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The K-ras gene is frequently mutated in lung and other cancers. K-ras protein includes two splice variants, K-ras 4A and 4B. While K-ras 4B is more widely expressed, recent evidence implicates K-ras 4A in lung tumorigenesis. We found that K-ras 4A protein has a wide range of expression in a large panel of human lung adenocarcinoma cell lines. In cell lines with mutant K-ras, but not those with wildtype K-ras, the K-ras 4A protein had a strong positive correlation with levels of cellular superoxide. We investigated whether K-ras 4A protein was involved in superoxide production, or alternatively was modulated by elevated superoxide. Experiments with small interfering RNA targeting K-ras 4A did not confirm its role in superoxide generation. However, decreasing cellular superoxide with the scavenger Tiron tended to reduce levels of K-ras 4A protein. K-ras 4A and 4B mRNA were also quantified in a number of NSCLC cell lines. 4A mRNA correlated with 4A protein only in K-ras-mutant cells. K-ras 4A mRNA also correlated with superoxide, but with no difference between cell lines with mutant or wildtype K-ras. K-ras 4B mRNA correlated with 4A mRNA and with superoxide, in both K-ras mutant and wildtype cells. The results are consistent with superoxide directly or indirectly up-regulating expression of all K-ras genes, and also increasing the stability of K-ras 4A mutant protein selectively.

Genomes are becoming recognized as dynamic molecules which modify their structures, mediated through epigenetic mechanisms, in response to intrinsic and extrinsic stimulants, and directed by cell development in single-cell organisms and mammalian cells, examples including V(D)J recombination during B-cell and T-cell lymphocyte maturation, and subsequent class switch recombination and somatic hypermutation in the constant segment of the IgH locus to enhance antibody affinity (Shiao, 2015). Ontogeny-driven rearrangement, methylation, and transcription in mouse tandemly repeated 45S rDNA suggests that dynamic genome restructuring plays a key role in embryonic development and tissue differentiation in mammals (Shiao et al., 2011). Any mistakes accompanying this genome restructuring process can introduce mutations, examples including oncogenic fusions to immunoglobulin, T-cell receptor, and 28S rDNA genes (Kobayashi et al., 2014), and the Robertsonian translocation known to increase risk of miscarriage, infertility, and congenital abnormalities (Alfarawati et al., 2012). Whole genome sequencing becomes a critical tool to determine the extent of restructuring and to assist in deciphering the mechanisms of genome rearrangements. In this Research Topic, a collection of eight articles touches upon various aspects of whole genome sequencing, encompassing challenges to obtain 100% genome sequence coverage or reliable reference genomes, mapping and assembly of short-read sequencing data, detection of copy number variation across large genome segments, progress in long-read single molecule sequencing technologies, and clinical applications of exome and genome sequencing. One major challenge is associated with sequence reads that are insufficiently long to span long repetitive sequences, which are interspersed in the genome or can be concentrated in telomere, centromere, and acrocentric regions. These articles provide some directions that should bring whole genome sequencing closer to the goal of 100% completion. Mascher and Stein recommend a workflow for creating a genetically anchored whole-genome shot gun (WGS) assembly: (i) construction of a WGS assembly from next-generation sequencing (NGS) data in individual genomes, (ii) mapping of the sequence reads of a segregating population to the assembly to allow detection of single-nucleotide polymorphisms (SNPs), (iii) building a genetic linkage map, and (iv) integration of the contigs harboring the WGS SNPs to the linkage map. The workflow is particularly useful for non-model but economically important species, for which high-quality reference genomes are still under construction. Li and Freudenberg employ a computational model to demonstrate that the read length of genome sequencing needs to be increased exponentially to convert unmappable regions/reads to mappable, especially in highly repetitive genome regions stretching across 10,000 or more bases, such as telomeres, centromeres, short-arms of acrocentric chromosomes, and large heterochromatic regions. Jiang et al. present an approach combining conventional bacterial artificial chromosome (BAC) end sequences with physical map contig-specific sequences (PMCSS) to increase the total length of genome contigs for scaffolding. PMCSSs are obtained by restriction digestion with two enzymes, followed by tagging of digested fragments with barcoded adaptors, amplification, sequencing, and decoding of each fragment for assembly. The approach is valuable for assembly of repetitive sequences and/or complex genomes which go through multiple rounds of genome duplication. Peters et al. propose a strategy for achieving an error-free “perfect genome” from inputs of 10 to 100 cells, by distribution of fragmented long DNAs into individual nano-compartments, followed by on-site fragmentation and tagging with compartment-specific barcoded sequences, pooling of tagged DNAs, pair-end massively parallel sequencing of about 300 bases, and unbiased phased de novo assembly of sequences. The strategy is capable of resolving paternal/maternal alleles and repetitive sequences, and is applicable to analyses of in vitro fertilized embryo biopsies, circulating tumor cells, and circulating fetal cells. Glusman et al. describe a method to identify copy number variants (CNVs) of 1–100 kb in individual genomes, based on a comparison to Reference Coverage Profiles (RCP) pre-computed from over 6000 high quality (>40X) genomes. Their strategy is to compress raw genome coverage data from short-read sequencing hundreds-fold, followed by scaling of the genome coverage stratified at 1 kb-resolution into 25-type %GC ranges to the total autosomal coverage and further to average coverage from a set of genomes obtained by specific technology. Next, 1-kb resolution RCF represented by median scaled coverage is generated to serve as an estimator of diploid genome, followed by normalization of individual scaled coverage in each kb-sized region to the corresponding RCF value to obtain a normalized coverage profile (NCP). Finally, segmentation of the NCP is performed using hidden Markov model (HMMSeg) for ploidy calling, followed by computation of CNVs frequencies. Wang et al. overview progress in protein and solid-state nanopore sequencing technologies, and offer many tips for improving such technologies. The areas to be improved include detection of ionic current blockage, reduction of signal interference from neighboring bases, discrimination of electronic tunneling signal by an electrode which can be further modified to enhance its interaction with bases, and transistor-based sequencing devices. Alvarez et al. introduce a statistical method to account for correlations between currents to reduce call error of transverse current-based DNA/RNA nanopore sequencing. The error comes from intrinsic noise in the current arising from interaction of molecular orbitals of neighboring bases along the chain of the DNA or RNA molecule. They also demonstrate that aluminum and graphene electrodes produce near 100% fidelity of base calling in 200-bp random sequences, human insulin and BRCA1 genes, and homopolymers after applying the statistical method. Shen et al. review clinical applications of massively parallel next-generation sequencing of exomes and/or genomes in cancer diagnosis and treatment. They provide examples of clinically relevant somatic mutations and structural rearrangements in several cancer types and compile a list of commercial laboratories which offer such exome- or genome-based sequencing service for clinical uses. These clinical applications will surely assist in development of personalized or precision medicine. Recently, reports of read length of over 50 kb using a nanopore sequencer in conjunction with short-read sequencing provide proof of the principle that nanopore sequencing has the potential to directly sequence entire regions containing long repetitive sequences (Goodwin et al., 2015; Madoui et al., 2015). The high error rate of the nanopore technology still represents a bottleneck for extending the read length. In sum, major progress in marching toward 100% genome sequencing has been made, but many challenges remain. These challenges include routinely obtaining long reads (preferably 100 kb or more) with high fidelity to encompass large repetitive sequences and CNVs, being able to handle the low quantities of native DNA/RNA typically employed for clinical or cell-specific diagnoses, and developing robust algorithms to assist in sequence call, genome assembly, and clinical interpretation from the torrents of sequence data that are increasingly becoming available. Novel technologies, strategies, and ingenuity are needed for further improvement of whole genome sequencing.

