Mutational analysis of pentabrominated diphenyl-induced hepatocellular tumors in rats and mice, tissue levels of PBDE congeners in rats and mice, and AhR genotyping of Wistar Han rats.

This article describes data related to the research article entitled "Carcinogenic activity of pentabrominated diphenyl ether mixture (DE-71) in rats and mice" (Dunnick et al., 2018). PBDE-induced hepatocellular tumors harbored Hras and Ctnnb1 mutations and the methods for these studies are provided. Tissue levels of PBDE congeners in rats and mice after oral exposure to PBDE mixture increased with increasing dose of PBDE. There was no correlation between AhR status and the incidence of hepatocellular tumors in female Wistar Han rats. This manuscript provides additional information on the methods for conducting mutational analysis, PBDE tissue level determinations, and AhR genotyping.

Carcinogenic activity of pentabrominated diphenyl ether mixture (DE-71) in rats and mice.

Pentabrominated diphenyl ether (PBDE) flame retardants have been phased out in Europe and in the United States, but these lipid soluble chemicals persist in the environment and are found human and animal tissues. PBDEs have limited genotoxic activity. However, in a 2-year cancer study of a PBDE mixture (DE-71) (0, 3, 15, or 50 mg/kg (rats); 0, 3, 30, or 100 mg/kg (mice)) there were treatment-related liver tumors in male and female Wistar Han rats [Crl:WI(Han) after in utero/postnatal/adult exposure, and in male and female B6C3F1 mice, after adult exposure. In addition, there was evidence for a treatment-related carcinogenic effect in the thyroid and pituitary gland tumor in male rats, and in the uterus (stromal polyps/stromal sarcomas) in female rats. The treatment-related liver tumors in female rats were unrelated to the AhR genotype status, and occurred in animals with wild, mutant, or heterozygous Ah receptor. The liver tumors in rats and mice had treatment-related Hras and Ctnnb mutations, respectively. The PBDE carcinogenic activity could be related to oxidative damage, disruption of hormone homeostasis, and molecular and epigenetic changes in target tissue. Further work is needed to compare the PBDE toxic effects in rodents and humans.

Tetrabromobisphenol A activates the hepatic interferon pathway in rats.

Tetrabromobisphenol A (TBBPA) is a widely used flame retardant in printed circuit boards, paper, and textiles. In a two-year study, TBBPA showed evidence of uterine tumors in female Wistar-Han rats and liver and colon tumors in B6C3F1 mice. In order to gain further insight into early gene and pathway changes leading to cancer, we exposed female Wistar Han rats to TBBPA at 0, 25, 250, or 1000mg/kg (oral gavage in corn oil, 5×/week) for 13 weeks. Because at the end of the TBBPA exposure period, there were no treatment-related effects on body weights, liver or uterus lesions, and liver and uterine organ weights were within 10% of controls, only the high dose animals were analyzed. Analysis of the hepatic and uterine transcriptomes showed TBBPA-induced changes primarily in the liver (1000mg/kg), with 159 transcripts corresponding to 132 genes differentially expressed compared to controls (FDR=0.05). Pathway analysis showed activation of interferon (IFN) and metabolic networks. TBBPA induced few molecular changes in the uterus. Activation of the interferon pathway in the liver occurred after 13-weeks of TBBPA exposure, and with longer term TBBPA exposure this may lead to immunomodulatory changes that contribute to carcinogenic processes.

In vivo molecular markers for pro-inflammatory cytokine M1 stage and resident microglia in trimethyltin-induced hippocampal injury.

Microglia polarization to the classical M1 activation state is characterized by elevated pro-inflammatory cytokines; however, a full profile has not been generated in the early stages of a sterile inflammatory response recruiting only resident microglia. We characterized the initial M1 state in a hippocampal injury model dependent upon tumor necrosis factor (TNF) receptor signaling for dentate granule cell death. Twenty-one-day-old CD1 male mice were injected with trimethyltin (TMT 2.3 mg/kg, i.p.) and the hippocampus was examined at an early stage (24-h post-dosing) of neuronal death. Glia activation was assessed using a custom quantitative nuclease protection assay. We report elevated mRNA levels for glia response such as ionizing calcium-binding adapter molecule-1 and glial fibrillary acidic protein (Gfap); Fas, hypoxia inducible factor alpha, complement component 1qb, TNF-related genes (Tnf, Tnfaip3, Tnfrsfla); interleukin-1 alpha, Cd44, chemokine (C-C motif) ligand (Ccl)2, Cc14, integrin alpha M, lipocalin (Lcn2), and secreted phosphoprotein 1 (Spp1). These changes occurred in the absence of changes in matrix metalloproteinase 9 and 12, neural cell adhesion molecule, metabotropic glutamate receptor (Grm)3, and Ly6/neurotoxin 1 (Lynx1), as well as, a decrease in neurotrophin 3, glutamate receptor subunit epsilon (Grin)-2b, and neurotrophic tyrosine kinase receptor, type 3. The M2 anti-inflammatory marker, transforming growth factor beta-1 (Tgfb1) was elevated. mRNAs associated with early stage of injury-induced neurogenesis including fibroblast growth factor 21 and Mki67 were elevated. In the "non-injured" temporal cortex receiving projections from the hippocampus, Lynx1, Grm3, and Grin2b were decreased and Gfap increased. Formalin fixed-paraffin-embedded tissue did not generate a comparable profile.

Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production.

Despite recent therapeutic advancements, multiple myeloma (MM) remains incurable and new therapies are needed, especially for the treatment of elderly and relapsed/refractory patients. We have screened a panel of 100 off-patent licensed oral drugs for anti-myeloma activity and identified niclosamide, an anti-helminthic. Niclosamide, at clinically achievable non-toxic concentrations, killed MM cell lines and primary MM cells as efficiently as or better than standard chemotherapy and existing anti-myeloma drugs individually or in combinations, with little impact on normal donor cells. Cell death was associated with markers of both apoptosis and autophagy. Importantly, niclosamide rapidly reduced free light chain (FLC) production by MM cell lines and primary MM. FLCs are a major cause of renal impairment in MM patients and light chain amyloid and FLC reduction is associated with reversal of tissue damage. Our data indicate that niclosamides anti-MM activity was mediated through the mitochondria with rapid loss of mitochondrial membrane potential, uncoupling of oxidative phosphorylation and production of mitochondrial superoxide. Niclosamide also modulated the nuclear factor-κB and STAT3 pathways in MM cells. In conclusion, our data indicate that MM cells can be selectively targeted using niclosamide while also reducing FLC secretion. Importantly, niclosamide is widely used at these concentrations with minimal toxicity.

Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences.

Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.

Association of c-myc overexpression and hyperproliferation with arsenite-induced malignant transformation.

Numerous studies link arsenic exposure to human cancers in a variety of tissues, including the liver. However, inorganic arsenic has never been unequivocally shown to be an animal carcinogen, and its carcinogenic mechanism remains undefined. Our previous studies indicate that chronic (> or =18 weeks), low-level (125 to 500 nM) exposure to arsenite induces malignant transformation in the normally nontumorigenic rat liver epithelial cell line (TRL 1215), and these chronic arsenic-exposed (CAsE) cells produce invasive and metastatic tumors upon inoculation into nude mice. In addition, a prior microarray screening analysis of aberrant gene expression showed several oncogenes were overexpressed in CAsE cells exposed to 500 nM arsenite, including a prominent overexpression of the protooncogene c-myc, as well as genes related to cell proliferation. Thus, to better understand the mechanism of arsenic carcinogenesis, we studied the role of c-myc overexpression in arsenite-induced cell transformation. The upregulation of c-myc was confirmed by RT-PCR at the transcription level and by Western blot analysis for the translation product. Further analysis showed that arsenite produced significant increases in the steady-state expression of c-myc in a time- and concentration-dependent manner during the malignant transformation process. The level of c-myc expression was highly correlated (r = 0.988) with tumor formation after inoculation of CAsE cells into nude mice and was also highly correlated (r = 0.997) with genomic DNA hypomethylation. CAsE cells showed a high cell proliferation rate in a fashion related to the level of arsenic exposure. The expression of c-myc was highly correlated with cellular hyperproliferation (r = 0.961). Consistent with the enhanced proliferation both proliferating cell nuclear antigen and cyclin D1 were overexpressed in CAsE cells. In summary, a prominent overexpression of c-myc, a gene frequently activated during hepatocarcinogenesis, is strongly correlated with several events possibly associated with arsenic-induced malignant transformation, including hyperproliferation, DNA hypomethylation and tumor formation upon inoculation into nude mice. These correlations provide convincing evidence c-myc overexpression is mechanistically important in arsenic-induced malignant transformation in this model system.

Site-specific phosphorylation of human p53 protein determined by mass spectrometry.

