The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia.

Cell movement is mediated by the protrusion of cytoplasm in the form of sheet- and rod-like extensions, termed lamellipodia and filopodia. Protrusion is driven by actin polymerization, a process that is regulated by signaling complexes that are, as yet, poorly defined. Since actin assembly is controlled at the tips of lamellipodia and filopodia [1], these juxtamembrane sites are likely to harbor the protein complexes that control actin polymerization dynamics underlying cell motility. An understanding of the regulation of protrusion therefore requires the characterization of the molecular components recruited to these sites. The Abl interactor (Abi) proteins, targets of Abl tyrosine kinases [2-4], have been implicated in Rac-dependent cytoskeletal reorganization in response to growth factor stimulation [5]. Here, we describe the unique localization of Abi proteins in living, motile cells. We show that Abi-1 and Abi-2b fused to enhanced yellow fluorescent protein (EYFP) are recruited to the tips of lamellipodia and filopodia. We identify the targeting domain as the homologous N terminus of these two proteins. Our findings are the first to suggest a direct involvement of members of the Abi protein family in the control of actin polymerization in protrusion events, and establish the Abi proteins as potential regulators of motility.

Localization and phosphorylation of Abl-interactor proteins, Abi-1 and Abi-2, in the developing nervous system.

Abl-interactor (Abi) proteins are targets of Abl-family nonreceptor tyrosine kinases and are required for Rac-dependent cytoskeletal reorganization in response to growth factor stimulation. We asked if the expression, phosphorylation, and cellular localization of Abi-1 and Abi-2 supports a role for these proteins in Abl signaling in the developing and adult mouse nervous system. In mid- to late-gestation embryos, abi-2 message is elevated in the central and peripheral nervous systems (CNS and PNS). Abi-1 mRNA is present, but not enhanced, in the CNS, and is not observed in PNS structures. Abi proteins from brain lysates undergo changes in apparent molecular weight and phosphorylation with increasing age. In the postnatal brain, abi-1 and abi-2 are expressed most prominently in cortical layers populated by projection neurons. In cultured neurons, Abi-1 and Abi-2 are concentrated in puncta throughout the cell body and processes. Both Abi and Abl proteins are present in synaptosomes and growth cone particles. Therefore, the Abi adaptors exhibit proper expression patterns and subcellular localization to participate in Abl kinase signaling in the nervous system.

Analysis of the biologic properties of p230 Bcr-Abl reveals unique and overlapping properties with the oncogenic p185 and p210 Bcr-Abl tyrosine kinases.

The reciprocal translocation between chromosomes 9 and 22 that fuses coding sequences of the Bcr and Abl genes is responsible for a remarkably diverse group of hematologic malignancies. A newly described 230-kd form of Bcr-Abl has been associated with an indolent myeloproliferative syndrome referred to as chronic neutrophilic leukemia. We have cloned the corresponding gene and examined the biologic and biochemical properties of p230 Bcr-Abl after retroviral-mediated gene transfer into hematopoietic cell lines and primary bone marrow cells. p230 Bcr-Abl-expressing 32D myeloid cells were fully growth factor-independent and activated similar signal transduction pathways as the well-characterized p210 and p185 forms of Bcr-Abl. In contrast, primary mouse bone marrow cells expressing p230 required exogenous hematopoietic growth factors for optimal growth, whereas p185- and p210-expressing cells were independent of growth factors. The 3 Bcr-Abl proteins exerted different effects on differentiation of bone marrow cells. p185 induced outgrowth of lymphoid precursors capable of tumor formation in immunodeficient mice. In contrast, p210- and p230-expressing bone marrow cells caused limited outgrowth of lymphoid precursors that failed to form tumors in immunodeficient mice. Removal of cytokines and autologous stroma from Bcr-Abl-expressing bone marrow cultures produced the expansion of distinct lineages by the various Bcr-Abl proteins. p185 drove expansion of cytokine-independent lymphoid progenitors, while p210 and p230 generated cytokine-independent monocyte/myeloid cells. These findings suggest that the different Bcr-Abl fusion proteins drive the expansion of different hematopoietic populations, which may explain the association of the various Bcr-Abl oncoproteins with different spectra of human leukemias. (Blood. 2000;95:2913-2921)

Oncogenic Abl and Src tyrosine kinases elicit the ubiquitin-dependent degradation of target proteins through a Ras-independent pathway.

