Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity

Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in human–hamster hybrid (AL) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5′,6′-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in AL cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.

The role of MHC class I glycoproteins in the regulation of induction of cell death in immunocytes by malignant melanoma cells

A deranged expression of MHC class I glycoproteins, characteristic of a variety of malignancies, contributes to the ability of cancer to avoid destruction by T cell-mediated immunity. An abrogation of the metastatic capacity of B16 melanoma cells has been achieved by transfecting an MHC class I-encoding vector into class I-deficient B16 melanoma clones [Gorelik, E., Kim, M., Duty, L. & Galili, U. (1993) Clin. Exp. Metastasis 11, 439–452]. We report here that the deranged expression of class I molecules by B16 melanoma cells is more than a mere acquisition of the capacity to escape immune recognition. Namely, cells of the B16 melanoma prompted splenic lymphocytes to commit death after coculture. However, a class I-expressing and nonmetastatic CL8-2 clone was found to be less potent as an inducer of apoptosis than class I-deficient and metastatic BL9 and BL12 clones. Both Thy1.2+ and Thy1.2− splenocytes underwent cell death when exposed to the class I-deficient BL9 clone. A proportion of CD4+ and CD8+ cells among splenocytes exposed to the BL9 clone was lower than that observed in a coculture with cells of the CL8-2 clone. Consistently, none of the melanoma clones studied produced a ligand to the FAS receptor (FAS-L). Thus, our results provide evidence that (i) the production of FAS-L may not be the sole mechanism by which malignant cells induce apoptosis in immunocytes, and (ii) absence of MHC class I glycoproteins plays an important role in preventing the elimination of potential effector immunocytes by tumor cells.

Induction of pre-B cell proliferation after de novo synthesis of the pre-B cell receptor

The assembly of a pre-B cell receptor (pre-BCR) composed of an Ig μ heavy chain (μH-chain), the surrogate light (SL) chain, and the Igα/β dimer is critical for late pro-B cells to advance to the pre-B cell stage. By using a transgenic mouse model, in which μH-chain synthesis is solely driven by a tetracycline-controlled transactivator, we show that de novo synthesis of μH-chain in transgenic pro-B cells not only induces differentiation but also proliferation. This positive effect of μH-chain synthesis on proliferation requires the presence of SL chain and costimulatory signals provided by stromal cells or IL-7. We conclude that pre-BCR signaling induces clonal expansion of early pre-B cells.

Requirement for natural killer T (NKT) cells in the induction of allograft tolerance

In this study, we investigated the role of Vα14 natural killer T (NKT) cells in transplant immunity. The ability to reject allografts was not significantly different between wild-type (WT) and Vα14 NKT cell-deficient mice. However, in models in which tolerance was induced against cardiac allografts by blockade of lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions, long-term acceptance of the grafts was observed only in WT but not Vα14 NKT cell-deficient mice. Adoptive transfer with Vα14 NKT cells restored long-term acceptance of allografts in Vα14 NKT cell-deficient mice. The critical role of Vα14 NKT cells to mediate immunosuppression was also observed in vitro in mixed lymphocyte cultures in which lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions were blocked. Experiments using IL-4- or IFN-γ-deficient mice suggested a critical contribution of IFN-γ to the Vα14 NKT cell-mediated allograft acceptance in vivo. These results indicate a critical contribution of Vα14 NKT cells to the induction of allograft tolerance and provide a useful model to investigate the regulatory role of Vα14 NKT cells in various immune responses.

Induction and repression of DAN1 and the family of anaerobic mannoprotein genes in Saccharomyces cerevisiae occurs through a complex array of regulatory sites

The DAN/TIR mannoprotein genes of Saccharomyces cerevisiae (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3 and TIR4) are expressed in anaerobic cells while the predominant cell wall proteins Cwp1 and Cwp2 are down-regulated. Elements involved in activation and repression of the DAN/TIR genes were defined in this study, using the DAN1 promoter as a model. Nested deletions in a DAN1/lacZ reporter pinpointed regions carrying activation and repression elements. Inspection revealed two consensus sequences subsequently shown to be independent anaerobic response elements (AR1, consensus TCGTTYAG; AR2, consensus AAAAATTGTTGA). AR1 is found in all of the DAN/TIR promoters; AR2 is found in DAN1, DAN2 and DAN3. A 120 bp segment carrying two copies of AR1 preferentially activated transcription of lacZ under anaerobic conditions. A fusion of three synthetic copies of AR1 to MEL1 was also expressed anaerobically. Mutations in either AR1 site within the 120 bp segment caused a drastic loss of expression, indicating that both are necessary for activation and implying cooperativity between adjacent transcriptional activation complexes. A single AR2 site carried on a 46 bp fragment from the DAN1 promoter activated lacZ transcription under anaerobic conditions, as did a 26 bp synthetic AR2 fragment fused to MEL1. Nucleotide substitutions within the AR2 sequence eliminated the activity of the 46 bp segment. Ablation of the AR2 sequences in the full promoter caused a partial reduction of expression. The presence of the ATTGTT core (recognized by HMG proteins) in the AR2 sequence suggests that an HMG protein may activate through AR2. One region was implicated in aerobic repression of DAN1. It contains sites for the heme-induced Mot3 and Rox1 repressors.