Human recombinant p53 (r-p53) protein was studied by mass spectrometry (MS) to determine site-specific posttranslational differences between basal and hyperphosphorylated r-p53. Wild-type p53 was basally expressed after baculovirus infection while a parallel preparation was treated with the phosphatase inhibitor okadaic acid during the terminal stages of expression to create a hyperphosphorylated form of p53 known for its higher DNA binding and transcriptional activation. After immunoaffinity and HPLC purification, MALDI/MS measured a higher molecular mass for r-p53 from okadaic acid treatment relative to control, suggesting a higher phosphorylation state. This was supported by an acidic shift of r-p53 isoforms separated by gel isoelectric focusing. Employing a variety of mass spectrometric analyses combined with separation and affinity techniques, six specific phosphorylation sites of p53 were identified. The MS data indicated that hyperphosphorylated p53 showed a higher degree of phosphorylation than basal p53 at specific amino- and carboxy-terminal sites. In particular, ESI-MS demonstrated that Ser(315) was entirely phosphorylated after okadaic acid treatment, as confirmed biochemically by CDK2 kinase assay and by isoelectric focusing. In summary, MS analysis uniquely revealed increased, site-specific phosphorylations on p53 after phosphatase inhibition, particularly at Ser(315), which may be critical molecular events in defining p53 activity.

Genetic events associated with arsenic-induced malignant transformation: applications of cDNA microarray technology.

Arsenic is a human carcinogen. Our recent work showed that chronic (>18 wk), low-level (125-500 nM) arsenite exposure induces malignant transformation in normal rat liver cell line TRL1215. In these arsenic-transformed cells, thecellular S-adenosylmethionine pool was depleted from arsenic metabolism, resulting in global DNA hypomethylation. DNA methylation status in turn may affect the expression of a variety of genes. This study examined the aberrant gene expression associated with arsenic-induced transformation with the use of Atlas Rat cDNA Expression microarrays. Poly(A(+)) RNA was prepared from arsenic-transformed cells and passage-matched control cells, and (32)P-labeled cDNA probes were synthesized with Clontech Rat cDNA Synthesis primers and moloney murine leukemia virus reverse transcriptase. The hybrid intensity was analyzed with AtlasImage software and normalized with the sum of the four housekeeping genes. Four hybridizations from separate cell preparations were performed, and mean and SEM for the expression of each gene were calculated for statistical analysis. Among the 588 genes, approximately 80 genes ( approximately 13%) were aberrantly expressed. These included genes involved in cell-cycle regulation, signal transduction, stress response, apoptosis, cytokine production and growth-factor and hormone-receptor production and various oncogenes. These initial gene expression analyses for the first time showed potentially important aberrant gene expression patterns associated with arsenic-induced malignant transformation and set the stage for numerous further studies. Mol. Carcinog. 30:79-87, 2001. Published 2001 Wiley-Liss, Inc.

Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.

Consecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, beta-casein and alpha-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation.

An intragenic deletion of nuclear localization signal-1 of p53 tumor suppressor gene results in loss of apoptosis in murine fibroblasts.

We established mouse lines containing either full-length wild-type p53 or nuclear localization signal-I (NLS-I) deleted p53 to study the role of NLS-I in p53 translocation and function. Induction of apoptosis in response to DNA damage, a primary function of p53, was tested in these cell lines. After exposure to gamma-ionizing radiation or hydrogen peroxide, DNA ladders and labeling of nucelosomal fragments were detected in cells with wild-type p53 gene, but not in p53 null cells or NLS-I deleted cells, suggesting that the NLS-I of p53 protein is necessary for apoptosis. Analysis of p53 protein from subcellular fractions indicated that NLS-I deprived p53 remained in the cytoplasmic fraction, which may explain why NLS-I deleted p53 failed to induce apoptosis.

p53-dependent p21 induction following gamma-irradiation without concomitant p53 induction in a human peripheral neuroepithelioma cell line.

We previously generated cell hybrids between a derivative of the E6-containing HeLa cell line and a p53 null peripheral neuroepithelioma (PNET) cell line. Although p53 protein from the hybrids was genotypically wild type, it did not demonstrate wild-type behavior. Therefore, in the present study, we introduced wild-type p53 into the PNET parent to investigate whether p53 retained wild-type function within this cell line. Although the p53 null PNET parent lacked detectable p21 protein, introduction of wild-type p53 resulted in a detectable expression of p21 protein in all clones tested, suggestive of wild-type p53 function. In addition, p53 expression was necessary for induction of p21 in response to irradiation, and, furthermore, we show this induction to occur at the transcriptional level. Although introduction of wild-type p53 seems to be responsible for p21 induction, the overall protein levels of p53 were not induced. The involvement of p53 in up-regulating p21 is further substantiated by the observation that p21 up-regulation was dependent on the introduction of the wild-type protein. Our results suggest that wild-type p53 is capable of up-regulating p21 in response to DNA damage in the absence of p53 induction.

