rates of detoxication between the species. Recent studies in mice have shown that the hepatic cytochrome P450 system detoxicates AA, and inducers of the arylhydrocarbon response protect mice from the nephrotoxic effects of AA. The purpose of this study was to determine the role of specific cytochrome P450 (P450) enzymes in AA metabolism in vivo. Of 18 human P450 enzymes we surveyed only two, CYP1A1 and CYP1A2, which were effective in demethylating 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI) to the nontoxic derivative 8-hydroxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAIa). Kinetic analysis revealed similar efficiencies of formation of AAIa by human and rat CYP1A2. We also report here that CYP1A2-deficient mice display increased sensitivity to the nephrotoxic effects of AAI. Furthermore, Cyp1a2 knockout mice accumulate AAI-derived DNA adducts in the kidney at a higher rate than control mice. Differences in bioavailability or hepatic metabolism of AAI, expression of CYP1A2, or efficiency of a competing nitroreduction pathway in vivo may explain the apparent differences between human and rodent sensitivity to AAI.

Detoxification of aristolochic acid I by O-demethylation: less nephrotoxicity and genotoxicity of aristolochic acid Ia in rodents.

Ingestion of aristolochic acids (AA) contained in herbal remedies results in aristolochic acid nephropathy (AAN), which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA I and AA II, primary components in AA, have similar genotoxic potential, whereas only AA I shows severe renal toxicity in rodents. AA I is demethylated to form 8‐hydroxy‐aristolochic acid I (AA Ia) as a major metabolite. However, the nephrotoxicity and genotoxicity of AA Ia has not yet been determined. AA Ia was isolated from urine collected from rats treated with AA I and characterized by NMR and mass spectrometry. The purified AA Ia was administered intraperitoneally to C3H/He male mice for 9 days and its toxicity was compared with AA I. Using 32P‐postlabeling/polyacrylamide gel electrophoresis, the level of AA Ia‐derived DNA adducts in renal cortex was ∼70–110 times lower than that observed with AA I, indicating that AA Ia has only a limited genotoxicity. Supporting this result, when calf thymus DNA was reacted with AA Ia in a buffer containing zinc dust, the formation of AA Ia‐DNA adducts was two‐orders of magnitude lower than that of AA I. Histopathologic analysis revealed that unlike AA I, no significant changes were detected in the renal cortex of mice treated with AA Ia. Therefore, the contribution of AA Ia to renal toxicity is minimum. We conclude the metabolic pathway of converting AA I to AA Ia functions as the detoxification of AA I.