Proline oxidase (POX) is a novel mitochondrial tumor suppressor that can suppress proliferation and induce apoptosis through the generation of reactive oxygen species (ROS) and decreasing hypoxia-inducible factor (HIF) signaling. Recent studies have shown the absence of expression of POX in human cancer tissues, including renal cancer. However, the mechanism for the loss of POX remains obscure. No genetic or epigenetic variation of POX gene was found. In this study, we identified the upregulated miR-23b in renal cancer as an important regulator of POX. Ectopic overexpression of miR-23b in normal renal cells resulted in striking downregulation of POX, whereas POX expression increased markedly when endogenous miR-23b was knocked down by its antagomirs in renal cancer cells. Consistent with the POX-mediated tumor suppression pathway, these antagomirs induced ROS, inhibited HIF signaling and increased apoptosis. Furthermore, we confirmed the regulation of miR-23b on POX and its function in the DLD1 Tet-off POX cell system. Using a luciferase reporter system, we verified the direct binding of miR-23b to the POX mRNA 3'-untranslated region. In addition, pairs of human renal carcinoma and normal tissues showed a negative correlation between miR-23b and POX protein expression, providing its clinical corroboration. Taken together, our results suggested that miR-23b, by targeting POX, could function as an oncogene; decreasing miR-23b expression may prove to be an effective way of inhibiting kidney tumor growth.

Trichloroethylene (TCE), an organic solvent commonly used for metal degreasing and as a chemical additive, is a significant environmental contaminant that poses health concerns in humans. The US Environmental Protection Agency (EPA) is currently revising the 2001 TCE human risk assessment draft. The next draft is expected to be ready in 2008. TCE metabolites are detectable in humans and carry varying potencies for induction of cancers in animals. Genomic mechanisms have been explored in animals and in humans to link TCE to carcinogenesis. DNA analysis provides an opportunity for detection of unique genetic alterations representing a signature of TCE exposure. These alterations can arise from genotoxic and non-genotoxic pathways at multiple points throughout tumorigenesis. Although fixation of alterations may require several stages of selection and modification, the spectra can be specific to TCE. Only a fraction of these alterations eventually lead to tumor formation and some contribute to tumor progression. Genetic events in two major TCE target organs are reviewed, including the VHL gene in kidney, and the Ras gene and genome-wide hypomethylation in liver. Attempts to identify a genetic signature of TCE exposure are challenged by inconsistent findings, lack of evidence of promutagenic lesions, biological relevance of specific genomic changes, and likelihood of coexposures. For human risk assessment, genome-wide screening is useful and is possible with the development of new DNA sequencing technologies. Genetic screening for preneoplastic and tumor tissues from high-risk population is proposed to exclude the noise of passenger mutations and genetic polymorphisms.

In cancer, gene silencing via hypermethylation is as common as genetic mutations in p53. Understanding the relationship between mutant p53 and hypermethylation of other tumor suppressor genes is essential when elucidate mechanisms of tumor development. In this study, two isogenic human B lymphoblast cell lines with different p53 status include TK6 containing wild-type p53 and WTK1 with mutant p53 were used and contrasted. Lower levels of p16(ink4A) protein were detected in WTK1 cells than in TK6 cells, which were accompanied by increased DNA (cytosine-5)-methyltransferase 1 (DNMT1) gene expression as well as hypermethylation of the p16 ( ink4A ) promoter. siRNA experiments to transiently knock down wild-type p53 in TK6 cells resulted in increase of DNMT1 expression as well as decrease of p16(ink4A) protein. Conversely, siRNA knockdown of mutant p53 in WTK1 cells did not alter either DNMT1 or p16(ink4A) protein levels. Furthermore, loss of suppression function of mutant p53 to DNMT1 in WTK1 was caused by the attenuation of its binding ability to the DNMT1 promoter. In summary, we provide evidences to elucidate the relationship between mutant p53 and DNMT1. Our results indicate that mutant p53 loses its ability to suppress DNMT1 expression, and thus enhances methylation levels of the p16 ( ink4A ) promoter and subsequently down-regulates p16(ink4A )protein.

Genome rearrangement, characterized by insertion, deletion, amplification, inversion, and/or transposition of DNA segments, during development has been observed in multiple cell types, such as, B-cell and T-cell lymphocytes, and non-mammalian organisms, such as ciliates. These genome restructuring processes are driven by epigenetic markings, including DNA methylation status, histone modification, and/or noncoding RNA transcription, which are modifiable by external and internal environments. In turn, the genome rearrangement establishes a new state of genomic control via epigenetic marks regulating expressions of genes in specific cell types or stages. Epigenetic reprogramming occurs during mammalian development. Direct evidence of ontogeny-driven genome rearrangement in mammals at organismal level is still missing. It is arguable that genome rearrangement, which introduces genome diversity, is a driving force of mammalian development. There are some indirect evidence supporting that such genome rearrangement occurs at organismal level.

Paternal exposure of mice to Cr(III) causes increased tumor risk in offspring; an epigenetic mechanism has been hypothesized. Representational difference analysis of gene methylation in sperm revealed hypomethylation in the 45S ribosomal RNA (rRNA) gene after Cr(III) exposure, compared with controls. The most striking effects were seen in the rRNA spacer promoter, a region in the intergenic region of rRNA gene clusters that can influence transcription. Methylation of the rRNA spacer promoter has not been studied heretofore. Sperm DNAs from Cr(III)-treated and control mice were modified by the bisulfite method followed by PCR amplification of the spacer promoter, including 27 CpG sites. Cloning and dideoxy sequencing identified sequence variants (T or G at base -2214) in the spacer promoter. The T allele had less DNA methylation than the G allele in control mice (17 of 17 clones vs. 42 of 72 clones, P = 0.0004). In spite of diversity of sperm DNA methylation patterns, the DNA clones from Cr(III)-exposed mice had fewer methylated CpG sites, by an average of 19% (P < 0.0001). This difference was limited to the G allele. The pyrosequencing technique was applied to quantify the percentage of methylation directly from amplified PCR products. Strikingly, for nine CpG sites including the spacer promoter core region, hypomethylation was highly significant in the Cr(III)-treated group (paired T test, P < 0.0001). Thus, one allele of the 45S rRNA spacer promoter is hypomethylated in sperm germ cells after Cr(III) exposure. This epimutation may lead to increase of tumor risk in the offspring.