Induction of novel Grp75 isoforms by 2-deoxyglucose in human and murine fibroblasts.

Grp75 is a stress-inducible mitochondrial chaperone which has a high homology to senescence-related protein, p66mot mortalin. In human cells the mortalin gene assigns to the locus of a putative tumor suppressor gene for myeloid malignancies. In order to study expression and localization of Grp75 and p66mot in human and murine fibroblast lines, polyclonal antibodies were raised to conserved portions of each sequence. HT1080 and C3H10T1/2 cells were treated with various Grp-inducing agents. A single 75 kDa band was detected by Western blot of cytoplasmic proteins which was not greatly altered after thermal stress or treatment with L-azetidine-2-carboxylic acid or nonactin. However, glucose deprivation by 2-deoxyglucose treatment induced five novel isoforms at 74-75 kDa mass. Mortalin at 66 kDa could not be detected under these treatment conditions.

Tumor suppressor p53 gene forms multiple isoforms: evidence for single locus origin and cytoplasmic complex formation with heat shock proteins.

The tumor suppressor protein p53 is a major cell cycle control factor, and mutations in p53 are the most common genetic lesion found in human tumors, resulting in loss of function and contributing to malignant transformation. This report reviews several studies which show that p53 protein appears as at least eleven isoforms having the same amino acid backbone but varying in charge by level of phosphorylation. All isoforms are derived from a single locus, which indicates that p53 activity is modulated by post-translational modification. In addition, mutant p53 forms hetero-oligomers with two families of proteins: HSP70 and a 90 kDa group similar to HSP90. Cytoplasmic complexes are most likely formed to protect p53 from proteolysis and are probably involved in translocation of activated p53 from the cytoplasm to the nucleus for transactivation of other cell cycle control genes.

HSP binding and mitochondrial localization of p53 protein in human HT1080 and mouse C3H10T1/2 cell lines.

In normal cells, the tumor suppressor actions of p53 protein are mediated by specific DNA binding and protein-protein interactions within the nucleus. Mutant p53 proteins, however, often assume an aberrant conformation devoid of tumor suppressor activity and newly capable of binding to the cognate or inducible HSP70. Recent reports from our laboratory and others show that additional unknown proteins may also complex with mutant p53. In this study, we characterize p53:HSP complexes and their subcellular location in the transformed cell lines, human HT1080 and murine C3H10T1/2, which both contain aberrant p53 conformers. Immunoprecipitation and SDS-PAGE of p53 from whole cell lysates revealed the additional presence of a broad 70 kDa band and a 90 kDa band in both lines, while p53 isolated from nuclear lysates was free from other proteins. 2D-PAGE was used to isolate and identify HSP members from cytoplasmic and nuclear lysates by immunoprecipitation, Western blotting and protein sequencing. Anti-p53 immune complexes from cytoplasmic lysates contained not only HSC70 but also GRP75, GRP78 and a weakly basic 90 kDa protein, which may be related to HSP90. The inducible form of HSP70 was not complexed to p53 protein, even though expressed in these cells. Analysis of anti-HSP70, anti-GRP75 and anti-HSP90 immune complexes suggests that HSP members exist as performed complexes in the cytoplasm, but not the nucleus. The presence of the mitochondrial and endoplasmic reticular chaperones, GRP75 and GRP78, in p53:HSP complexes suggested that p53 might be found in these cytoplasmic organelles which was confirmed in mitochondria by biochemical and immunoelectron microscopic evidence. These studies suggest that newly identified members of p53:HSP complexes represent components of a chaperone program which affects the subcellular distribution of p53 protein in these transformed lines.

Human p53 expressed in baculovirus-infected Sf9 cells displays a two-dimensional isoform pattern identical to wild-type p53 from human cells.