Oncogenic forms of the Abl and Src tyrosine kinases trigger the destruction of the Abi proteins, a family of Abl-interacting proteins that antagonize the oncogenic potential of Abl after overexpression in fibroblasts. The destruction of the Abi proteins requires tyrosine kinase activity and is dependent on the ubiquitin-proteasome pathway. We show that degradation of the Abi proteins occurs through a Ras-independent pathway. Significantly, expression of the Abi proteins is lost in cell lines and bone marrow cells isolated from patients with aggressive Bcr-Abl-positive leukemias. These findings suggest that loss of Abi proteins may be a component in the progression of Bcr-Abl-positive leukemias and identify a novel pathway linking activated nonreceptor protein tyrosine kinases to the destruction of specific target proteins through the ubiquitin-proteasome pathway.

Reduction of chromium(VI) by ascorbate leads to chromium-DNA binding and DNA strand breaks in vitro.

Chromium(VI) is a known human carcinogen which requires intracellular reduction for activation. Ascorbate (vitamin C) has been reported to function as a major reductant of Cr(VI) in animals and cell culture systems. The reaction of Cr(VI) with varying concentrations of ascorbate was studied under physiological conditions in vitro in order to determine the types of reactive intermediates produced and to evaluate the reactivity of these intermediates with DNA. Reactions of 1.8 mM Cr(VI) with 0-18 mM ascorbate at pH 7.0 in N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES; 0.10 M) and tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl; 0.050 M) buffers were studied by electron paramagnetic resonance and UV/visible spectroscopy. Cr(V) and carbon-based free radical adducts of 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were observed at 0.5 to 1 and 1 to 1 reactions of ascorbate to Cr(VI). Levels of Cr(V) were higher for reactions in HEPES buffer, and levels of carbon-based radicals were higher in Tris.HCl buffer. Levels of Cr(IV) and Cr(III) increased with increasing concentration of ascorbate in both buffers. Reaction of Cr(VI) with varying ascorbate in the presence of calf thymus DNA or pBR322 DNA resulted in Cr-DNA adducts and plasmid relaxation, respectively. Maximum binding of Cr to DNA was observed for the 1:1 reaction ratio of Cr(VI) with ascorbate in both HEPES and Tris.HCl buffers, but total Cr bound to DNA was 8-fold lower in Tris.HCl than HEPES buffer. Preincubation of Cr(VI) with ascorbate before reaction with DNA decreased Cr-DNA binding to background levels. Preincubation of Cr(III) with ascorbate resulted in only low Cr-DNA binding. Levels of Cr-DNA binding were higher with single-stranded vs double-stranded DNA. Reactions with 14C-labeled ascorbate produced no cross-linking of ascorbate to DNA. Maximum plasmid relaxation was observed for the 1:1 ascorbate to Cr(VI) ratio in both buffers; however, single-strand breaks were 2-fold higher in Tris.HCl than HEPES buffer. Reactions with plasmid in the presence of DMPO quenched formation of single-strand breaks. Interpretation of these results in light of the spectroscopic studies suggested that Cr(V) and carbon-based radicals were responsible for Cr-DNA adducts and DNA single-strand breaks, respectively.

Chromium(VI) reduction by ascorbate: role of reactive intermediates in DNA damage in vitro.

