In vivo bioluminescent monitoring of chemical toxicity using heme oxygenase-luciferase transgenic mice.

Transgenic mice expressing the luciferase (luc) gene under the control of the heme oxygenase-1 promoter (Ho1) were used to measure the induction of heme oxygenase in response to known toxicants. Transgenic Ho1-luc expression was visualized in vivo using a low-light imaging system (IVIS). Ho1-luc activation was compared to Ho1-luc expression, HO1 protein levels, standard markers of toxicity, and histology. Male and female Ho1-luc transgenic mice were exposed to acute doses of cadmium chloride (CdCl2, 3.7 mg/kg), doxorubicin (15 mg/kg), and thioacetamide (300 mg/kg). These agents induced the expression of Ho1-luc in the liver and other tissues to varying degrees. The greatest increase in Ho1-luc activity was observed in the liver in response to CdCl2; intermediate responses were observed for doxorubicin and thioacetamide. Induction of the Ho1-luc transgene by these agents was similar to endogenous protein levels of heme oxygenase as assessed by Western blotting, and generally correlated with plasma levels of circulating enzymes reflecting hepatic or general tissue damage. Histopathology confirmed the toxic effects of CdCl2 on liver and kidney; doxorubicin on kidney, liver, and intestine; and thioacetamide on the liver. Tissue damage was much more pronounced than the luciferase expression following thioacetamide treatment when compared with tissue damage and bioluminescence of the other toxicants. Nevertheless, the induction of Ho1-luc expression following exposure to these agents suggests that the Ho1-luc transgenic mouse may prove useful as a model for in vivo screening of compounds that induce luciferase expression as a marker of toxicity.

Inhibitors of histone deacetylases promote hematopoietic stem cell self-renewal.

BACKGROUND: Histone deacetylases (HDAC) are associated with a variety of transcriptional repressors that control cellular differentiation and proliferation. HDAC inhibitors such as trichostatin A, trapoxin and chlamydocin could be useful tools to modulate these cellular processes. We investigated their effect on the self-renewal of hematopoietic stem cells (HSC) during ex vivo culture. METHODS: Purified murine HSC with the phenotype c-Kit+,Thy-1.1(lo), Lin(-/lo), Sca-1+ were cultured for 4 days with IL-3, IL-6 and c-Kit ligand without or with HDAC inhibitors, after which their degree of phenotypic differentiation in culture was assessed by flow cytometric analysis. To explore whether HDAC inhibitors could have a beneficial role in human HSC transplantation, mobilized peripheral blood CD34+ cells were cultured with thrombopoietin mimetic peptide, flt3 ligand, and c-Kit ligand, without or with various HDAC inhibitors. The fluorescent dye, carboxyfluorescein-diacetate succinimidylester (CFSE), was used to track division of cell subsets, and engrafting ability was evaluated in a non-obese diabetic (NOD) -SCID xenotransplantation model. RESULTS: Murine HSC cultured with HDAC inhibitors maintained a more primitive phenotype than control cultures. The number of human HSC expressing Thy-1 increased up to seven-fold during a 5-day culture with HDAC inhibitors compared with control cultures. Chlamydocin was the most effective of the HDAC inhibitors tested at promoting Thy-1 expression on human cells. CFSE tracking showed that the increase in Thy-1+ cells resulted from cell division. In a NOD-SCID repopulation assay, cells exposed to chlamydocin for 24 h displayed an average four-fold higher engrafting ability over control cells. DISCUSSION: Our studies suggest that HDAC inhibitors can induce ex vivo expansion of human HSC, and may improve engraftment in hematopoietic transplant patients when cell dose is limiting.

Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects.

