Arsenic-induced congenital malformations in genetically susceptible folate binding protein-2 knockout mice.

Arsenic is a well-known carcinogen, which has been suspected of being a human teratogen, although there is currently insufficient and inadequate supportive data to make any definitive judgments. In addition, the significance of individual genetic differences on pregnancy outcomes following in utero exposure to arsenic is currently unknown. In order to better understand the role of folate transport mechanisms in arsenic-induced neural tube defects, we examined the effect of in utero exposure to sodium arsenate in a genetically altered murine model in which the folate binding protein 2 (Folbp2) gene has been inactivated by homologous recombination. In utero sodium arsenate exposure induced exencephaly in 40.6% of Folbp2(-/-) embryos compared with 24.0% in control Folbp2(+/+) embryos. The differences in response frequencies were further exacerbated when the dams were fed a folate-deficient diet. Under these conditions, exencephaly was observed in 64.0% of Folbp2(-/-) embryos compared with 25.7% in control Folbp2(+/+) embryos. Analysis of arsenic metabolites excreted in the urine following sodium arsenate injection to Folbp2(-/-) and Folbp2(+/+) mice indicated that there were no significant differences in arsenic metabolism between the two groups. Thus, the increased susceptibility of Folbp2(-/-) mice to arsenate-induced teratogenicity may not be due to differences in biomethylation and exposure. In conclusion, the data suggest that impaired folate transport in the developing mouse embryo increases the risk for developmental defects following in utero exposure to sodium arsenate and that these differences are not due to differences in metabolism of arsenic.

Folic acid protects SWV/Fnn embryo fibroblasts against arsenic toxicity.

It has been proposed that arsenic exerts its toxic effects, in part, by perturbing cellular methyl metabolism. Based on the hypothesis that folic acid treatment will attenuate the cytotoxic and growth inhibitory effects of arsenic, SWV/Fnn embryo fibroblasts were cultured in media supplemented with various concentrations of folic acid during treatment with sodium arsenite or dimethylarsinic acid (DMA). It was found that folic acid protects SWV/Fnn embryo fibroblasts from sodium arsenite and DMA cytotoxicity in a dose-dependent manner. In contrast, folic acid supplementation has no effect on toxicity resulting from treatment with ethanol or staurosporine, suggesting that folic acid is not generally protective against necrosis and apoptosis. Although folic acid protects against acute arsenic toxicity, this agent shows a modest and delayed ability to attenuate the growth inhibitory effect of arsenic on these cells. These results support a model in which perturbations of methyl metabolism contribute to the acute cytotoxicity of arsenic.

Sodium arsenite inhibits terminal differentiation of murine C3H 10T1/2 preadipocytes.

Cancer represents an imbalance between cell proliferation and differentiation, two processes that are coordinately and antagonistically regulated. Aberrant cell proliferation is considered to be an important etiological factor in the development of arsenic-induced cancer, suggesting that arsenic also dysregulates differentiation. Based on evidence that arsenic modulates mitogenic events that antagonize the process of differentiation, this study addresses the hypothesis that sodium arsenite inhibits insulin/dexamethasone-induced differentiation of C3H 10T1/2 preadipocytes; it was further postulated that arsenic-treated cells retain mitogenic responsiveness under differentiating conditions. To test this hypothesis, the differentiation capacity of C3H 10T1/2 preadipocytes was examined in control cells and cells treated with sodium arsenite. Differentiation was assessed morphologically and quantified by Oil Red-O staining of accumulated lipids. The effect of long-term arsenic exposure on mitogenic competence was quantified by flow cytometry, [(3)H]thymidine incorporation, and cell counting under conditions favorable for adipocyte differentiation. Results indicate that arsenic inhibits morphological differentiation of wild-type C3H 10T1/2 preadipocytes. Short-term arsenic exposure inhibits differentiation in a dose-dependent manner, with arsenic concentrations > or = 3 microM producing a significant inhibition of dexamethasone/insulin-induced lipid accumulation. Furthermore, arsenic-treated cells exhibit an accentuated response to mitogenic stimulation under differentiating conditions. These data suggest that arsenic exposure results in the inhibition of cellular programming required for terminal differentiation of C3H 10T1/2 preadipocytes and that cells acquire mitogenic hyperresponsiveness. The ability of arsenic to dysregulate the balance between proliferation and differentiation is proposed to be one mechanism by which this metalloid causes cancer in humans.