Transgenerational carcinogenesis refers to transmission of cancer risk to the untreated progeny of parents exposed to carcinogens before mating. Accumulated evidence suggests that the mechanism of this process is epigenetic, and might involve hormonal and gene expression changes in offspring. To begin to test this hypothesis, we utilized a mouse model (NIH Swiss) in which exposure of fathers to Cr(III) chloride 2 wk before mating can alter incidence of neoplastic and nonneoplastic changes in offspring tissues. Utilizing a MS-RDA approach, we found that the sperm of these fathers had a significantly higher percentage of undermethylated copies of the 45S ribosomal RNA gene (rRNA); this finding was confirmed by bisulfite sequencing. Because gene methylation is a known mechanism of expression control in germ cells, and ribosomal RNA levels have been linked to cancer, these findings are consistent with the hypothesis. Secondly, we observed that offspring of Cr(III)-treated fathers were significantly heavier than controls, and had higher levels of serum T3. Possible effects of T3 levels on gene expression in the offspring were examined by microarray analysis of cDNAs from liver. A total of 58 genes, including 25 named genes, had expression ratios that correlated significantly with serum T3 ratios at P

VHL down-regulation and differential localization as mechanisms in tumorigenesis.BackgroundThe von Hippel-Lindau (VHL) gene has been widely analyzed in many tumors. Early studies in animal tumors suggest that changes in VHL protein level and localization may be also important in tumorigenesis. In this study, we determined the role of VHL protein in human renal cell carcinomas.MethodsSeventy-five human renal cell carcinomas, predominantly of clear cell type (60 of 75), were examined for VHL protein by immunohistochemistry. The level and pattern of protein expression were then compared to VHL mutations and tumor characteristics.ResultsAn apparent decline of VHL level (positive in

Potassium bromate (KBrO3) is a rat renal carcinogen and a major drinking water disinfection by-product in water disinfected with ozone. Clear cell renal tumors, the most common form of human renal epithelial neoplasm, are rare in animals but are inducible by KBrO3 in F344 rats. Detection of cytoplasmic periodic acid-Schiff-positive granules in clear cell tumors, indicative of glycogen accumulation, provides evidence of their biochemical similarity to human counterparts. Mutation in the coding region of the von Hippel–Lindau (VHL) gene is frequently detected in human clear cell renal carcinomas. Detection of VHL mutations in KBrO3-induced rat renal tumors could enhance the relevancy of these rat renal tumors for human health risk assessment. Formalin-fixed paraffin-embedded control tissues and renal tumors from male F344 rats exposed to KBrO3 in the drinking water for 2 years were examined microscopically and were microdissected for DNA extraction. The coding sequence and a promoter region of the VHL gene were examined by polymerase chain reaction-single strand conformation polymorphism and/or DNA sequencing. Two of nine clear cell renal tumors carried the same C to T mutation at the core region of the Sp1 transcription factor binding motif in the VHL promoter and one of four untreated animals had C to T mutation outside the highly conserved core region. Mutation in the VHL coding sequence was only detected in one tumor. No VHL mutations were observed in three chromophilic tumors. KBrO3-induced rat renal tumors are morphologically similar to their human counterpart but the genetic basis of tumorigenesis is different. q 2002 Elsevier Science Ireland Ltd. All rights reserved.

Mice heterozygous for deletion of the transforming growth factor beta1 (TGF-beta1) gene show an enhanced rate of lung tumorigenesis following carcinogen treatment. Since the growth inhibitory activity of TGF-beta1 in epithelial cells is associated with K-ras p21, and K-ras mutations commonly occur in chemically-induced mouse lung tumors, we postulated that tumors in heterozygous TGF-beta1 mice might be more likely to have K-ras mutations compared with tumors in wildtype TGF-beta1 mice. Urethane-induced lung tumors in AJBL6 TGF-beta1 +/- and +/+ mice were examined for K-ras mutations by polymerase chain reaction/single strand conformation polymorphism analysis and sequencing. Mutation frequencies were similar in both genotypes: 12/18 +/- tumors (67%) and 10/16 +/+ tumors (62%). Mutations occurred in 80% +/- and 75% +/+ carcinomas, but in only 50% of the adenomas of both TGF-beta1 genotypes. Codon 61 A--G transition mutations were predominant, occurring in 61% +/- and 44% +/+ tumors. Three +/- (17%) and three +/+ (19%) tumors showed codon 12 mutations, mostly G--A transitions. Two +/- tumors had both codon 61 and codon 12 mutations. Interestingly, carcinomas with mutations in codon 61 were larger than those with codon 12 changes. It appears that the mechanism of enhanced susceptibility of TGF-beta1+/- mice to urethane-induced lung carcinogenesis does not involve selective development of tumors with K-ras mutations.

Codon 12 mutations are frequent in the Ki-ras oncogene in human lung adenocarcinomas, but the effects of these alterations have not been well characterized in lung epithelial cells. Murine primary lung tumors derived from peripheral epithelial cells also may present Ki-ras mutations and are useful models for study of early phases of tumor development. One hypothesis is that Ki-ras mutation and/or a Ki-ras p21 increase could enhance Ki-ras p21-GTP and cell-cycle stimulation through raf-1 and extracellularly regulated protein kinases (Erks). We examined lung tumors 1–7 mm in largest dimension initiated in male Swiss mice by N-nitrosodimethylamine for pathologic type, Ki-ras mutations and levels of total Ki-ras p21, Ki-ras p21 bound to GTP, raf-1, Erk1 and Erk2 and their phosphorylated (activated) forms, and proliferating cell nuclear antigen. Total Ki-ras p21 and activated ras-GTP were not significantly greater in tumors than in normal lung or in tumors with versus those without Ki-ras mutations. Carcinomas with Ki-ras mutations were significantly smaller than those without mutations. Carcinomas were significantly larger than adenomas only for tumors without mutations. High levels of Erk2 and correlation of Erk2 amount with ras-GTP were specific characteristics of tumors with Ki-ras mutations. Size of all tumors correlated with ras-GTP but not with proliferating cell nuclear antigen. Raf-1 was expressed mainly in alveolar macrophages in normal lung but was focally upregulated in papillary areas of some tumors. The results indicate that Ki-ras influences the characteristics of lung tumors, but a linear ras–raf–Erk–cell-cycle control sequence does not adequately characterize tumorigenic events in this model. Mol. Carcinog. 28:156–167, 2000. © 2000 Wiley-Liss, Inc.