Histone acetylation is emerging as a major regulatory mechanism thought to modulate gene expression by altering the accessibility of transcription factors to DNA. In this study, treatment of human tumor cells with the histone deacetylase inhibitor, trapoxin (TPX), resulted in selective changes in genes that control the cell cycle. TPX activated p21(waf1) transcription that led to elevated p21(waf1) protein levels in three human tumor cell lines without altering the protein levels of cdk2, cdk4, or cyclin B. In addition, TPX increased cyclin E transcription without increasing the levels of Rb, E2F, dihydrofolate reductase, or glyceraldehyde-3-phosphate dehydrogenase. The elevated levels of p21(waf1) protein led to decreased Rb phosphorylation and cdk2 activity. These effects resulted in G(1) and G(2) cell cycle arrest in H1299 human lung and MDA-MB-435 breast carcinoma cells and apoptosis in A549 lung carcinoma cells. Chromatin immunoprecipitation assays revealed that TPX increased the level of chromatin acetylation associated with histone H3 in the trapoxin-responsive region of the p21(waf1) promoter. This study demonstrates that inhibition of HDAC by TPX increases acetylation of H3-associated chromatin and alters gene expression with marked selectivity.

Influence of some novel N-substituted azoles and pyridines on rat hepatic CYP3A activity.

A series of N-substituted heteroaromatic compounds structurally related to clotrimazole was synthesized, and the effects of these compounds on ethosuximide clearance in rats were determined as a measure of their abilities to induce cytochrome P4503A (CYP3A) activity. Ethosuximide clearance and in vitro erythromycin N-demethylase activity were shown to correlate. In this series, imidazole or other related heteroaromatic "head groups" were linked to triphenylmethane or other phenylmethane derivatives. Within the series, it was found that 1-triphenylmethane-substituted imidazoles elicited the greatest increase in CYP3A activity, and that among the triphenylmethyl-substituted imidazoles, the highest activities were achieved by the substitution of F- or Cl- in either the meta or para position of one of the phenyl rings. Diphenylmethyl-substituted pyridine was effectively devoid of activity. Compounds eliciting the largest increase in CYP3A activity (viz. 1-[(3-fluorophenyl)diphenylmethyl]imidazole, 1-[(4-fluorophenyl)diphenylmethyl]imidazole, and 1-[tri-(4-fluorophenyl)methyl]imidazole) produced little or no increase in ethoxyresorufin O-dealkylase (EROD) activity (i.e. CYP1A), whereas benzylimidazole, which elicited only a small increase in CYP3A activity, produced an almost 9-fold increase in CYP1A activity. For a series of eleven compounds exhibiting a wide range of influence on CYP3A activity, a positive correlation was found between ethosuximide clearance and hepatic CYP3A mRNA levels.

NF-kappa B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation.

The expression of cytomegalovirus alpha (immediate early) genes is under control of an enhancer that carries signals for strong constitutive expression as well as response elements for transactivation by viral proteins. We have used synthetic oligonucleotides representing the 16, 18 and 19 bp repeat elements within the enhancer to investigate the role of virus-induced cellular transcription factors in enhancer activation. We show that the transcription factor NF-kappa B, which binds to the 18 bp repeat, plays a central role in enhancer activation in infected human fibroblasts and that activation is mediated by the product of the viral gene ie1. The simian immunodeficiency virus kappa B site can functionally substitute for the 18 bp element in transient transactivation assays and can also compete efficiently for specific binding to the 18 bp repeat element in vitro. Point mutations in the NF-kappa B site within the 18 bp element disrupt ie1-mediated transactivation and binding. We have found that the characteristics of the 18 bp binding factor from human fibroblasts are indistinguishable from NF-kappa B induced by phorbol ester plus mitogen treatment of T lymphocytes, as determined by gel mobility shift assay as well as protection of the binding site from chemical cleavage. Furthermore, T cell stimulation mediates activation of the viral enhancer via kappa B sites, an observation that may be important in the interaction of cytomegalovirus with the naturally infected human host. Thus, NF-kappa B plays a central role as a target for enhancer activation via viral and cellular factors.

Common DNA binding site for Fos protein complexes and transcription factor AP-1.