Sodium arsenite-induced dysregulation of proteins involved in proliferative signaling.

It is well accepted that arsenic is a human carcinogen, yet its mechanism of action is not defined. Arsenic cannot be classified as an initiating agent or as a promoter, although altered proliferative responsiveness has been proposed as a mechanism by which arsenic exerts its carcinogenic effects. Based on the hypothesis that arsenic exposure results in modulation of both positive and negative regulators of cell proliferation, this study examined physiological and biochemical changes in the proliferative response of murine fibroblasts grown long-term in the maximum tolerated concentration of sodium arsenite. In response to EGF stimulation, DNA synthesis and the proportion of cells entering S phase of the cell cycle both were increased in cells grown long-term in arsenic compared to control cells. Analysis of positive proliferative regulators revealed an increase in the expression of c-myc and E2F-1, thereby supporting the hypothesis that arsenic increases activity of positive growth modulators. In contrast, the activity and expression of ERK-2 were unchanged, as was the expression of EGF-receptor and mSOS. When negative regulators of proliferation were examined, expression levels of MAP kinase phosphatase-1 and p27(Kip1) were found to be lower in arsenic-treated cells compared to control cells; this result supports a model in which arsenic disinhibits normal regulation of cell proliferation. Taken together, these data indicate that long-term exposure to sodium arsenite creates conditions within the cell consistent with sensitization to mitogenic stimulation. It is further postulated that the observed changes in mitogenic signaling proteins contribute to the carcinogenic property of arsenic.

Identification of an NPY-Y1 receptor subtype in bovine chromaffin cells.

Bovine chromaffin cells have been used in a variety of studies designed to reveal different aspects of neuropeptide Y (NPY) action. Pharmacological data have defined five NPY receptor subtypes, only one of which (Y3) has not been cloned. Some studies with bovine chromaffin cells have concluded that the effects of NPY on this cell type are mediated by the Y3 subtype. Previous work from our laboratory demonstrates that a Y1 subtype mediates the effect of NPY in this tissue. In the current studies we provide further evidence for the existence of the Y1 subtype in bovine chromaffin cells. BIBP3226, the selective Y1 antagonist, potently displaces [125I]NPY from its binding site IC50 = 1.91 x 10(-9) M. Moreover, [125I]BIBP3226 binds to bovine chromaffin cell membranes with high affinity (IC50 = 5.9 x 10(-8) M). Examination of BIBP3226 antagonism of NPY inhibition of forskolin stimulated cyclic AMP accumulation reveals that it is a competitive antagonist with a K(B) similar to the IC50 for [125I]BIBP3226 binding. Northern blot analysis using a porcine cDNA clone for the Y1 subtype demonstrates a 3.5-kb mRNA species in chromaffin cells. These data identify the bovine chromaffin cell NPY receptor as a Y1 subtype.

Wild-type and Ras-transformed fibroblasts display differential mitogenic responses to transient sodium arsenite exposure.

