Sandra Leone-Kabler

Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aliphatic aldehydes in V79 cells.

Toxic medium chain length alkanals, alkenals, and 4-hydroxyalkenals that are generated during lipid peroxidation are potential substrates for aldehyde dehydrogenase (ALDH) isoforms. We have developed transgenic cell lines to examine the potential for either human ALDH1A1 or ALDH3A1 to protect against damage mediated by these toxic aldehydes. Using crude cytosols from stably transfected cell lines, these aldehydes were confirmed to be excellent substrates for ALDH3A1, but were poorly oxidized by ALDH1A1. Expression of ALDH3A1 by stable transfection in V79 cells conferred a high level of protection against growth inhibition by the medium-chain length aldehyde substrates with highest substrate activity, including hexanal, trans-2-hexenal, trans-2-octenal, trans-2-nonenal, and 4-hydroxy-2-nonenal (HNE). This was reflected in a parallel ability of ALDH3A1 to prevent depletion of glutathione by these aldehydes. Expression of hALDH3 completely blocked the potent induction of apoptosis by HNE in both V79 cells and in a RAW 264.7 murine macrophage cell line, consistent with the observed total prevention of HNE-protein adduct formation. Structure-activity studies indicated that the rank order of potency for the contributions of HNE functional groups to toxicity was aldehyde >/=C2=C3 double bond>>C4-hydroxyl group. Oxidation of the aldehyde moiety of HNE to a carboxyl by ALDH3A1 expressed in stably transfected cell lines drastically reduced its potency for growth inhibition and apoptosis induction. In contrast, ALDH1A1 expression provided only moderate protection against trans-2-nonenal (t2NE), and none against the other six-nine carbon aldehydes. Neither ALDH1A1 nor ALDH3A1 conferred any protection against acrolein, acetaldehyde, or chloroacetaldehyde. A small degree of protection against malondialdehyde was afforded by ALDH1A1, but not ALDH3A1. Paradoxically, cells expressing ALDH3A1 were 1.5-fold more sensitive to benzaldehyde toxicity than control V79 cells. These studies demonstrate that expression of class 3 ALDH, but not class 1 ALDH, can be an important determinant of cellular resistance to toxicity mediated by aldehydes of intermediate chain length that are produced during lipid peroxidation.

Differential sensitivity to lung tumorigenesis following transplacental exposure of mice to polycyclic hydrocarbons, heterocyclic amines, and lung tumor promoters

Research conducted by this laboratory over the past decade has demonstrated the high susceptibility of the fetus to lung tumor formation following in utero exposure of the resistant C57BL/6 and DBA/2N strains of mice to 3-methylcholanthrene (MC). In this review, we describe our more recent studies on the effects of MC and cotreatment with the lung tumor promoter, butylated hydroxytoluene (BHT), on lung tumor formation in the intermediately susceptible BALB/c strain of mice, and the determination of the potential carcinogenicity of the heterocyclic amine, 2-amino3-methylimidazo[4,5-f]quinoline (IQ) in resistant mouse strains. BALB/c mice showed a similar incidence of lung tumors, both in terms of percentage of mice with tumors and number of tumors per mouse, as found in the resistant [D2 B6D2F1]F2 mice. Ki-ras point mutations were found in 56% (20/36) of BALB/c lung lesions compared with an incidence of 79% in [D2 B6D2F1]F2 mice. BALB/clung lesions demonstrated a similar association of Ki-ras