BACKGROUND In the general population, Helicobacter pylori (H. pylori), particularly the cagA positive strain, has been associated with intestinal-type gastric carcinoma. Gastric carcinomas are rarely observed in patients age ≤40 years. Host-related factors have been thought to be more important than environmental agents in these early-onset cancers. The aim of this study was to ascertain the possible role of H. pylori infection and that of cagA positive strains in the development of gastric carcinoma in these young patients. METHODS In this case−control study, 105 gastric carcinoma patients (male-to-female ratio = 1.1; mean age, 34.4 years; range, 16−40 years) and an equal number of controls (matched for gender and age) were retrospectively selected from the same geographic area. The phenotypes of gastritis and H. pylori were histologically assessed, and the presence of the ureC gene, which is indicative of H. pylori infection, and the cagA genotype were determined by polymerase chain reaction. Gastric carcinoma risk was calculated by both univariate and multivariate statistical methods, taking into account the cancer phenotype, the gastritis phenotype detected in both patients and controls, and the H. pylori genotype. RESULTS For 74 diffuse and 31 intestinal gastric carcinomas, multivariate logistic regression analysis produced results consistent with those of univariate statistical tests, showing a significant association between gastric carcinoma and both H. pylori infection (odds ratio [OR] = 2.79; 95% confidence interval [CI] = 1.52−5.11) and cagA positive status (OR = 2.94; 95% CI = 1.56−5.52). CONCLUSIONS In young Italian patients with gastric carcinoma, the significant association with cagA positive H. pylori infection suggests that the bacterium has an etiologic role in both diffuse-type and intestinal-type gastric carcinoma. Cancer 1999;85:2506–11. © 1999 American Cancer Society.

Examination of p53 mutation spectra may provide clues to molecular mechanisms involved in different histologic types of gastric cancer. A total of 105 gastric cancer cases classified according to the Laurén's system were selected from a high-risk area around Florence, Italy. Exons 5-8 of the p53 gene were examined for mutations by the polymerase chain reaction-single strand conformation polymorphism technique and DNA sequencing, using DNA from formalin-fixed paraffin-embedded tissues. Mutation frequency was similar in intestinal-type (12/28) and unclassified tumors (9/18), but was significantly lower in diffuse cancers (12/57, P0.05). A similar frequency of p53 mutations was observed among tumor stages in both intestinal-type and unclassified cancers, but in diffuse tumors mutations tended to be associated with invasion beyond the muscularis propria. When base changes were considered, G:C--A:T transitions at CpG sites were the most common mutations for all the three tumor types with 6 of 11 (55%) in intestinal type, 8 of 12 (67%) in diffuse type, and 5 of 8 (63 %) in unclassified tumors. Frequent p53 mutations in both intestinal-type and unclassified tumors support the hypothesis that unclassified tumors represent variants of the intestinal type and suggest that unclassified tumors, like the intestinal type, may also associate with environmental exposures. The predominance of G:C--A:T transitions at CpG sites, which are associated with methyltransferase-induced DNA methylation at carbon 5 of cytosine, in all three tumor types suggests that the status of DNA methylation may be the major determinant for p53 mutations and may be also equally important in gastric carcinogenesis regardless of histology.

Von Hippel-Lindau (VHL) gene mutations occur throughout three exons including the exon-intron boundaries in human VHL disease–associated and sporadic renal cell carcinomas. To explore the possible role of the VHL gene in chemically induced rat kidney tumors originating from various cell types, more than 150 bp of Fischer 344 and Noble rat VHL intron sequences flanking the three exons was determined by dideoxy sequencing. Five primer sets were selected for polymerase chain reaction amplification of the coding regions of rat VHL exons 1–3 and the exon-intron boundaries. Tissues from 10 renal eosinophilic epithelial tumors induced by N-nitrosoethyl(2-hydroxyethyl)amine, 10 nephroblastomas induced by N-nitroso-N-ethylurea, and seven renal mesenchymal tumors induced by N-nitrosomethyl(methoxymethyl)amine were examined for VHL mutations by polymerase chain reaction–single-strand conformation polymorphism analysis. No mutation was detected in any tumor type, indicating that VHL mutations are not involved in the pathogenesis of rat kidney tumors arising from the distal region of the renal tubules, the metanephric blastema, or stromal tissues of the cortex. Mol. Carcinog. 19:230–235, 1997. © 1997 Wiley-Liss, Inc. This article is a US Government work and, as such, is the public domain in the United States of America.

Helicobacter hepaticus is a recently discovered bacterium that invades mouse liver causing chronic active hepatitis followed by development of preneoplastic hepatocellular foci, hepatocellular adenomas and carcinomas. This establishes a unique animal model for study of the mechanisms of cancer development due to a chronic bacterial infection. A possible mechanism of bacteria-associated tumorigenesis is mutation of oncogenes or tumor suppressor genes. Since mutations in ras oncogenes have been widely detected in a variety of chemically induced and spontaneous mouse liver tumors and specific mutations in the p53 tumor suppressor gene have been associated with human bladder cancers attributed to chronic schistosomal infection, we studied exons 1 and 2 of the N-, K- and H-ras genes and exons 5-8 of the p53 gene for the presence of point mutations in 25 liver tumors from 10 naturally infected A/JCr mice, ranging in age from 16 to 24 months. The 20 adenomas and five carcinomas varied in size from 0.1 to 2.3 cm and arose in livers characterized by a wide assortment of pathological profiles, including hepatitis, inflammation, hyperplasia, hypertrophy, leukocyte infiltration, necrosis and focal phenotypic alteration. DNA samples extracted from formalin-fixed paraffin-embedded tissues were screened by PCR/SSCP analysis and showed no mutations in the analyzed genes. Complete absence of mutations in ras genes in 25 mouse liver tumors is unusual. Other genes may be targeted or H. hepaticus infection causes liver cancer through other pathways than direct damage to DNA.

Both gene methylation changes and genetic instability have been noted in offspring of male rodents exposed to radiation or chemicals, but few specific gene targets have been established. Previously, we identified the gene for ribosomal RNA, rDNA, as showing methylation change in sperm of mice treated with the preconceptional carcinogen, chromium(III) chloride. rDNA is a critical cell growth regulator. Here, we investigated the effects of paternal treatments on rDNA in offspring tissue. A total of 93 litters and 758 offspring were obtained, permitting rigorous mixed-effects models statistical analysis of the results. We show that the offspring of male mice treated with Cr(III) presented increased methylation in a promoter sequence of the rDNA gene, specifically in lung. Furthermore polymorphic variants of the multi-copy rDNA genes displayed altered frequencies indicative of structural changes, as a function of both tissue type and paternal treatments. Organismal effects also occurred: some groups of offspring of male mice treated with either Cr(III) or its vehicle, acidic saline, compared with those of untreated mice, had altered average body and liver weights and levels of serum glucose and leptin. Males treated directly with Cr(III) or acidic saline presented serum hormone changes consistent with a stress response. These results establish for the first time epigenetic and genetic instability effects in a gene of central physiological importance, in offspring of male mice exposed preconceptionally to chemicals, possibly related to a stress response in these males.