Arsenic is a human carcinogen whose mechanism of action remains undefined. Based on the hypothesis that arsenic sensitizes cells to mitogenic stimulation by affecting the receptor tyrosine kinase (RTK) signal transduction pathway, these studies first examined the response of fibroblasts to specific mitogens using a defined media system. In both rodent and human fibroblasts, DNA synthesis was found to be stimulated in cells exposed to a transient, sub-lethal concentration of sodium arsenite followed by stimulation with known RTK pathway activators. This effect is observed for up to 32 h after removal of arsenic, suggesting that the RTK pathway is affected in a sustained manner. In contrast, transient arsenic exposure of ras-transformed cells results in decreased mitogen-stimulated DNA synthesis. Flow cytometry indicates that arsenic increases the percentage of wild-type cells in the S-phase of the cell cycle; conversely, the percentage of ras-transformed cells in S-phase is decreased by arsenic. No evidence of arsenic-induced cytotoxicity was detected using the neutral red assay, ensuring that decreased DNA synthesis in ras-transformed cells is not due to cell death. Taken together, the results of experiments presented herein indicate that arsenic produces sustained alterations in the growth characteristics of rodent and human fibroblasts. It is postulated that the proliferation-enhancing effect of arsenic on wild-type cells contributes to its ability to cause cancer.

Pyrethroid insecticides as phosphatase inhibitors.

In this study we tested the hypothesis that pyrethroid insecticides inhibit calcineurin directly and that inhibition is unaffected by the immunophilin cofactors necessary for calcineurin inhibition by cyclosporin A and FK506. The type II pyrethroid insecticides cis-cypermethrin (c-Cyp), trans-cypermethrin, deltamethrin (Delt), and fenvalerate A alpha (Fen), as well as the type I pyrethroid insecticides cis- and trans-permethrin and S-bioallethrin, were unable to inhibit the phosphatase activity of purified calcineurin under conditions of maximal activation by Ca2+ and calmodulin. Furthermore, c-Cyp, Delt, and Fen did not affect the Ca2+ dependence of calcineurin at 0.1 microM of calmodulin, indicating that Ca2+ binding to calmodulin was not affected by these agents. c-Cyp, Delt, and Fen also failed to inhibit calcineurin phosphatase activity in rat brain supernatant and cultured IMR-32 cells, although potent inhibition was displayed by both cyclosporin A and FK506 in each of these systems. Neither the Ca2+-dependent nor the okadaic acid-inhibitable phosphatase activity toward a 24-amino acid 32P-phospho-peptide substrate was affected by any of the pyrethroid insecticides, indicating that neither type-1 or type-2A phosphatase nor calcineurin is inhibited by pyrethroids. To determine if these results were dependent upon experimental conditions, experiments were repeated using polyethylene glycol-treated glass tubes in place of the standard polypropylene tubes. Regardless of the type of tube, no inhibition of calcineurin by any of the pyrethroid insecticides was observed. These data indicate that the pyrethroid insecticides are not effective inhibitors of calcineurin or other phosphatases.

Cyclosporin A has low potency as a calcineurin inhibitor in cells expressing high levels of P-glycoprotein.

Cyclosporin A (CsA) is a widely-used immunosuppressant drug whose therapeutic and toxic actions are mediated through inhibition of calcineurin (CN), a calcium- and calmodulin-dependent phosphatase. Inhibition of CN by CsA requires drug binding to its protein cofactor in the inhibition, cyclophilin. Because cyclophilin is a high affinity target for CsA it is expected that this protein can act as a reservoir for the drug in the cell and may be able to inhibit cellular efflux of CsA. P-glycoprotein (P-gp) is known to increase the rate of CsA efflux from CsA loaded cells but it is not clear if the P-gp drug efflux pump can compete effectively with cyclophilin at therapeutically relevant concentrations of CsA. To test the hypothesis that increased expression of P-gp confers protection against CsA-dependent inhibition of CN phosphatase activity, KB-V cells expressing varying levels of P-gp were analyzed to determine the potency of CsA as a CN inhibitor. When intact cells were treated with CsA, a positive correlation was observed between P-gp expression and resistance to CsA-dependent inhibition of CN: the IC50 is approximately 20-fold higher in the multidrug resistant epidermal carcinoma cell line, KB-V, which expresses P-gp at a high level than in the parental, KB, cell line expressing very low levels of P-gp. The resistance displayed by KB-V cells is abrogated by co-administration of the P-gp inhibitor verapamil, whereas verapamil has no effect on CsA potency in control KB cells. In cell lysates from KB-V cells with different amounts of P-gp CsA exhibits equivalent potency, indicating that the difference in sensitivity to CsA among the cell types requires maintenance of cell integrity. These observations support the view that resistance to CN inhibition by CsA occurs in cells with moderately elevated P-gp activity. Therefore, P-gp activity appears to be an important determinant of CsA cellular specificity for both therapeutic and toxic effects.