Role of tumor suppressor genes in transplacental lung carcinogenesis

Most human cancers involve multiple genetic changes, including activation of oncogenes such as Ki‐ras‐2 (Kras2) and inactivation of any one of a number of tumor suppressor genes such as p53 and members of the retinoblastoma (Rb) regulatory axis. As part of an ongoing project to determine how in utero exposure to chemical carcinogens affects the molecular pathogenesis of murine lung tumors, the p53 and p16Cdkn2a genes were analyzed by using paraffin‐embedded lung tissues from mice treated transplacentally with 3‐methylcholanthrene. Single‐strand conformation polymorphism analysis of exons 5–8 of the p53 gene, as well as their flanking introns, demonstrated an absence of mutations at this gene locus. However, a genetic polymorphism was identified at nt 708 in intron 4 of the DBA/2 strain of mice 5 bp downstream of a 3′ branching‐point splice signal. Analysis of exons 1 and 2 of the Cdkn2a gene by single‐strand conformation polymorphism and sequence analyses revealed mutations in exon 2 in 7% of the tumors examined. Tumor 23‐1 exhibited a CAC→TAC transition at nt 301 (His74→Tyr74), and tumor 36‐1 exhibited a GGG→GAG transition at nucleotide 350 (Gly90→Glu 90). Northern blot analysis of 14 of the larger tumors showed a marked decrease in the levels of Rb RNA expression. Immunohistochemical analysis revealed a spectrum of pRb expression, with the smaller adenomas showing moderate numbers of nuclei with heterogeneous staining for pRb in contrast with a highly reduced or near‐complete absence of expression in the nuclei of larger tumors with features of adenocarcinomas. The low incidence of mutations at tumor suppressor loci suggested that inactivation of tumor suppressor genes was a late event in murine lung tumor pathogenesis. The identification of both mutations at the Cdkn2a gene locus and reduced levels of Rb expression combined with previous studies demonstrating a high incidence of mutated Kras2 alleles in these tumors implies that alterations of the Rb regulatory axis, in combination with mutation of Kras2, may be the preferred pathway for the pathogenesis of pulmonary tumors in transplacentally exposed mice. Mol. Carcinog. 21:177–184, 1998.

Molecular pathogenesis of transplacentally induced mouse lung tumors

Previous studies from this and other laboratories have shown that treatment of pregnant mice with 3-methylcholanthrene (MC) caused lung tumors in the offspring, the incidence of which correlated with fetal inducibility of Cyp1a1. Analysis of paraffin-embedded lung tissue for Ki-ras-2 mutations indicated that 79% of the lesions examined contained point mutations in codons 12 and 13 of the Ki-ras-2 gene locus, the majority of which (84%) were G-->T transversions. The mutational spectrum was dependent on the tumor stage, as both the incidence of mutation and type of mutation produced correlated with malignant progression of the tumor. Mutations occurred in 60% of the hyperplasias, 80% of the adenomas, and 100% of the adenocarcinomas. In the tumors with mutations, GLY12-->CYS12 transversions occurred in 100% of the hyperplasias, 42% of the adenomas, and 14% of the adenocarcinomas. GLY12-->VAL12 transversions were not observed in hyperplasias and occurred in 42% of the adenomas and 57% of the adenocarcinomas. The remaining ASP12 and ARG13 mutations occurred only in adenomas (17%) and adenocarcinomas (29%). The tumors were also analyzed for alterations in the structure or function of the tumor suppressor genes Rb, p53, and Cdkn2a. No mutations were observed in exons 5-8 of the p53 gene. SSCP analysis demonstrated that 2 of 15 lung tumors contained shifted bands at the Cdkn2a gene locus. Sequence analysis had identified these as mutations in exon 2, with a CAC-->TAC transition at base 301 (HIS74-->TYR74) in tumor 23-1 and GGG-->GAG transition at base 350 (GLY90-->GLU90) in tumor 36-1. Northern blot analysis of the larger tumors revealed that 14 of 14 of these large lung tumors exhibited markedly decreased expression of Rb gene transcripts. These results were confirmed by immunohistochemistry. The larger tumors, which exhibited features of adenocarcinomas, showed a marked reduction or almost complete absence of nuclear pRb staining compared with smaller adenomas and normal lung tissue. The results suggest that Ki-ras-2 mutations are an early and frequent event in lung tumorigenesis, and that the type of mutation produced by environmental chemicals can influence the carcinogenic potential of the tumor. The results obtained with the Cdkn2a and Rb genes suggest that alterations in the Rb regulatory axis may play a key role in the pathogenesis of the pulmonary tumors and appear to occur later in the neoplastic process. It appears from these experiments that the combination of mutated Ki-ras-2 and alterations in the Rb regulatory gene locus, which are frequent alterations in human lung tumors, may be the preferred pathway for lung tumor pathogenesis in mice exposed transplacentally to environmental carcinogens.