mutations, happening spontaneously or from environmental insult. Mammalian brains are somatic mosaics, due to aneuploidy and genomic copy number variations. Recently, it also has been found that active transposable elements, long interspersed nuclear elements 1 (LINE-1) in neuronal tissue, contribute to the mosaicism, with tissue specificity [4,5]. LINE-1 elements influence chromosome integrity and gene expression. Retrotransposition activity for the LINE-1 elements was strong during neuronal different iation and occurred with high frequency. Activity was also found in the adult brain. Regulatory events leading to LINE-1 activation were elucidated [6]. These observations led to the proposition that LINE-1 element transposition has a purpose: adaptive increase in neuronal variation and plasticity and in brain-controlled phenotypes [7]. However, specific sites of insertion will need to be located to confirm this idea. Another line of evidence, implicating the occurrence of developmentally-programmed genomic structural changes, comes from a recent study of the gene for ribosomal DNA (rDNA) during ontogeny in the mouse [8]. There are hundreds of copies of rDNA in each cell on several chromosomes. The rDNA of the mice was found to present several SNPs in promoter regions of the gene. The relative percentages of these variant SNPs, indicative of rDNA structural status, were determined in sperm, embryos and two differentiated tissues, lung and liver, using a highthroughput quantitative pyrosequencing technique. The percentage of the variants changed in the differentiated tissues: in the males they differed significantly in lung and liver compared with sperm, and in the females they differed in lung compared with liver. Second, within-litter Differentiated tissues are elaborated during mammalian ontogeny by the coordinated sequential execution of cell type-specific gene expression programs. A common supposition is that basic inherited genomic structure remains constant during this process. This supposition is critically important for the recently developed procedures for obtaining induced pluripotent stem cells from somatic tissues, with great potential for clinical applications. Is this supposition comprehensively true? Would it not make sense, in terms of energy conservation, to program the necessary changes permanently through alterations in genome structure? Several special-case examples of such purposeful, site-specific manipulation of primary genome structure have long been known to exist [1,2] and have obvious functional consequences during a specific life-cycle stage. These are most common in unicellular organisms but also occur in higher organisms, for example, amplification of specific genes for rapid production of proteins, such as those of the chorion of insect eggs, and rearrangements to produce diversification of variant surface glycoprotein in trypanosomes. Vertebrates also utilize genomic re arrangement to enhance diversity of antigen receptors, through the action of the DNA sequence-specific V(D)J recombinases. Evidence is emerging that programmatic modification of the primary structure of the inherited genome may in fact be common, during development and in adult tissues. Structural genetic differences are known to occur between cell populations within an individual, including individual humans, constituting somatic mosaicism [3]. These differences, though frequent, have been interpreted as abnormalities, resulting from uncorrected somatic

Improved therapies are needed for non-small cell lung cancer. Diazeniumdiolate-based nitric oxide (NO)–releasing prodrugs are a growing class of promising NO-based therapeutics. Recently, we have shown that O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, 1) is effective against non-small cell lung cancer (NSCLC) cells in culture and in vivo. Here we report mechanistic studies with compound 1 and its homopiperazine analogue, and structural modification of these into more stable prodrugs. Compound 1 and its homopiperazine analogue were potent cytotoxic agents against NSCLC cells in vitro and in vivo, concomitant with activation of the SAPK/JNK stress pathway and upregulation of its downstream effector ATF3. Apoptosis followed these events. An aryl-substituted analogue, despite extended half-life in the presence of glutathione, did not activate JNK or have anti-tumor activity. The data suggest that rate of reactivity with glutathione and activation of JNK/ATF3 are determinants of cancer cell killing by these prodrugs.

Pyrosequencing technique has been widely used to perform both single-nucleotide polymorphism detection and quantitative DNA methylation analysis. Simplex Pyrosequencing is sufficient to interrogate more than one polymorphic site if these gene variants are within the reach of the sequencing reaction. For polymorphisms far apart from each other or located on different genes, multiple simplex analyses are required. To reduce the number of simplex reactions, multiplex Pyrosequencing becomes a useful alternative method. The multiplex reaction is performed in the presence of single or multiple templates with several sequencing primers. Factors such as primer selection for the PCR and Pyrosequencing reaction, generation of optimal nucleotide dispensation order, use of internal and external controls, preparation of instrumentation, and Pyrogram interpretation are essential to the success of the multiplexing. In this chapter, the mouse 45S rRNA gene is used to present two general multiplex Pyrosequencing protocols for determining DNA methylation and allele frequency in the spacer promoter region of this gene.

Pyrosequencing technique has been widely used to perform both single-nucleotide polymorphism detection and quantitative DNA methylation analysis. Simplex Pyrosequencing is sufficient to interrogate more than one polymorphic site if these gene variants are within the reach of the sequencing reaction. For polymorphisms far apart from each other or located on different genes, multiple simplex analyses are required. To reduce the number of simplex reactions, multiplex Pyrosequencing becomes a useful alternative method. The multiplex reaction is performed in the presence of single or multiple templates with several sequencing primers. Factors such as primer selection for the PCR and Pyrosequencing reaction, generation of optimal nucleotide dispensation order, use of internal and external controls, preparation of instrumentation, and Pyrogram interpretation are essential to the success of the multiplexing. In this chapter, the mouse 45S rRNA gene is used to present two general multiplex Pyrosequencing protocols for determining DNA methylation and allele frequency in the spacer promoter region of this gene.

Selenium is an essential nutrient, a component of several anti-oxidant enzymes, and a possible factor in cancer risk, including lung cancer. We determined the subtoxic range of selenium concentration (as sodium selenite) required to increase and maintain the expression of anti-oxidant selenoproteins gluthathione peroxidases GPX1 and GPX4 at a constant level in cultures of human lung adenocarcinoma cell lines (H460, H1703 and H1944) and in HPL1D, a non-transformed lung epithelial cell line. Selenium dose-dependently increased GPX1 protein expression 1.8-fold in HPL1D cells and approximately 40-fold in H460 and H1944 cancer cells, with maximum effects at 20-40 nM. GPX4 protein was also increased, but more so in HPL1D (five-fold) than in H460 or H1944 cells (two- to three-fold). GPX1 mRNA showed similar patterns but differences of lesser magnitude. GPX1 protein and activity level was not consistently detectable in H1703 cells, with or without Se supplementation; its mRNA was present but very low. GPX4 protein level was also low in H1703 cells, but was markedly increased by selenium supplementation (48-fold). These results confirm a role for selenium in risk of lung cancer and the independent regulation of GPX1 and GPX4. Characterization of individual tumors with regard to GPX1 and GPX4 levels and regulation might be useful for interpretation of clinical studies on effects of selenium in lung cancer risk.

Paternal exposure of mice to Cr(III) causes increased tumor risk in offspring; an epigenetic mechanism has been hypothesized. Representational difference analysis of gene methylation in sperm revealed hypomethylation in the 45S ribosomal RNA (rRNA) gene after Cr(III) exposure, compared with controls. The most striking effects were seen in the rRNA spacer promoter, a region in the intergenic region of rRNA gene clusters that can influence transcription. Methylation of the rRNA spacer promoter has not been studied heretofore. Sperm DNAs from Cr(III)-treated and control mice were modified by the bisulfite method followed by PCR amplification of the spacer promoter, including 27 CpG sites. Cloning and dideoxy sequencing identified sequence variants (T or G at base -2214) in the spacer promoter. The T allele had less DNA methylation than the G allele in control mice (17 of 17 clones vs. 42 of 72 clones, P = 0.0004). In spite of diversity of sperm DNA methylation patterns, the DNA clones from Cr(III)-exposed mice had fewer methylated CpG sites, by an average of 19% (P0.0001). This difference was limited to the G allele. The pyrosequencing technique was applied to quantify the percentage of methylation directly from amplified PCR products. Strikingly, for nine CpG sites including the spacer promoter core region, hypomethylation was highly significant in the Cr(III)-treated group (paired T test, P0.0001). Thus, one allele of the 45S rRNA spacer promoter is hypomethylated in sperm germ cells after Cr(III) exposure. This epimutation may lead to increase of tumor risk in the offspring.