Determinants of species- and placenta-specific expression of p100 GAP.

This study was based on the hypothesis that both primary sequence and methylation status of the GTPase activating protein (GAP) gene limits expression of p100 GAP to primate placenta. Due to alternate splicing, a 65-bp insert appears between the first and second coding exons of p100 GAP mRNA, and translation of p100 GAP initiates within this insert. Examination of the sequence surrounding the 65-bp insert revealed that the monkey GAP gene contained both the 3' splice donor site and the internal start codon, whereas the mouse GAP gene contained neither. To address p100 GAP tissue specificity, the methylation status of the GAP gene was examined. Site-specific demethylation was found to correlate with synthesis of p100 GAP, suggesting that methylation regulates the expression of different GAP isoforms. The results of this study provide a mechanistic basis for the observation that p100 GAP is synthesized only in primate placenta and suggest that its expression is regulated, in part, by gene methylation.

Use of an alternate splice donor site in the human GAP gene is responsible for synthesis of the p100 isoform.

GAP (GTPase-activating protein), a negative regulator of the receptor tyrosine kinase signal transduction pathway, exists as two isoforms: a ubiquitous, p120 form and a primate placenta-specific p100 form lacking the N-terminal hydrophobic domain. The cDNA species encoding p120 and p100 GAP are identical except that p100 GAP cDNA contains a 65-bp insert not present in p120 cDNA. The purpose of this study was to locate the 65-bp insert in the genomic GAP sequence, thereby determining the mechanism by which alternate splicing produces the two mRNA species. It was found that the 65-bp insert is located just 3' to the sequence encoding the hydrophobic domain, indicating that the p100 form of GAP results from utilization of an alternate splice donor site. In addition, the sequence encoding the hydrophobic domain was found to be contained within a single large exon. The intron separating this exon from the exon encoding the 5'-portion of the SH2-N domain was determined to be at least 40 kb in length. Finally, it was found that the sequence encoding the SH2-N domain contains an intron 1006 bp long, and the sequence of this intron has been deduced. It is anticipated that the data presented in this paper will provide the basis for elucidating RNA processing mechanisms responsible for preferential expression of p100 GAP in the human placenta.

Compensatory modulation of GAP activity in response to oncogenic stimulation.

GAP is a key negative regulator of the receptor tyrosine kinase (RTK) signal transduction pathway. The purpose of this study was to determine if expression or activity of GAP is modulated by hyperstimulation of the RTK pathway. It was found that cells forced to express wild-type Ha-ras, viral Ha-ras, or v-src exhibit increased GAP activity as compared to control cells. In addition, a novel GAP isoform appears in all ras-expressing NIH3T3 cell clones. These data indicate that there is compensatory regulation of GAP in response to an increase in RTK pathway activity.

Methylation- and mutation-dependent stimulation of Alu transcription in vitro.

Alu genes are GC-rich, highly repetitive genetic elements whose functions remain unknown. Members of this family are readily transcribed in vitro by RNA polymerase III, but RNA corresponding to only a small sub-set of Alu elements has been found in vivo. Based on the hypothesis that methylation of Alu elements affects their transcription, the transcriptional activity of unmethylated and methylated template DNA was assessed in vitro. It was found that methylation of a single CG site just 5' to Alu functions to stimulate transcription; the base composition in this region also affects transcriptional activity. These results indicate that the methylation state and sequence of DNA flanking Alu elements influence its transcription rate.