Effects of prior oral contraceptive use and soy isoflavonoids on estrogen-metabolizing cytochrome P450 enzymes

Estrogen exposure and metabolism may play an important role in the development of estrogen-sensitive cancers in postmenopausal women. In this study we investigated whether past oral contraceptive (OC) administration or current dietary isoflavonoids (IF) affected expression and/or activity of steroid hormone-metabolizing cytochrome P450 (CYP) enzymes using complementary primate and cell culture models. One-hundred-eighty-one female cynomolgus macaques were randomized to receive OC or nothing for 26 months premenopausally, then ovariectomized and randomized to one of three diets for 36 months: an IF-depleted soy protein isolate (Soy-) diet, a Soy diet with IF (Soy+), or a Soy- diet supplemented with conjugated equine estrogens (CEE). Prior OC-treatment significantly reduced CYP gene expression in the mammary gland (< or =60% of OC-). Dietary IFs had no effect on CYP expression, while CEE-treatment decreased CYP1A1 and increased CYP3A4 mRNA in a tissue-specific manner. For in vitro studies, we measured effects of the isoflavonoids genistein, daidzein and equol on CYP activity using intact V79 cells stably transfected to express CYP1A1, CYP1B1, or CYP3A4. All three IFs significantly altered CYP activity in a dose-dependent and isoform-specific manner (20-95% inhibition versus controls). These results suggest potential mechanisms for prior OC and dietary IF effects on cancer risk in estrogen-responsive tissues.

Oxathiolene oxides: a novel family of compounds that induce ferritin, glutathione S-transferase, and other proteins of the phase II response

Compounds that induce the synthesis of cytoprotective phase II enzymes have shown promise as cancer chemopreventive agents. Although chemically diverse, phase II enzyme inducers are capable of participating in Michael reaction chemistry. We have synthesized a novel class of organosulfur compounds, termed oxathiolene oxides (OTEOs). Based on their chemical properties, we hypothesized that these compounds could function as phase II enzyme inducers. Northern blot analysis showed that oxathiolene oxides induce the phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), and ferritin H and L mRNA in a concentration-dependent fashion in a normal embryonic mouse liver cell line, BNLCL.2. OTEO-562 (3-cyclohexenyl-4-methyl-1,2-oxathiol-3-ene-2-oxide) was the strongest inducer. Western blot analysis demonstrated that GST-alpha and ferritin H protein levels were also induced in cells treated with OTEO-562, as was total GST and NQO1 enzyme activity. Further, induction of NQO1 activity by OTEO-562 was equivalent in aromatic hydrocarbon (Ah) receptor wild-type and Ah receptor mutant cell lines, suggesting that oxathiolene oxides activate phase II enzymes by an Ah receptor-independent mechanism. Consistent with this observation, OTEO-562 failed to induce cytochrome P450 1A1 mRNA. These results suggest that oxathiolene oxides may merit further investigation as candidate chemopreventive agents.

CB1 cannabinoid receptor-mediated increases in cyclic AMP accumulation are correlated with reduced Gi/o function

Abstract Background: CB1 cannabinoid receptors (CB1Rs) stimulate Gi/o-dependent signaling pathways. CB1R-mediated cAMP increases were proposed to result from Gs activation, but CB1R-stimulated GTPγS binding to Gs has not heretofore been investigated. Methods: Three models of CB1R-stimulated cAMP production were tested: pertussis toxin disruption of Gi/o in N18TG2 cells; L341A/A342L-CB1R expressed in Chinese hamster ovary (CHO) cells; and CB1 and D2 dopamine receptors endogenously co-expressed in MN9D cells. cAMP was assayed by [3H]cAMP binding competition. G protein activation was assayed by the antibody-targeted scintillation proximity assay. Results: In L341A/A342L-CB1-CHO cells, cannabinoid agonists significantly stimulated cAMP accumulation over vehicle; (–)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxyl propyl] cyclohexan-1-ol (CP55940)-stimulated [35S]GTPγS binding to Gi1/2/3 was reversed, whereas binding to Gs was not different from CB1R. In MN9D cells, CB1 agonist HU210 or D2 agonist quinpirole alone inhibited forskolin-activated cAMP accumulation, whereas HU210 plus quinpirole increased cAMP accumulation above basal. HU210 alone stimulated [35S]GTPγS binding to Gi1/2/3, whereas co-stimulation with quinpirole reversed HU210-stimulated [35S]GTPγS binding to Gi1/2/3. Conclusions: CB1R couples to Gs but with low efficacy compared to Gi/o. The L341A/A342L mutation in CB1R reversed CP55940 activation of Gi to an inhibition, but had no effect on Gs. Combined CB1 plus D2 agonists in MN9D cells converted the CB1 agonist-mediated activation of Gi to inhibition of Gi. In these models, the CB1 agonist response was converted to an inverse agonist response at Gi activation. Cannabinoid agonist-stimulated cAMP accumulation can be best explained as reduced activation of Gi, thereby attenuating the tonic inhibitory influence of Gi on the major isoforms of adenylyl cyclase.