Background Reverse transcription-polymerase chain reaction (RT-PCR) is a very sensitive technique to measure and to compare mRNA levels among samples. However, it is extremely difficult to maintain linearity across the entire procedure, especially at the step of PCR amplification. Specific genes have been used as baseline controls to be co-amplified with target genes to normalize the amplification efficiency, but development or selection of reliable controls itself has created a new challenge. Results Here, we describe a new quantitative RT-PCR to compare two mRNA samples directly without the requirement of synthetic control DNAs for reference. First, chimeric RT primers carrying gene-specific and universal PCR priming sequences with or without a linker for size distinction were utilized to generate cDNAs. The size-different cDNAs were then combined in a single reaction for PCR amplification using the same primer set. The two amplified products were resolved and detected with gel electrophoresis and fluorescence imaging. Relative abundance of the two products was obtained after a baseline correction. Conclusion This methodology is simple and accurate as indicated by equal amplification efficiency throughout PCR cycling. It is also easily implemented for many existing protocols. In addition, parameters affecting RT linearity are characterized in this report.

The von Hippel-Lindau (VHL) protein is able to suppress tumor growth and to down-regulate many angiogenic factors, and is ubiquitously detected in adult and fetal tissues. This makes VHL an excellent target for therapeutic intervention. Observation of VHL alterations in sporadic tumors has been increasing as a result of examination of abnormalities other than intragenic mutations. These abnormalities include loss of chromosome 3p25, changes in the promoter, down-regulation of transcript, and changes in protein level. This article also presents the finding of differential expression of two common VHL proteins among rat tissues, suggesting tissue- and development-dependent functional difference between these two isoforms. Molecular pathways linking VHL to angiogenesis have been extensively characterized and mechanisms have been proposed to explain how altered VHL leads to tumorigenesis. VHL functions in the presence of oxygen and/or oxygen species. Two strategies are proposed here for anti-tumor and anti-angiogenic treatments of VHL-deficient tumors and those without detectable VHL intragenic mutations. One is to restore wild-type VHL function in VHL-deficient tumors and the other is to enhance wild-type VHL expression and activity in tumors under hypoxic conditions.

Non-clear cell rat kidney tumors, inducible by N-nitroso compounds but lacking mutations in the von Hippel–Lindau (VHL) coding sequence, were examined for other VHL alterations. Neither mutations nor DNA methylation was detected in a putative promoter region. By immunohistochemistry, however, VHL protein level was evidently reduced in six of the eight eosinophilic renal epithelial tumors and in all the ten nephroblastomas. Immunoblotting of normal kidney detected two VHL proteins of 20 and 22 kDa in a 16-day-old fetal rat but only 20 kDa protein in an adult rat. This is the first demonstration of VHL alteration at the protein level.

Preconceptional exposure of male NIH Swiss mice to chromium(III) chloride resulted in increased incidence of neoplastic and non-neoplastic changes in their progeny, including lung tumors in females [Toxicol. Appl. Pharmacol. 158 (1999) 161-176]. Since mutations in the K-ras protooncogene are frequent, early changes in mouse lung tumors, we investigated possible mutational activation of this gene as a mechanism for preconceptional carcinogenesis by chromium(III). These offspring had lived until natural death at advanced ages (average 816+/-175 days for controls, 904+/-164 for progeny of chromium-treated fathers). Mutations of K-ras, analyzed by single-strand conformation polymorphism and sequencing, were, in codon 12, wild type GGT (glycine), to GAT (aspartic acid); to GTT (valine); and to CGT (arginine); and in codon 61, wild-type CAA (glutamine), to CGA (arginine). K-ras mutation frequencies in lung tumors were very similar in control progeny (4/14) and in progeny of chromium-treated fathers (5/15). Thus, germline mutation or tendency to spontaneous mutation in K-ras does not seem to be part of the mechanism of preconceptional carcinogenesis here. However, an additional interesting observation was that K-ras mutations were much more frequent in lung carcinomas (8/16) than in adenomas (1/13) (P=0.02), for all progeny combined. This was not related to age of the tumor-bearing mice or the size of the tumors. K-ras mutations may contribute to malignant tumor progression during aging, of possible relevance to the putative association of such mutations with poor prognosis of human lung adenocarcinomas.

Cytochrome P450 2E1 (CYP2E1) metabolizes low molecular weight toxicants. CYP2E1 gene polymorphisms have been linked to risk of various cancers and liver disease in humans. Since the patas monkey is a promising model for study of cancer-related alcohol/nitrosamine interactions, we examined CYP2E1 in this monkey for characteristics of two regions that are polymorphic in humans, an RsaI site in the 5' promoter region and a DraI site in intron 6. Another monkey species often used in biomedical research, the cynomolgus monkey, was also examined. Human DNA primers used to amplify a 413 bp segment around the RsaI site also amplified a segment of similar size (409 bp) from DNA of 25 patas monkeys, whereas a product of approximately 800 bp was amplified from DNA of eight cynomolgus monkeys. RsaI did not cut the amplified DNA product from either monkey species. Sequencing revealed that the patas RsaI site was identical to that in humans with the c2c2 CYP2E1 genotype, GTAT. The equivalent cynomolgus sequence, CTAC, has not been observed in humans. Thus, the patas monkey appears to be a useful model for CYP2E1 c2c2 humans, and this genotype, present in 2-25% of humans, may be more primitive than c1c1. For the DraI site, the human primers amplified DNA products similar in size to those from humans, from all patas and cynomolgus monkey DNA samples; none were cut by DraI. Thus, both monkey species appeared to be generally similar to humans of CYP2E1 CC DraI genotype, which is the rarer form of the gene.

A rat model of colon cancer in which tumors are induced by azoxymethane (AOM) is frequently used to study putative environmental agents that may modify the risk of human colon cancer development. In order to evaluate the usefulness of this model for human risk assessment, a comparison of the molecular changes associated with tumorigenesis in the rat model with those in human colon cancer is desirable. Microsatellite instability (MSI), an alteration in length of short repetitive DNA sequences associated with defective DNA mismatch repair, is an important molecular characteristic of many human colon tumors. Intestinal tumors were induced in male Fischer 344 rats injected with 15 mg/kg body wt AOM in four weekly doses. Thirteen intestinal tumors were examined for MSI at 10 different microsatellite loci, using a capillary electrophoresis (CE) method for accurate assessment of DNA length. This method was shown to have a resolution of 1 bp for a 140-bp PCR product and to be capable of detecting one mutant sequence within a background of 10 wild-type sequences. The CE method also readily distinguished a known MSI-positive human tumor sample from its matching control sample. Among the 13 rat intestinal tumors examined, only one had MSI, which was present at only a single locus. We conclude that, unlike sporadic human colon tumors in which 15-30% of tumors have MSI (usually at multiple loci), MSI is very rare in AOM-induced rat intestinal tumors.