Reaction of chromium(VI) with one equivalent of ascorbate was studied by electron paramagnetic resonance spectroscopy in the presence of 0.10 M 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) at room temperature in 0.10 M (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (HEPES) and 0.05 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffers (pH 7.0 room temperature). Chromium(V), ascorbyl radical, and carbon-based DMPO-radical adducts were observed. A higher level of Cr(V) was observed in HEPES buffer and a higher level of the DMPO-radical adducts was observed in Tris-HCl buffer. Chromium-DNA binding studies were carried out in vitro for calf thymus DNA incubated with Cr(VI) and ascorbate in both buffers at 37 degrees C. Higher Cr-DNA binding was observed in HEPES buffer. DNA strand-break studies were carried out in vitro on pBR322 DNA incubated with Cr(VI) and ascorbate in both buffers at 37 degrees C. Higher percent nicking was observed in Tris-HCl buffer. Addition of DMPO decreased nicking levels in Tris-HCl buffer. These results suggest that free radicals are more reactive than Cr(V) in producing DNA strand breaks and that Cr(V) will react with DNA to produce Cr-DNA adducts. The fact that buffer affects the nature of the reactive intermediates produced upon reduction of Cr(VI) may be related to differences in intracellular metabolism of Cr(VI) and resulting DNA damage observed in various cell culture systems and animal tissues in vivo.

Hexachlorobenzene-induced hyperparathyroidism and osteosclerosis in rats.

Hexachlorobenzene (HCB) exposure has been shown to alter the normal concentrations of parathyroid hormone and 1,25-dihydroxyvitamin D3 in rats and to result in osteoporosis in humans. Experiments were undertaken to investigate the effects of HCB on the homeostatic mechanism of calcium metabolism and to determine its effect on bone in rats. Fischer 344 rats were dosed 5 days/week for 5, 10, or 15 weeks with 0, 0.1, 10.0, or 25.0 mg HCB/kg body wt. Body weight was not affected by any of the exposure conditions. Liver weight was significantly elevated above control values at the two higher dose levels at all three time periods. Kidney weight and kidney-to-body weight ratio were significantly elevated at the highest dose level after 10 weeks and at the two higher dose levels after 15 weeks of exposure. Serum alkaline phosphatase was significantly decreased at the two higher dose levels after both 10 and 15 weeks of exposure. 1,25-Dihydroxyvitamin D3 was measured in the 5-week exposure group only and was significantly elevated in the three higher dose levels. After 5 and 15 weeks of HCB exposure, parathyroid hormone concentration was significantly elevated at the two higher dose levels at both time periods. Wet femur density was significantly increased at the two higher dose levels of HCB after 10 weeks of exposure and the three higher dose levels after 15 weeks of exposure. Dry femur density was also increased in the cases where wet femur density was increased. However, femur weight was not affected at any dose level. The results from this study indicate that HCB induces hyperparathyroidism in rats, as demonstrated by increased serum parathyroid hormone levels and osteosclerosis of the femur.(ABSTRACT TRUNCATED AT 250 WORDS)

Impairment of calcium homeostasis by hexachlorobenzene (HCB) exposure in Fischer 344 rats.

Human exposure to hexachlorobenzene (HCB) has resulted in demineralization of bone and development of osteoporosis. Experiments were undertaken to investigate the effects of HCB on the homeostatic mechanism of calcium metabolism. Fischer 344 rats were dosed with 0, 0.1, 1.0, 10.0, or 25.0 mg HCB/kg body weight 5 d/wk for 5 wk while being fed normal rat diet or vitamin D3-deficient diet. Rats receiving the normal diet had a dose-related decrease in body weight gain and increased liver weight when compared to their controls. Serum cholesterol, alanine aminotransferase (ALT), 1, 25-dihydroxy-vitamin D3 [1,25-(OH)2D3], and parathyroid hormone (PTH) were significantly elevated when compared to control values. In the vitamin D3-deficient diet group, there was a dose related increase in liver weight, liver-to-body weight ratio and kidney-to-body weight ratio. Serum cholesterol and 1,25-(OH)2D3 were significantly elevated. Urinary calcium decreased significantly with increasing HCB dosage, indicating conservation of calcium. The data from this study indicate that HCB does affect calcium metabolism by altering the concentrations of two primary controlling factors in calcium homeostasis.