The RNA polymerase III terminator used by a B1-Alu element can modulate 3' processing of the intermediate RNA product.

The dispersion of short interspersed elements (SINEs) probably occurred through an RNA intermediate. B1 is a murine homolog of the human SINE Alu; these elements are composed of 5' G + C-rich regions juxtaposed to A-rich tracts and are flanked by direct repeats. Internal promoters direct RNA polymerase III to transcribe B1 and Alu elements and proceed into the 3' flanking DNA until it reaches a (dT)4 termination signal. The resulting transcripts contain 3'-terminal oligo(U) tracts which can presumably base pair with the A-rich tract to form self-primed templates for reverse transcriptase and retrotransposition. Nuclear extracts from mouse tissue culture cells contain an RNA processing activity that removes the A-rich and 3'-terminal regions from purified B1 RNAs (R. Maraia, Nucleic Acids Res. 19:5695-5702, 1991). In this study, we examined transcription and RNA processing in these nuclear extracts. In contrast to results with use of purified RNA, nascent transcripts synthesized in nuclear extract by RNA polymerase III are not processed, suggesting that the transposition-intermediate-like RNA is shielded from processing by a protein(s). Alteration of an AATTTT TAA termination signal to a GCTTTTGC signal activated processing by greater than 100-fold in coupled transcription/processing reactions. A similar difference was found when expression was compared in frog oocytes. No difference in processing was found if the transcripts were made by T7 RNA polymerase in the presence of the nuclear extract, indicating that the different processing effects of the two terminators were dependent on synthesis by polymerase III. The modulation of processing of B1-Alu transcripts and the potential for retrotransposition of B1 and Alu DNA sequences are discussed.

Differential DNase I hypersensitivity of ras oncogenes in B6C3F1, C3H/He, and C57BL/6 mouse liver.

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, whereas the paternal C3H/He strain and the maternal C57BL/6 strain exhibit a 60% and a negligible incidence, respectively. In addition, both male and female B6C3F1 mice are extremely sensitive to chemical induction of hepatocarcinogenesis. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently, Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors. The objective of this study was to examine a possible point of transcriptional control of Ha-ras, Ki-ras, and myc in all three mouse strains, our hypothesis being that these oncogenes may be primed for expression in the nascent liver of those strains exhibiting a high spontaneous hepatoma incidence. A positive correlation has been established between gene expression and the presence of DNase I hypersensitive sites. DNase I hypersensitive sites were observed in the Ha-ras and myc oncogenes in the three mouse strains. However, Ha-ras appears to possess an additional site in B6C3F1 and C3H/He as compared to C57BL/6. Similarly, the Ki-ras oncogene exhibited a DNase I hypersensitive site only in B6C3F1 and C3H/He mouse liver. These results indicate that the hepatoma-prone strains (B6C3F1 and C3H/He) may have a greater potential for Ha- and Ki-ras expression than does the non-hepatoma-prone strain (C57BL/6).

Hypomethylation of ras oncogenes in chemically induced and spontaneous B6C3F1 mouse liver tumors.

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, and both males and females of this strain are sensitive to chemical induction of liver tumors. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors, and such activated oncogenes frequently contain a particular point mutation. In light of indications that the transforming capacity of some oncogenes is directly related to the level of the gene product, we hypothesized that transcriptional control of Ha-ras, Ki-ras, and myc is compromised in B6C3F1 mouse liver tumors. A positive correlation has been established between gene expression and hypomethylation. Therefore, the methylation states of these genes were examined in spontaneous liver tumors and in tumors induced by two diverse hepatocarcinogens: phenobarbital and chloroform. Ha-ras was found to be hypomethylated in all tumors examined, whereas Ki-ras was sometimes hypomethylated; such hypomethylation might play a role in the promotion stage of carcinogenesis. The methylation state of myc was unaltered, although this gene appeared to be amplified in tumors. These results suggest that a component of the mechanism by which these oncogenes are activated in B6C3F1 mouse liver tumors involves loss of stringent control of expression, via hypomethylation of the ras oncogenes and, possibly, amplification of myc. These results support the assertion that tumors induced by different classes of carcinogens or arising spontaneously share common biochemical pathways of oncogene activation during tumorigenesis.