In probing the possible non-genotoxic molecular mechanism(s) of nickel(II)-induced carcinogenesis, we performed a non-radioactive mRNA differential display analysis for nickel(II) acetate-treated Chinese hamster ovary cells (CHO-K1-BH4). Three out of thirty differentially expressed cDNAs had sequences highly similar to known genes. Down-regulation of vimentin and a hSNF2H homologue and up-regulation of ferritin heavy chain were confirmed by Northern blot analysis. The expression of these mRNAs was time- and nickel(II) concentration-dependent. For vimentin, the decrease in mRNA level was concurrent with a decrease in the protein level. For ferritin, the increase in mRNA had no effect on the protein level. Dysregulation of these gene products signifies their involvement in the epigenetic effects of carcinogenic nickel(II) compounds.

Gastric cancers are rarely diagnosed before the age of 40 years and the incidence reaches a peak during the 7th decade in the general population. A molecular mechanism of early tumor onset may be determined by comparing microsatellite instability (MSI), indicative of error-prone mismatch repair, and loss of heterozygosity (LOH) between gastric cancers in patients < or = 40 years of age and those of older ages. Three to 5 chromosomal loci, where MSI and/or LOH are commonly found in gastric cancers in the general population, were examined in formalin-fixed, paraffin-embedded samples from 102 patients < or = 40 years of age using a polymerase chain reaction-based non-radioactive screening method. MSI and/or LOH at a minimum of 1 locus were detected in 11/102 patients. The frequency of MSI and/or LOH at the D11S904 locus was significantly higher than that at the D2S119, D2S123, D5S409 and IFNA regions. No preferential genetic changes at the D11S904 locus were observed in elderly patients. Among several clinicopathological variables, a statistically significant association with MSI and/or LOH was observed only for tumors located at the cardia, compared with tumors at the antrum and the corpus. Our findings suggest that a unique mechanism may be involved in increasing the susceptibility of the D11S904 locus for either MSI or LOH, especially for cardia tumors in young patients. Early onset of gastric cancers in patients < or = 40 years of age is associated with genetic changes at preferential chromosomal loci, including D11S904.

Background Mutations in the von Hippel-Lindau (VHL) gene are common in human clear cell kidney cancers. Carcinogens in cigarette smoke, especially nitrosamines, are known to induce kidney tumors of a variety of histologic types in rodents--but with no evidence of VHL mutations; however, none of these tumors resembled human clear cell carcinomas. We examined N-nitrosodimethylamine-induced kidney tumors of the clear or mixed clear/granular cell type in Wistar rats to assess the presence of VHL mutations. Methods Sections of eight clear or mixed clear/granular cell kidney tumors that had been formalin fixed and paraffin embedded were microdissected. DNA was extracted from the microdissected tissue, and exons 1-3 of the rat VHL gene were examined by use of polymerase chain reaction and cycle sequencing techniques. Results Four VHL gene mutations (three G:C to A:T and one A:T to G:C) were detected in three of the tumors in contrast to no mutations in 40 previously reported rat kidney tumors of other histologic types (three of eight tumors versus none of 40; two-sided Fisher's exact test; P=.003). Only tumors showing prominent swollen clear cell cytology with a signet-ring appearance had VHL mutations. Conclusions To our knowledge, this is the first report of VHL mutations in kidney tumors after direct chemical exposure and provides a possible molecular pathway linking tobacco smoking to kidney cancer.

Nickel(II) compounds are known human and animal carcinogens. In this study, the effects of nickel(II) acetate on cell cycle, apoptosis and p53 expression were investigated in order to unveil the elements of early cellular responses to the metal. Chinese hamster ovary (CHO) cells were grown for 72 h in Ham's F-12 medium containing 0, 40, 80, 160, 240, 320, 480 or 640 microM nickel(II) acetate. DNA fragmentation, representative of apoptosis, was examined by agarose gel electrophoresis. The distribution of cells among various phases of cell cycle was determined by DNA flow cytometry. Expression of p53 protein was measured by the Western blotting technique. DNA fragmentation was detectable in cells treated with > or = 160 microM nickel(II) and its intensity increased with increasing nickel(II) concentration. The proportion of cells at S phase declined in a nickel(II) concentration-dependent manner. The decline was accompanied by an increase of cell proportion in G2/M phase and the increase became statistically significant in cells exposed to at least 480 microM nickel(II). Expression of p53 protein was not different from that in the control among samples treated with < or = 480 microM nickel(II). However, an extra fraction that migrated close to the p53 protein fraction was detected in cells treated with 640 microM nickel(II). Our findings suggest that nickel(II) modulates cellular response through effectors involved in both G2/M arrest and apoptosis regulatory pathways. The proportion of cells arrested at G2/M phase or undergoing apoptosis depends directly on nickel(II) concentration. High concentration of nickel(II) appears to up-regulate protein(s) other than the common form of p53 protein.

BACKGROUND: Upregulation of the cell cycle associated genes, p16/CDKN2 and the retinoblastoma susceptibility gene (Rb), is commonly seen during the proliferation of normal cells. An inverse relation between the expression of p16/CDKN2 and Rb has been noted in many tumours, but has not yet been determined in oesophageal squamous carcinoma. AIMS: To investigate p16/CDKN2 genetic alterations and both the p16/CDKN2 and the Rb protein (pRb) immunophenotypes in oesophageal squamous carcinoma. METHODS: Twenty primary oesophageal squamous carcinomas were examined for mutations in p16/CDKN2 by the polymerase chain reaction, single stranded conformational polymorphism, and DNA sequencing. Synthesis of p16/CDKN2 and pRb proteins was determined by immunohistochemistry in 19 specimens of formalin fixed, paraffin wax embedded tissues. RESULTS: Mutations of p16/CDKN2 were not detected in exons 1 and 2. In only one case, G to C and C to T base changes were detected in a non-coding region of exon 3. Expression of p16/CDKN2 and Rb was observed in both normal and neoplastic areas of tissue sections, indicating neither consistent homozygous deletion nor consistent hypermethylation of the genes in tumours. Fourteen tumours showed an inverse expression of p16/CDKN2 and Rb. An increased percentage of cells that immunostained positively for p16/CDKN2 but not for pRb was observed in eight tumours, five of which had no detectable pRb, suggesting defective Rb expression in these oesophageal squamous carcinomas. CONCLUSIONS: These results indicate that p16/CDKN2 mutations occur infrequently in oesophageal squamous carcinoma. The alteration of the Rb gene is suggested as an important step in the development of these tumours.

