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.

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.

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.

Cell cycle activation in lung adenocarcinoma cells by the ErbB3/phosphatidylinositol 3-kinase/Akt pathway.

Although ErbB3, a member of the epidermal growth factor receptor family, has been implicated in mammary tumorigenesis, investigation of its role in lung tumorigenesis has been limited. We found that ErbB3 was present at high levels in five of seven human lung adenocarcinoma cell lines examined, along with its ligands, heregulins alpha and beta, whereas ErbB3 was absent from HPL1D, a non- transformed cell line from human pulmonary peripheral epithelium. Interactions and effects of ErbB3 were studied in detail in adenocarcinoma lines H441 and H1373. Complexes containing phosphorylated ErbB2, phosphorylated ErbB3 and the p85 regulatory subunit of phosphoinositidyl 3-kinase were detected by co-immunoprecipitation experiments and were present constitutively even in the absence of serum-stimulated cell division. Serum treatment increased the pErbB3/p85 complexes and also stimulated phosphorylation of Akt and GSK3beta, increase in cyclin D1 and cell cycle progression, and these events were blocked by the Akt activation inhibitor LY294002. An ErbB3-specific antisense oligonucleotide reduced amounts of ErbB3 protein and p85 complex in both cell lines, and significantly suppressed cell proliferation. These results together suggest involvement of ErbB3 in growth of lung adenocarcinomas, through activation of phosphoinositidyl 3 kinase and Akt, inactivation of GSK3beta and stabilization of cyclin D1 for cell cycle maintenance. It could be a useful therapeutic target.

Increased K-ras protein and activity in mouse and human lung epithelial cells at confluence.

Although K-ras is frequently mutated in lung adenocarcinomas, the normal function of K-ras p21 in lung is not known. In two mouse (E10 and C10) and one human (HPL1D) immortalized lung cell lines from peripheral epithelium, we have measured total K-ras p21 and active K-ras p21-GTP during cell proliferation and at growth arrest caused by confluence. In all three cell types, total K-ras p21 increased 2- to 4-fold at confluence, and active K-ras p21-GTP increased 10- to 200-fold. It was estimated that 0.03% of total K-ras p21 was in the active GTP-bound state at 50% confluence, compared with 1.4% at postconfluence. By contrast, stimulation of proliferation by serum-containing medium did not involve K-ras p21 activation, even though a rapid, marked activation of both Erk1/2 and Akt occurred. At confluence, large increases, up to 14-fold, were seen in Grb2/Sos1 complexes, which may activate K-ras p21. In sum, increased protein expression and activity of K-ras p21 are associated with growth arrest, not with proliferation, in mouse and human lung cell lines.

Decrease in K-ras p21 and increase in Raf1 and activated Erk 1 and 2 in murine lung tumors initiated by N-nitrosodimethylamine and promoted by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Recent evidence suggests that K-ras protooncogene protein p21 may have a tumor-suppressive role in the context of development of lung adenocarcinoma. Levels of K-ras p21, raf-1, mitogen-activated protein kinases Erk 1 and 2, the phosphorylated-activated forms of Erk 1 and 2 (Erk 1P and 2P), and proliferating cell nuclear antigen (PCNA) were measured by immunoblotting in mouse lung tumors (5 to 9 mm in size) caused by N-nitrosodimethylamine (NDMA) and in control lungs. In tumors compared with normal lung, cell membrane-associated K-ras p21 was significantly decreased and cytosolic K-ras p21 increased. Total, membrane, and cytosolic raf-1 and Erk 1P and 2P were increased in tumors compared with normal lung. A single dose of 5 nmol/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) given after NDMA resulted in a significant 2.4-fold increase in tumor multiplicity. A significantly greater decrease in membrane-associated K-ras p21 and increase in total and membrane associated raf-1 occurred in the NDMA/TCDD tumors compared with the NDMA-only tumors. PCNA levels increased in tumors, a finding confirmed by immunohistochemistry, and correlated with tumor size after NDMA/TCDD treatment but not after NDMA only. The increase in raf-1 in the tumors was confirmed by immunohistochemistry, which also revealed an increase in raf-1-positive alveolar macrophages specifically associating with tumors from the earliest stages. These results suggest a possible tumor-suppressive function for K-ras p21 in lung and a positive role for raf-1 and Erk 1/2 in lung tumorigenesis. TCDD may promote tumors by contributing to downregulation of K-ras and stimulation of raf-1.