Baculovirus expression of human p53 protein, a nuclear cell cycle regulator, was examined in Sf9 cells and compared to native p53 synthesized in primary human cells. Maximum expression of the recombinant p53 protein occurred 48 h postinfection. De novo synthesis of the protein was evident for only 2 days postinfection; however, in pulse-chase studies, 30% of the synthesized protein remained stable up to 5 days. Seventy-seven percent of immunoprecipitated, [35S]-methionine-labeled, recombinant p53 protein resided in the cytoplasm of Sf9 cells, while 15% localized to the nucleus and 8% was released extracellularly. Separation of modified p53 protein, by charge and molecular weight, was accomplished by two-dimensional PAGE, and the electrophoretic pattern of the recombinant protein was identical to the wild-type protein from primary human mammary epithelial cells, indicating that the posttranslational modifications of the recombinant protein in this system are similar to those in primary human cells. Eleven isoforms focused between pI 5.75 and pI 6.5. The recombinant p53 isoforms were phosphorylated by 32P-labeling. Phosphatase digestion of immunoprecipitated p53 effectively removed phosphorous groups from the recombinant protein, reducing the number of isoforms from 11 to 2, demonstrating that phosphorylation is the major posttranslational event in the recombinant protein.

HSP70-2 is part of the synaptonemal complex in mouse and hamster spermatocytes.

Mouse spermatogenic cells are known to express HSP70-2, a member of the HSP70 family of heat-shock proteins. The purpose of the present study was to characterize further the expression and localization of HSP70-2 in meiotic cells of mice and hamsters. After separating mouse spermatogenic cells into cytoplasmic and nuclear fractions, proteins were separated by two-dimensional gel electrophoresis and detected with HSP-specific antibodies. Of several HSP70 proteins identified in the cytoplasm, only HSC70 and HSP70-2 were also detected in the nucleus. Immunocytological analyses of spermatocyte prophase cells revealed that HSP70-2 was associated with the synaptonemal complex. Surface-spread synaptonemal complexes at pachytene and diplotene stages labeled distinctly with the antiserum to HSP70-2. Synaptonemal complexes from fetal mouse oocytes failed to show any evidence of HSP70-2. Reverse-transcriptase-polymerase chain reaction (RT-PCR) analyses of gene expression confirmed this sex specificity; Hsp70-2 mRNA was detected in mouse testes, but not ovaries. These findings are suggestive of a previously unsuspected sexual dimorphism in structure and/or function of the synaptonemal complex.

Regulation of microfilament organization and anchorage-independent growth by tropomyosin 1.

Variants of chemically immortalized Syrian hamster embryo cells that had either retained (supB+) or lost (supB-) the ability to suppress tumorigenicity when hybridized with a fibrosarcoma cell line were subcloned. Both supB cell types are nontumorigenic; however, the supB- but not supB+ cells exhibit conditional anchorage-independent growth. Alterations of actin microfilament organization were observed in supB- but not supB+ cells that corresponded to a significant reduction of the actin-binding protein tropomyosin 1 (TM-1) in subB- cells. To examine the possibility of a direct relationship between TM-1 expression and the subB- phenotype, subB+ cells were transfected with an expression vector containing the TM-1 cDNA in an antisense orientation. The antisense-induced reduction of TM-1 levels in supB+ clones caused a microfilament reorganization and conferred anchorage-independent growth potential that were indistinguishable from those characteristic of supB- cells. These data provide direct evidence that TM-1 regulates both microfilament organization and anchorage-independent growth and suggest that microfilament alterations are sufficient for anchorage-independent growth.

Gel electrophoretic analysis of cellular and secreted proteins from resting and activated rat alveolar macrophages treated with pentamidine isethionate.

Pneumocystic carinii pneumonia, which is a major cause of death among patients suffering from acquired immunodeficiency syndrome, has often been treated successfully with pentamidine isethionate. This study examines pentamidine effects on cellular and secreted proteins from rat alveolar macrophages by two-dimensional gel electrophoresis and computerized image analysis. Over 100 secreted proteins were detected by fluorography. Fluorography showed pentamidine diminished tumor necrosis factor and interleukin-1 release along with other proteins. Effects of combined bacterial lipopolysaccharide and pentamidine were more pronounced on secreted versus cellular proteins in protein amount and pattern difference. Thus pentamidine exhibited a general repressive effect on cellular and secreted protein expression in resting and activated macrophages.

Baculovirus and insect cell gene expression: review of baculovirus biotechnology.

The BEVS continues to evolve as a powerful, flexible tool for molecular biology, protein function, and biomedical research. Future developments offer the promise of replacement of hazardous chemical insecticides with environmentally safe biopesticides, construction of baculovirus vectors which encode genes for specific post-translational modifications, and establishment of efficient, stably transformed insect cell lines. FDA approval of BEVS-produced products offer the prospect of new biopharmaceuticals, in particular human therapeutics and vaccines, to improve human health and increase the quality of life for millions of people.