Bidirectional non-protein-coding RNAs are ubiquitously transcribed from the genome. Convergent sense and antisense transcripts may regulate each other. Here, we examined the convergent cis-noncoding rRNAs (nc-rRNAs) in A5 and E9 lung cancer models. Sense nc-rRNAs extending from rDNA intergenic region to internal transcribed spacer of around 10 kb in length were identified. nc-rRNAs in sense direction exhibited in vitro characteristics of ribozymes, namely, degradation upon incubation with MgCl(2) and stabilization by complementary oligonucleotides. Detection of endogenous cleavage-ligation products carrying internal deletion of hundreds to thousands nucleotides by massively parallel sequencing confirmed the catalytic properties. Transfection of oligonucleotides pairing with antisense nc-rRNAs stabilized both target and complementary transcripts, perturbed rRNA biogenesis, and induced massive cell death via apoptotic and/or nonapoptotic mechanisms depending on cell type and treatment. Oligonucleotides targeting cellular sense transcripts are less responsive. Spontaneously detached cells, though rare, also showed accumulation of nc-rRNAs and perturbation of rRNA biogenesis. Direct participation of nc-rRNAs in apoptotic and nonapoptotic death was demonstrated by transfection of synthetic nc-rRNAs encompassing the rDNA promoter. In sum, convergent cis-nc-rRNAs follow a feed-forward mechanism to regulate each other and rRNA biogenesis. This opens an opportunity to disrupt rRNA biogenesis, commonly upregulated in cancers, via inhibition of ribozyme-like activities in nc-rRNAs.

Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures.

Environmental and dietary factors modify genomes and phenotypes. The extents of these modifications are not clear. DNA methylation and genotypes of the multi-copy 45S rRNA gene were quantified in 4 tissues at 3 developmental stages (sperm of 128 treated males, 98 litters of 876 day-8 embryos, and lung, liver, and a subset of sperm from 93 litters of 758 6-week adult offspring) in 3 treatment groups. Mixed models for adjusting confounding factors were used for all statistical analyses. Single injections of 1 mmol/kg chromium(III), [Cr(III)], an environmental agent and a dietary supplement, or acid saline (AS) vehicle were given to male mice only, which were then euthanized or bred 2 weeks later. A trend toward hypomethylation in the rRNA spacer promoter region but no frequency differences in 5 sequence variants of the rRNA, defined by 4 single nucleotide polymorphisms, were observed in sperm 2 weeks after Cr(III) treatment as compared to AS and untreated (UT) groups. This epigenetic trend disappeared at the day-8 embryo stage for both genders after paternal treatments with Cr(III) and/or AS. In offspring at 6 weeks of age, significant hypermethylation of the rRNA spacer-promoter was detected in the lung of the male offspring from Cr(III)-treated fathers, reversing the hypomethylation trend seen in the sperm 2 weeks after Cr(III) treatment. There was also a change in genotype: a significant reduction of the rRNA CGC variant in Cr(III) and/or AS groups in lung and liver in males, and in lung in females. Further examinations of the regressions of individual sequence variants on DNA methylation and on other variants revealed significant modifications to those correlates by Cr(III) and/or AS but to different degrees depending on the developmental stages, thus supporting the hypothesis of paternal exposure-induced epigenetic and genetic reprogramming. These genomic reprogrammings were accompanied by phenotypic changes related to paternal Cr(III) or AS treatment, including increased body and liver weights and alterations in serum glucose and leptin. Male mice treated with either Cr(III) or AS demonstrated a typical chemical stress response, as indicated by acute reduction in serum insulin and leptin, followed later by increases in these hormones. Taken together, this multi-faceted cross-generational study uncovers modifiable epigenetic and genetic reprogramming during development and differentiation. Paternal stress apparently had multiple effects on genomes and phenotypes, in their offspring. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 184.

Increase in ribosome biogenesis is a characteristic of proliferating cells and may be causative in tumorigenesis. Significant increases in basal levels of several primary precursor transcripts during ribosomal RNA (rRNA) processing and of a cis antisense noncoding rRNA (AS-ncrRNA) near the main transcription start site were detected in human lung adenocarcinoma cells (H441 and A549) compared with non-transformed peripheral lung epithelial cells (HPL). A cis sense noncoding rRNA (S-ncrRNA) was also discovered but its levels showed cell-specific changes. It was hypothesized that a high level of the AS-ncrRNA is required to maintain the tumor phenotype. The functions of the AS-ncrRNA and S-ncrRNA were assessed using complementary oligonucleotides, termed −86S and −84AS (negative numbers correspond to the target locations in reference to the primary transcription start site) and intended for inhibiting the above targets, respectively. The levels of precursor rRNAs and both ncrRNAs in cells transfected with the −86S and −84AS oligonucleotides were measured using reverse transcription and quantitative polymerase chain reaction. Cell phenotypes and apoptotic cell death were also examined. A two-hit treatment with −86S 24 hrs apart induced cell detachment predominantly in H441 and A549 cancer cells. Detached cancer cells showed several hundred- to a thousand-fold upregulation of S-ncrRNA over the vehicle-treated controls while the S-nrcRNA in rare floating HPL cells was only increased by about 20 fold. Precursor rRNAs and AS-ncrRNA were also upregulated but to a lesser extent after −86S treatment in both adherent and detached cells. For the −84AS treatment, detached cells were fewer than after the −86S treatment; however those few floating cancer cells also exhibited several hundred-fold increase of the S-ncrRNA. Although the mechanism of upregulation is unclear, the S-ncrRNA may possibly act as a cell death regulator. Flow cytometric assays to detect fragmented DNA in cells after 24-hr and 48-hr treatments showed an apparent increase in apoptosis (2- to 4-fold) for the −86S treatment compared to −84AS and scrambled control. Further morphological analysis by transmission electron microscopy identified large and abundant autophagosomes and vacuoles preferentially in cancer cells, suggesting another possible cell death mechanism mediated by the oligonucleotide targeting AS-ncrRNA. In sum, rRNA processing and cis bidirectional noncoding rRNAs may form an interconnected network to maintain tumor phenotype. Interruption of this network by oligonucleotides targeting AS-ncrRNA activates massive elevation of the S-ncRNA level and sensitizes cancer cells preferentially for apoptotic and autophagic cell death. Targeting of AS-ncrRNA could have potential for human lung cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4085.

Background: Reverse transcription-polymerase chain reaction (RT-PCR) is a very sensitive technique to measure and to compare mRNA levels among samples. However, it is extremely difficult to maintain linearity across the entire procedure, especially at the step of PCR amplification. Specific genes have been used as baseline controls to be co-amplified with target genes to normalize the amplification efficiency, but development or selection of reliable controls itself has created a new challenge. Results: Here, we describe a new quantitative RT-PCR to compare two mRNA samples directly without the requirement of synthetic control DNAs for reference. First, chimeric RT primers carrying gene-specific and universal PCR priming sequences with or without a linker for size distinction were utilized to generate cDNAs. The size-different cDNAs were then combined in a single reaction for PCR amplification using the same primer set. The two amplified products were resolved and detected with gel electrophoresis and fluorescence imaging. Relative abundance of the two products was obtained after a baseline correction. Conclusion: This methodology is simple and accurate as indicated by equal amplification efficiency throughout PCR cycling. It is also easily implemented for many existing protocols. In addition, parameters affecting RT linearity are characterized in this report.