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIα promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [3H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68–79% of pyramidal neurons in the CA1–3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.

Mutations Affecting Induction of Glycolytic and Fermentative Genes during Germination and Environmental Stresses in Arabidopsis1

Expression of the alcohol dehydrogenase gene (ADH) of Arabidopsis is known to be induced by environmental stresses and regulated developmentally. We used a negative-selection approach to isolate mutants that were defective in regulating the expression of the ADH gene during seed germination; we then characterized three recessive mutants, aar1–1, aar1–2, and aar2–1, which belong to two complementation groups. In addition to their defects during seed germination, mutations in the AAR1 and AAR2 genes also affected anoxic and hypoxic induction of ADH and other glycolytic genes in mature plants. The aar1 and aar2 mutants were also defective in responding to cold and osmotic stress. The two allelic mutants aar1–1and aar1–2 exhibited different phenotypes under cold and osmotic stresses. Based on our results we propose that these mutants are defective in a late step of the signaling pathways that lead to increased expression of the ADH gene and glycolytic genes.

Adventitious Root Growth and Cell-Cycle Induction in Deepwater Rice1

Deepwater rice (Oryza sativa) is adapted to survive conditions of severe flooding over extended periods of time. During such periods adventitious roots develop to provide water, nutrients, and anchorage. In the present study the growth of adventitious roots was induced by treatment with ethylene but not auxin, cytokinin, or gibberellin. Root elongation was enhanced between 8 and 10 h after submergence. The population of cells in the S phase and expression of the S-phase-specific histone H3 gene increased within 4 to 6 h. Within 6 to 8 h the G2-phase population increased. Cell-cycle activation was accompanied by sequential induction of a cdc2-activating kinase homolog, R2, of two cdc2 genes, cdc2Os-1 and cdc2Os-2, and of three cyclin genes, cycA1;3, cycB2;1, and cycB2;2, but only induction of the R2 gene expression preceded the induction of the S phase, possibly contributing to cell-cycle regulation in the G1 phase. Both cdc2 genes were expressed at slightly higher levels during DNA replication. Transcripts of the A-type cyclin accumulated during the S and G2 phases, and transcripts of the B-type cyclins accumulated during the G2 phase. Cyclin expression was induced at all nodes with a similar time course, suggesting that ethylene acts systemically and that root primordia respond to ethylene at an early developmental stage.

Ccm1, a regulatory gene controlling the induction of a carbon-concentrating mechanism in Chlamydomonas reinhardtii by sensing CO2 availability

Aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii, induce a set of genes for a carbon-concentrating mechanism (CCM) to acclimate to CO2-limiting conditions. This acclimation is modulated by some mechanisms in the cell to sense CO2 availability. Previously, a high-CO2-requiring mutant C16 defective in an induction of the CCM was isolated from C. reinhardtii by gene tagging. By using this pleiotropic mutant, we isolated a nuclear regulatory gene, Ccm1, encoding a 699-aa hydrophilic protein with a putative zinc-finger motif in its N-terminal region and a Gln repeat characteristic of transcriptional activators. Introduction of Ccm1 into this mutant restored an active carbon transport through the CCM, development of a pyrenoid structure in the chloroplast, and induction of a set of CCM-related genes. That a 5,128-base Ccm1 transcript and also the translation product of 76 kDa were detected in both high- and low-CO2 conditions suggests that CCM1 might be modified posttranslationally. These data indicate that Ccm1 is essential to control the induction of CCM by sensing CO2 availability in Chlamydomonas cells. In addition, complementation assay and identification of the mutation site of another pleiotropic mutant, cia5, revealed that His-54 within the putative zinc-finger motif of the CCM1 is crucial to its regulatory function.

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-γ production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-α (TNF-α), IL-6, IL-12, and IFN-γ mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-α mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-α/β. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

Carcinogen-specific induction of genetic instability

It has been proposed recently that the type of genetic instability in cancer cells reflects the selection pressures exerted by specific carcinogens. We have tested this hypothesis by treating immortal, genetically stable human cells with representative carcinogens. We found that cells resistant to the bulky-adduct-forming agent 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) exhibited a chromosomal instability (CIN), whereas cells resistant to the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) exhibited a microsatellite instability (MIN) associated with mismatch repair defects. Conversely, we found that cells purposely made into CIN cells are resistant to PhIP, whereas MIN cells are resistant to MNNG. These data demonstrate that exposure to specific carcinogens can indeed select for tumor cells with distinct forms of genetic instability and vice versa.

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply

Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.