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.

Anti-tumor efficacy of naked siRNAs for ERBB3 or AKT2 against lung adenocarcinoma cell xenografts.

The use of siRNAs against specific molecular targets has potential for cancer therapy but has been thought to be limited by the need for formulation to improve cellular uptake. Lung adenocarcinoma cells are markedly suppressed in culture by siRNAs to the receptor ERBB3 or its downstream signaling partner AKT2. We now demonstrate that naked, unformulated siRNAs to ERBB3 or AKT2, administered i.v. as saline solutions, 2 µg/g five times per week for 3 weeks (total dose 30 µg/g), were effective suppressors of growth of A549 human lung adenocarcinoma cell xenografts in athymic mice, 12 mice per group, in four different experiments. ERBB3 and AKT2 siRNAs each inhibited growth by 70-90% on average, compared to saline-treated or untreated controls; a nonsilencing siRNA was without significant effect. Lesser but significant effects were noted with a total dose of 12 µg/g. With the higher dose, effects persisted for several weeks after the end of treatment. Expected reductions of ERBB3 and AKT2 mRNAs and proteins occurred and correlated with decrease in tumor volume. There were no significant changes in serum cytokines. These results show that naked siRNAs to ERBB3 or AKT2 may have potential for lung cancer therapy.

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.

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.

DNA damage, superoxide, and mutant K-ras in human lung adenocarcinoma cells.

DNA single-strand breaks (quantitative comet assay) were assessed to indicate ongoing genetic instability in a panel of human lung adenocarcinoma cell lines. Of these, 19/20 showed more DNA damage than a nontransformed cell line from human peripheral lung epithelium, HPL1D. DNA damage was significantly greater in those derived from pleural effusates vs those from lymph node metastases. DNA strand breaks correlated positively with superoxide (nitroblue tetrazolium reduction assay), and negatively with amount of OGG1, a repair enzyme for oxidative DNA damage. Levels of CuZn superoxide dismutase varied moderately among the lines and did not correlate with other parameters. A role for mutant K-ras through generation of reactive oxygen species was examined. Cells with mutant K-ras had significantly lower amounts of manganese superoxide dismutase (MnSOD) vs those with wild-type K-ras, but MnSOD protein correlated positively with superoxide levels. In a subset of cell lines with similar levels of MnSOD, comparable to those in HPL1D cells, K-ras activity correlated positively with levels of both superoxide and DNA strand breaks. These results suggest that persistent DNA damage in some lung adenocarcinoma cells may be caused by superoxide resulting from mutant K-ras activity, and that OGG1 is important for prevention of this damage.

Effects of selenium supplementation on expression of glutathione peroxidase isoforms in cultured human lung adenocarcinoma cell lines.

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.

Alternate paths from epidermal growth factor receptor to Akt in malignant versus nontransformed lung epithelial cells: ErbB3 versus Gab1.

In many human lung adenocarcinoma cell lines, a pathway involving epidermal growth factor receptor (EGFR), ErbB2 and ErbB3 receptors, phosphatidyl inositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3-beta (GSK3-beta), and cyclin D1 controls cell growth, survival, and invasiveness. We have investigated this pathway in paired transformed/nontransformed cell lines from murine peripheral lung epithelium, E9/E10 and A5/C10. The E9 and A5 carcinoma lines expressed ErbB3 and transforming growth factor-alpha (TGF-alpha) and responded to TGF-alpha stimulation with protein complex formation including the p85 regulatory subunit of PI3K, activation of Akt, phosphorylation of GSK3-beta, and increased cyclin D1 protein and the cell cycle. ErbB3 and TGF-alpha were not detected in the nontransformed E10 and C10 cell lines. Nevertheless, exposure of E10 or C10 cells to TGF-alpha activated PI3K and Akt and increased cyclin D1 and cell growth. The effector pathway from the EGFR to PI3K in these nontransformed cells included the adaptor Grb2, the docking protein Gab1, and the phosphatase Shp2. Gab1 was highly expressed in E10 and C10 cells but not in the malignant E9 and A5 sister lines. Complexes of EGFR/Grb2/Gab1/Shp2 after TGF-alpha stimulation were prominent only in E10 and C10 cells. Thus, alternate pathways downstream of EGFR regulate mitosis in these paired malignant versus nontransformed lung cell lines.

Inactivation of ErbB3 by siRNA promotes apoptosis and attenuates growth and invasiveness of human lung adenocarcinoma cell line A549.

The ErbB3 receptor and the downstream signaling kinase Akt are implicated in proliferation of lung adenocarcinoma cells. Inhibition by siRNAs to ErbB3 and Akt isoforms 1, 2 and 3 was utilized to investigate the contribution of these molecules to tumor survival, spreading and invasiveness, and the roles of specific Akt isoforms. ErbB3 siRNA stably and dose-dependently suppressed ErbB3 protein for 2 days or more, and reduced cell numbers, by both suppressing cell cycle and causing apoptosis and necrosis. It also inhibited soft agar growth, cell motility and migration, and invasiveness. Akt1, 2 and 3 siRNAs had similar suppressive effects on cell number, apoptosis/necrosis and soft agar growth. However, although Akt1 siRNA had no effect on cell migration or invasion, Akt2 siRNA effectively suppressed both activities, and Akt3 siRNA had moderate effectiveness. In A549 cells, ErbB3 is indicated as having major effects on cell division, survival, motility, migration and invasiveness. All three Akt isoforms are to varying degrees involved in these cell behaviors, with Akt2 especially implicated in migration and invasion. ErbB3 and the Akts are promising targets for therapy, and siRNAs may be useful for this purpose.

Recombinant lemA without adjuvant induces extensive expansion of H2-M3-restricted CD8 effectors, which can suppress primary listeriosis in mice.

Mice infected with Listeria monocytogenes (LM) produce large numbers of H2-M3-restricted CD8 T cells directed against the formylated peptides, f-MIGWII and f-MIVIL. To examine responsiveness to these epitopes in the absence of infection, we inoculated mice with recombinant lemA (r-lemA) containing f-MIGWII or r-vemA (a variant of r-lemA containing f-MIVIL in place of f-MIGWII) without adjuvant. To monitor responses, we measured peptide-specific cytoplasmic IFN-gamma production ex vivo by freshly harvested splenocytes at varying times post-inoculation. B6 mice inoculated with r-lemA produced substantial numbers of epitope-specific CD8 cells with peak levels on day 7 when there were 1.1 x 10(6) f-MIGWII-specific CD8 cells in the spleen (8.2% of total CD8 splenocytes). The r-vemA-treated animals accumulated 0.25 x 10(6) cells (1.8% of total CD8 cells) at this time point. Comparable responses were observed after rechallenge of immunized animals. Other elements in the lemA moiety distinct from the immunogenic peptide were required since mice did not respond to equimolar amounts of synthetic f-MIGWII or f-MIVIL alone. In comparative studies, B6 and C3H/HeJ mice responded to r-lemA much more vigorously than BALB/c animals. When r-lemA- or r-vemA-treated B6 animals were challenged i.v. with LM 7 days later, they suppressed splenic accumulation of bacteria much more effectively than controls. On the other hand, antigen-treated animals were not protected against infection 1 month later. Thus, responsive strains of mice respond vigorously to H2-M3-restricted epitopes, even in the absence of bacterial infection or adjuvant. The resulting effectors acutely enhance antimicrobial resistance but do not confer long-term memory protection.