The adipocyte P2 (aP2) gene contains a regulatory element, FSE2, that functions during adipocyte differentiation and binds a protein complex containing the product of the fos proto-oncogene (Fos). We show here that the quantitative and qualitative nature of the FSE2 binding complex closely reflects the status of Fos expression within a given cell type. There is a dramatic increase in the FSE2 binding complex when Fos levels are induced with serum, benzodiazepine, and nerve growth factor or are expressed from a v-fos gene. Immunoblotting analysis of DNA retardation gels indicates a comigration of FSE2 complex with the predominant Fos species. Using a combination of mutational analyses of FSE2 and competition for binding with related sequences, we show that the Fos complex recognizes DNA containing the sequence TGACTCA, previously identified as the consensus binding site for the transcription factors AP-1 in mammalian cells and GCN4 in yeast. The simultaneous presence in cell extracts of proteins related to both AP-1 and Fos with similar sequence recognition properties was demonstrated by photo-cross-linking to FSE2 DNA and immunoprecipitating with antibodies directed toward c-fos or v-jun. These results suggest a functional relationship between Fos and AP-1.

Analysis of Fos protein complexes and Fos-related antigens by high-resolution two-dimensional gel electrophoresis.

Protein complexes containing the c-fos protein (Fos) and (Fos)-related antigens were isolated from serum-stimulated fibroblasts and from nerve growth factor plus benzodiazepine-treated pheochromocytoma (PC12) cells, and investigated by high-resolution two-dimensional gel electrophoresis. The results show that Fos is complexed with a basic 39-kDa protein (p39) in fibroblasts, and primarily with an acidic 40-kDa protein (p40) in PC12 cells. Whole cells lysates from both cell types contain p40, suggesting that the interaction of Fos and other cellular proteins is dependent on the differentiated state of the cell. In addition to p39 and p40, a heterogeneous population of polypeptides of approximately 48 kDa are present in Fos complexes isolated from non-denatured extracts of either cell type. These proteins may represent a minor class of Fos-binding proteins. Analysis of extracts prepared under denaturing conditions using antisera raised against a Fos peptide (amino acids 127-152) reveals a series of Fos-related antigens. These antigens are induced, some with a slower kinetics than Fos, in fibroblasts and PC12 cells. Thus, c-fos may represent a marker for a family of genes, some of which are antigenically related, that are part of an early cellular transcriptional response to diverse extracellular stimuli.

Isolation and characterization of the c-fos(rat) cDNA and analysis of post-translational modification in vitro.

c-fos mRNA accumulates to a level of 0.2% of cellular poly(A)-containing RNA 45 min after treatment of rat pheochromocytoma (PC12) cells with 1 mM barium chloride. Several clones of the c-fos(rat) cDNA were isolated from a cDNA library constructed from this RNA population. Nucleotide sequence analysis of a full-length cDNA clone reveals striking conservation among the c-fos genes isolated from rat, mouse and human cells, and confirms the c-fos gene structure predicted from an analysis of c-fos genomic clones. Translation of an SP6-derived transcript of the c-fos(rat) cDNA in a messenger-dependent rabbit reticulocyte lysate yields the complete c-fos protein. It undergoes extensive post-translational modification in the lysate, particularly in the presence of additional cAMP. The c-fos protein synthesized in vitro appears to be phosphorylated by the cAMP-dependent protein kinase.

The Fos protein complex is associated with DNA in isolated nuclei and binds to DNA cellulose.

The properties of the viral and cellular fos proteins (Fos) were investigated as a first step toward understanding the function of the fos gene. Treatment of nuclei with salt and nonionic detergents solubilized a complex that contained Fos together with several other cellular proteins. The majority of the Fos protein complex was released from isolated nuclei incubated in the presence of deoxyribonuclease I or micrococcal nuclease but not with ribonuclease A, suggesting that Fos is associated with chromatin. This hypothesis is supported by the finding that Fos protein from native or denatured nuclear extracts exhibited DNA-binding activity in vitro. These results suggest that Fos is involved in the regulation of gene expression.

The fos gene product undergoes extensive post-translational modification in eukaryotic but not in prokaryotic cells.

