Yih-Horng Shiao

Epigenetic and gene expression changes related to transgenerational carcinogenesis.

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 ≤ 0.001. Some of these genes have potential roles in growth and/or tumor suppression. These results also support the hypothesis of an epigenetic and/or gene expression–based mechanism for transgenerational carcinogenesis. Published 2004 Wiley‐Liss, Inc.

Ontogeny-Driven rDNA Rearrangement, Methylation, and Transcription, and Paternal Influence

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.

Polymerase chain reaction–single‐strand conformation polymorphism analysis for the VHL gene in chemically induced kidney tumors of rats using intron‐derived primers

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.

An intergenic non-coding rRNA correlated with expression of the rRNA and frequency of an rRNA single nucleotide polymorphism in lung cancer cells.

Background Ribosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics. Methodology/Principal Findings Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately −1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from −388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5′ leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5′-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences. Conclusions/Significance The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5′ leader sequence of the primary rRNA transcript.

Heterozygous inactivation of TGF-β1 increases the susceptibility to chemically induced mouse lung tumorigenesis independently of mutational activation of K-ras

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.

K-ras mutations in mouse lung tumors of extreme age: independent of paternal preconceptional exposure to chromium(III) but significantly more frequent in carcinomas than adenomas

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.

Down-regulation of von Hippel–Lindau protein in N-nitroso compound-induced rat non-clear cell renal tumors

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 22kDa in a 16-day-old fetal rat but only 20kDa protein in an adult rat. This is the first demonstration of VHL alteration at the protein level.

K-ras 4A and 4B mRNA levels correlate with superoxide in lung adenocarcinoma cells, while at the protein level, only mutant K-ras 4A protein correlates with superoxide

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.

Molecular and organismal changes in offspring of male mice treated with chemical stressors

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.

Structural genomic changes during mammalian ontogeny: a new dimension

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