Assessment of teratogenic potential of trichlorfon in mice and rats.

Trichlorfon was evaluated for its teratogenic potential in the CD-1 mouse at doses of 200, 300 or 400 mg/kg/day administered by gavage on days 7-16 of gestation. In the CD-1 mouse, TCF was teratogenic, fetotoxic and lethal at the two highest dose levels which were also maternally lethal. At the lowest dose level which was not maternally lethal, there was a significant decrease in the number of calcified centers in the forepaws and hindpaws indicating fetotoxicity and a delay in maturation. TCF was administered at doses of 50, 100 or 200 mg/kg/day to CD rats by gavage on gestational days 7-19 (study I) or 8-20 (study II). In the CD rat in body study I and II, the highest dose level was maternally lethal. In study I, TCF was teratogenic with a shift in rib profile. In study II, TCF was teratogenic with an increased incidence in malformations of the urinary system. Additionally, TCF was fetotoxic with reduced ossification of the skulls at the lowest and highest dose levels.

A perinatal study of toluene in CD-1 mice.

Toluene administered by inhalation at 400 ppm to CD-1 mice from Days 6 to 16 of gestation was teratogenic but not fetotoxic resulting in a significant shift in the fetal rib profile. At the lower concentration of 200 ppm, there was an increase in dilated renal pelves which might reflect desynchronization of maturation with respect to development and growth. No other effects were noted at the 200-ppm concentration. At 400 ppm, toluene also produced an increased body weight in the neonates on Day 1 postpartum following in utero exposure. Activity of lactic dehydrogenase (LDH) was significantly increased in the brains of dams exposed to 400 ppm during gestation while nonpregnant adult mice studied concurrently had significant increased activities of LDH in the liver and kidneys of the 400-ppm group. The only change in the isozyme profiles was in the kidneys of the nonpregnant adult mice in which a slight decrease in LDH-2 was observed. No other changes were noted in the dams or pups.

Hexachlorobenzene-induced renal maldevelopment in CD-1 mice and CD rats.

Enlarged kidneys and hydronephrosis were observed in day-15 post-partum (pp) CD-1 mouse pups from dams treated with 10 or 50 mg hexachlorobenzene (HCB) per kg body weight (bw) on days 6-16 of gestation. Additional studies showed that enlarged kidneys occurred also on days 1 and 20 pp. CD rat pups from dams exposed to 10 mg HCB per kg bw on days 15-20 of gestation had enlarged kidneys on day 5 pp but not on days 10 or 20. In the CD rat, there was a significant increase in the kidney:bw ratio and the liver:bw ratio for the HCB-exposed pups at all three time periods. Pre- and postnatal exposure to HCB resulted in renal maldevelopment in CD-1 mice and CD rats in terms of enlarged kidneys and hydronephrosis.

Teratogenic evaluation of the pesticides baygon, carbofuran, dimethoate and EPN.

Baygon was administered IG once daily to CD rats (5 to 50 mg/kg), on the 7th-19th day of gestation or to CD-1 mice (5 to 60 mg/kg) on days 6-16 of gestation. Baygon, at dose levels which were not maternally lethal, did not produce fetotoxicity, fetal lethality or malformations in the fetuses. Baygon was not teratogenic in the CD rat or CD-1 mouse at maternally nontoxic dose levels. Carbofuran was administered IG once daily to CD rats (0.05 to 5.0 mg/kg), on the 7th-19th day of gestation or to CD-1 mice (0.1 to 20 mg/kg) on days 6-16 of gestation. At dose levels which were not maternally lethal, carbofuran did not produce fetotoxicity, fetal lethality or malformations in the fetuses. Carbofuran was not teratogenic in the CD rat or CD-1 mouse at maternally nontoxic dose levels. Dimethoate was administered IG once daily to CD-1 mice (10 to 80 mg/kg), on the 6th-16th day of gestation. At dose levels which were not maternally lethal, dimethoate did not produce fetotoxicity, fetal lethality or malformations in the fetuses. Dimethoate was not teratogenic in the CD-1 mouse at maternally nontoxic dose levels. EPN was administered IG once daily to CD-1 mice (1.0 to 12.0 mg/kg) on the 6th-16th day of gestation. EPN, at dose levels up to those which were maternally lethal, did not produce fetotoxicity, fetal lethality or an increase in malformations. EPN was not teratogenic in the CD-1 mouse at maternally nontoxic dose levels.