To investigate the properties of the fos oncogene, we have constructed bacterial and yeast vectors which express the entire fos-coded protein (Fos) and two C-terminal deletion products. In Escherichia coli, Fos proteins were expressed from the phage lambda pL promotor under the control of the temperature-sensitive lambda repressor. In vitro transcription/translation studies indicate that these vectors produce Fos proteins of the expected sizes. However, in vivo, Fos protein accumulation is observed only in hosts with the Lon- phenotype. In Saccharomyces cerevisiae, the fos gene was expressed from the PHO5 promoter which is induced under low-phosphate conditions. In contrast to the situation in E. coli, in which the heterologous proteins appeared as single major products when subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis, the Fos proteins in S. cerevisiae displayed extensive Mr heterogeneity. Pulse-chase analyses indicated that this heterogeneity was a consequence of extensive post-translational modification. These modifications occurred to an equivalent extent on the products coded by the fos gene with C-terminal deletions and thus appear not to be controlled by the missing domain.

Synthesis of outer membrane proteins in cpxA cpxB mutants of Escherichia coli K-12.

Two major proteins, the murein lipoprotein and the OmpF matrix porin, are deficient in the outer membrane of cpxA cpxB mutants of Escherichia coli K-12. We present evidence that the cpx mutations prevent or retard the translocation of these proteins to the outer membrane. The mutations had no effect on the rate of lipoprotein synthesis. Mutant cells labeled for 5 min with radioactive arginine accumulated as much lipoprotein as otherwise isogenic cpxA+ cpxB+ cells. This lipoprotein accumulated as such; no material synthesized in mutant cells and reactive with antilipoprotein antibodies had the electrophoretic mobility of prolipoprotein. Hence, the initial stages of prolipoprotein insertion into the inner membrane leading to its cleavage to lipoprotein appeared normal. However, after a long labeling interval, mutant cells were deficient in free lipoprotein and lacked lipoprotein covalently bound to peptidoglycan, suggesting that little if any of the lipoprotein synthesized in mutant cells reaches the outer membrane. Immunoreactive OmpF protein could also be detected in extracts of mutant cells labeled for 5 min, but the amount that accumulated was severalfold less in mutant cells than in cpxA+ cpxB+ cells. Analysis of beta-galactosidase synthesis from ompF-lacZ fusion genes showed this difference to be the result of a reduced rate of ompF transcription in mutant cells. Even so, little or none of the ompF protein synthesized in mutant cells was incorporated into the outer membrane.

Recombination-induced suppression of cell division following P1-mediated generalized transduction in Klebsiella aerogenes.

Klebsiella aerogenes recombinants resulting from bacteriophage P1-mediated generalized transduction failed to increase in number for approximately six generations after transduction. Nevertheless these recombinants continued to grow and became sensitive to penicillin after a transient resistance, suggesting that the cells were growing as long, non-dividing filaments. When filamentous cells were isolated from transduced cultures by gradient centrifugation, recombinants were 1000-fold more frequent among the filaments than among the normal-sized cells. The suppression of cell-division lasted for six generations whether markers near the origin (gln, ilv) or terminus (his, trp) of chromosome replication were used, despite a 50-fold difference in transduction frequencies for these markers. The suppression of cell division was a host response to recombination rather than to P1 invasion since cells lysogenized by P1 in these same experiments showed only a short (two generation) suppression of cell division. We speculate that the suppression of cell-division is an SOS response triggered by the degraded DNA not incorporated in the final recombinant. We demonstrate that both the filamentation and the transient penicillin resistance of recombinant cells can be exploited to enrich greatly for recombinants, raising transduction frequencies to as high as 10(-3).

Spectral characteristics of human leukocytes and their relevance to automated cell identification. III. Eosinophils, neutrophils and lymphocytes.

The spectral characteristics of various normal cell types from human blood were examined in respect to an automated, rapid-flow photometric identification system after staining for peroxidase activity. It was found that a plot of light loss against light scatter gave a separation of eosinophils, neutrophils and lymphocytes comparable in accuracy to visual separations. This staining was not useful for the segregation of monocytes and basophils, for which other stains have proven useful in the sytsem under study. It is concluded that automated, rapid-flow photometric identification of blood cell types can produce differential cell counts with good confidence levels.