DNA mutagenesis and recombination.

The polymerase chain reaction is used for site-specific mutagenesis and for DNA recombination without any enzymatic reaction in vitro, apart from DNA amplification.

Altered methylation of ras oncogenes in benzidine-induced B6C3F1 mouse liver tumors.

The B6C3F1 mouse is a hybrid strain which exhibits a high (30%) spontaneous hepatoma incidence and sensitivity to chemical induction of liver tumors. The spontaneous hepatoma incidence of the paternal C3H/He strain is approximately 60%, while that of the maternal C57BL/6 strain is very low. The presence of activated oncogenes, primarily Ha-ras, and to a lesser extent, Ki-ras, has been reported in B6C3F1 mouse liver tumors. Because alterations in a gene's capacity for expression, as well as mutation, may be involved in oncogene activation, this investigation was directed toward an examination of a putative control point for transcription, i.e., the methylation state of a gene. Hypomethylation is believed to be necessary, but not sufficient, for transcription. It was therefore hypothesized that alterations in the methylation state of the Ha-ras and Ki-ras oncogenes may facilitate the aberrant expression of these genes in B6C3F1 mouse liver. Restriction enzyme analysis (MspI, HpaII, and HhaI) was used to assess the extent of DNA methylation. MspI digestion of B6C3F1 and C3H/He DNA revealed the absence of a 15-kb Ha-ras band present in MspI-digested C57BL/6 DNA, suggesting that the Ha-ras oncogene of B6C3F1 and C3H/He mouse liver lacks a methylated site. In other respects, the Ha-ras and Ki-ras oncogenes are methylated to a degree which suggests that these oncogenes have a low potential for expression in normal mouse liver. The methylation state of the Ha-ras and Ki-ras oncogenes was also assessed in benzidine-induced hepatomas and adjacent nontumor tissue from B6C3F1 mice. In four out of four cases, the Ha-ras oncogene was hypomethylated in tumor as compared to nontumor tissue and increased expression of the gene was detected in three out of four hepatomas; the Ki-ras oncogene was hypomethylated in two out of four cases. These results suggest that hypomethylation of oncogenes may provide an epigenetic mechanism for facilitating their aberrant expression. The lack of a methylated site observed in the Ha-ras oncogene in B6C3F1 and C3H/He mouse liver may indicate an increased potential for its expression which could, in part, account for the high propensity for hepatoma development in these two strains.

Differential DNase I hypersensitivity of ras oncogenes in B6C3F1, C3H/He, and C57BL/6 mouse liver.

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, whereas the paternal C3H/He strain and the maternal C57BL/6 strain exhibit a 60% and a negligible incidence, respectively. In addition, both male and female B6C3F1 mice are extremely sensitive to chemical induction of hepatocarcinogenesis. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently, Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors. The objective of this study was to examine a possible point of transcriptional control of Ha-ras, Ki-ras, and myc in all three mouse strains, our hypothesis being that these oncogenes may be primed for expression in the nascent liver of those strains exhibiting a high spontaneous hepatoma incidence. A positive correlation has been established between gene expression and the presence of DNAase I hypersensitive sites. DNase I hypersensitive sites were observed in the Ha-ras and myc oncogenes in the three mouse strains. However, Ha-ras appears to possess an additional site in B6C3F1 and C3H/He as compared to C57BL/6. Similarly, the Ki-ras oncogene exhibited a DNase I hypersensitive site only in B6C3F1 and C3H/He mouse liver. These results indicate that the hepatoma-prone strains (B6C3F1 and C3H/He) may have a greater potential for Ha- and Ki-ras expression than does the non-hepatoma-prone strain (C57BL/6).