Placental transfer and fetal deposition of hexachlorobenzene in the hamster and guinea pig.

Hexachlorobenzene (HCB) was administered at dose levels of 0, 1.0, 10.0, or 50.0 mg HCB/kg body wt by gavage to pregnant hamsters and guinea pigs for 6 days up to the time of liver development in the fetus. Samples of maternal fat, thymus, skin, liver, lung, brain, spleen, urinary bladder, muscle, plasma, and blood were analyzed for HCB concentration. Additionally, fetuses, placentas, and yolk sacs were analyzed for HCB content. HCB was found in all tissues assayed. The highest concentrations of HCB were found in hamster tissues, with the hamster fetuses having a fivefold greater concentration of HCB than the guinea pig fetuses. Comparisons were made of the hamster and guinea pig data with similar data in the mouse and rat.

Neonatal and maternal body burdens of hexachlorobenzene (HCB) in mice: gestational exposure and lactational transfer.

Hexachlorobenzene (HCB), a ubiquitous lipophilic pollutant, was readily transferred in the milk of lactating dams to their suckling neonates. Pregnant CD-1 mice were treated during gestation, and the body burdens of HCB in the neonates and the dams were determined during lactation. Also, neonates from dams treated with HCB during gestation were cross-fostered at birth to dams treated with corn oil during gestation. The body burdens of HCB were greater in the neonates exposed to HCB by lactational transfer than the neonates exposed only by gestational transfer. In many tissues, the concentration of HCB in the pups from full litter was similar to that in pups from litters reduced to two pups per litter. Lactational transfer of HCB from the dams to the pups was a major route of excretion in that 95% of HCB was depleted during 20 days of lactation. HCB depletion was similar in dams with whole litters, and those with litters reduced to two pups.

Postnatal effects of hexachlorobenzene (HCB) on cardiac lactic dehydrogenase (LDH) and creatine kinase (CK) isozymes in CD-1 mice.

Pregnant CD-1 mice were treated with hexachlorobenzene (HCB) by gavage at doses of 0, 1, 10 and 50 mg HCB/kg body weight on days 6-17 of gestation and studied on day 1 or 21 postpartum (pp). Hearts of the dams and pups were assayed for lactic dehydrogenase (LDH) and creatine kinase (CK) activities and isozyme profiles. LDH and CK activities and CK isozyme profiles were not affected in the dams by HCB treatment, but isozyme LDH-5 was significantly depressed and isozyme LDH-3 significantly increased at the 50 mg/kg dose of HCB on day 1 pp. Hearts of the pups were studied on days 1, 8, 10, 15 and 21 pp. The 50 mg/kg dose level resulted in a statistically significantly increased mortality in the pups. The mortality at the other doses showed a dose-related effect. LDH and CK activities and CK isozyme profiles were not affected by HCB exposure in utero. The only isozyme affected was LDH-5 with a statistically significant increase in the 50 mg/kg dose group on day 21 pp.

Teratogenic evaluation and fetal deposition of hexabromobenzene (HBB) and hexafluorobenzene (HFB) in CD-1 mice.

HBB (hexabromobenzene) and HFB (hexafluorobenzene) were tested for their teratogenic potential in CD-1 mice. HBB and HFB were administered to pregnant mice from the 6th to the 16th day of gestation by gastric intubation. Neither HBB nor HFB were teratogenic or fetotoxic at doses up to 98.6 mg HBB/kg and 65.3 mg HFB/kg. No maternal toxicity was noted. HBB concentration in the fetuses indicated little, if any accumulation. No HFB was detected in the fetal or maternal tissues 24 hours after the last dose.

The effects of ethylene chlorohydrin on fatty acid synthesis.

Male chicks weighing 700 to 900 g. received an acute or eight doses IG of 60 or 40 mg/kg ethylene chlorohydrin (ECH) respectively and were sacrificed eighteen hours after the last dose. Mitochondrial elongation of fatty acids was decreased significantly while fatty acid synthetase activity was not significantly affected by ECH treatment. Cytochrome c oxidase activity in fresh whole liver homogenate was significantly higher in chicks subjected to acute exposure with ECH when compared to the controls. Upon freezing and thawing of homogenates, cytochrome c oxidase activity increased significantly in the control group but was unchanged in the ECH group which suggests that the mitochondrial membrane integrity is compromised by the ECH treatment. Serum and liver triglyceride levels were significantly elevated in both the single and multiple ECH dose groups. Liver to body weight ratios were significantly higher in both treatment groups when compared to their controls. Histological examination of the liver of ECH-treated chicks showed cytoplasmic clearing of the cells but no vacuolization or centrilobular necrosis. Serum isocitrate dehydrogenase levels were significantly higher in the multiple treatment ECH group than in the control group.

Teratogenic evaluation of ethylene chlorhydrin (ECh, 2-chlorethanol) in mice.

ECh administered intragastrically to pregnant CD-1 mice from the 6th to the 16th day of gestation at a dose of 100 mg/kg produced a significant reduction in maternal weight gain and a decrease in fetal body weight and liver weight. A lower dose of 50 mg/kg had no consistent effect and a higher dose of 150 mg/kg was maternally lethal. Administration of ECh in the drinking water at doses of 16, 43, 77, or 227 mg/kg did not produce any adverse effects on maternal or fetal body weight, viability, or fetal anatomical development.

2,4,5-T effects on cardiac and serum lactic dehydrogenase (LDH) and creatine kinase (CK) isozymes. II. Neonatal enzyme activities and isozyme profiles.

Neonates from CD-1 mice, which were treated during gestation with 100 mg/kg of 2,4,5-T, were studied from day 1 to 30 postpartum (pp) for effects on cardiac development by determining total cardiac activities and isozyme profiles of LDH and CK. On day 1pp, the total activities and isozyme profiles of LDH of the neonatal hearts were normal. During the developmental period of day 7 through 15, changes were noted in the developmental pattern of LDH isozymes some of which continued to day 30. The CK isozyme profile on day 1pp showed a significant change and changes continued throughout the lactational period. During this period, the normal developmental isozyme patterns of LDH and CK were altered by prenatal exposure to 2,4,5-T suggesting metabolic derangement or pathological changes in the neonatal heart.

2,4,5-T effects on cardiac and serum lactic dehydrogenase (LDH) and creatine kinase (CK) isozymes. I. Maternal enzyme activities and isozyme profiles.

Pregnant mice were exposed to 100 mg/kg 2,4,5-T, IG, on gestation days 6 through 17, and sacrificed on postpartum day 1 or 21. Toxicologic evaluation showed no changes in body weight, heart weight, heart to body weight ratio, or cardiac supernate protein between corn oil control and 2,4,5-T treated mice on days 1 or 21 postpartum (pp). There were no effects of 2,4,5-T treatment on the total LDH enzyme activity of cardiac supernate on day 1 or 21pp. Serum LDH total activity was depressed on day 1pp and comparable to control values on day 21pp. Cardiac CK total activity was elevated on day 1pp, but not on day 21pp. Serum CK total activity on day 1 was comparable to control values; however, on day 21, a significant decrease in activity was observed. Cardiac LDH and CK isozyme profiles were normal on days 1 and 21pp. The serum LDH isozyme profile was normal at both times. The serum CK isozyme profile on Day 1 was markedly altered by athe appearance of two aberrant isozyme bands while on day 21, there was a profile shift with an increase in the BB band and a compensatory decrease in the MM band. These changes in creatine kinase suggest metabolic or pathologic changes in the cardiac muscle.