Cannabinoid Receptor Interacting Protein 1a Competition with β-Arrestin for CB1 Receptor Binding Sites

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminal-associated protein that alters CB1R interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CB1R interactions with β-arrestin proteins that interact with the CB1R at the C-terminus. Coimmunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or β-arrestin, but CRIP1a and β-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and β-arrestin binding to the CB1R, which we hypothesized could attenuate the action of β-arrestin to mediate CB1R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB1Rs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB1R. CRIP1a overexpression attenuated CP55940-mediated GFP-CB1R as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented β-arrestin redistribution to punctae. Peptides mimicking the CB1R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB1R distal C-terminus 14-mer peptide reduced CB1R-CRIP1a association. Coimmunoprecipitation of CB1R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB1R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with β-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with β-arrestin 2. Thus, CRIP1a can compete with β-arrestins for interaction with C-terminal CB1R domains that could affect agonist-driven, β-arrestin-mediated internalization of the CB1R.

Cannabinoid receptor interacting protein suppresses agonist-driven CB1 receptor internalization and regulates receptor replenishment in an agonist-biased manner

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C‐terminus‐associated protein that modulates CB1R signaling via G proteins, and CB1R down‐regulation but not desensitization (Blume et al. [2015] Cell Signal., 27, 716–726; Smith et al. [2015] Mol. Pharmacol., 87, 747–765). In this study, we determined the involvement of CRIP1a in CB1R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB1Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1R and CRIP1a, and exhibits a well‐characterized endocannabinoid signaling system. We developed stable CRIP1a‐over‐expressing and CRIP1a‐siRNA‐silenced knockdown clones to investigate gene dose effects of CRIP1a on CB1R plasma membrane expression. Results indicate that CP55940 or WIN55212‐2 (10 nM, 5 min) reduced cell surface CB1R by a dynamin‐ and clathrin‐dependent process, and this was attenuated by CRIP1a over‐expression. CP55940‐mediated cell surface CB1R loss was followed by a cycloheximide‐sensitive recovery of surface receptors (30–120 min), suggesting the requirement for new protein synthesis. In contrast, WIN55212‐2‐mediated cell surface CB1Rs recovered only in CRIP1a knockdown cells. Changes in CRIP1a expression levels did not affect a transient rimonabant (10 nM)‐mediated increase in cell surface CB1Rs, which is postulated to be as a result of rimonabant effects on ‘non‐agonist‐driven’ internalization. These studies demonstrate a novel role for CRIP1a in agonist‐driven CB1R cell surface regulation postulated to occur by two mechanisms: 1) attenuating internalization that is agonist‐mediated, but not that in the absence of exogenous agonists, and 2) biased agonist‐dependent trafficking of de novo synthesized receptor to the cell surface.

Tpcn2 knockout mice have improved insulin sensitivity and are protected against high-fat diet-induced weight gain

Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, two-pore channel 2 ( Tpcn2) knockout mice exhibit a decreased insulin response but normal glucose clearance, suggesting they have improved insulin sensitivity compared with wild-type mice. We tested the hypothesis that improved insulin sensitivity in Tpcn2 knockout mice would protect against the negative effects of a high fat diet. Male and female Tpcn2 knockout (KO), heterozygous (Het), and wild-type (WT) mice were fed a low-fat (LF) or high-fat (HF) diet for 24 wk. HF diet significantly increases body weight in WT mice relative to those on the LF diet; this HF diet-induced increase in body weight is blunted in the Het and KO mice. Despite the protection against diet-induced weight gain, however, Tpcn2 KO mice are not protected against HF-diet-induced changes in glucose or insulin area under the curve during glucose tolerance tests in female mice, while HF diet has no significant effect on glucose tolerance in the male mice, regardless of genotype. Glucose disappearance during an insulin tolerance test is augmented in male KO mice, consistent with our previous findings suggesting enhanced insulin sensitivity in these mice. Male KO mice exhibit increased fasting plasma total cholesterol and triglyceride concentrations relative to WT mice on the LF diet, but this difference disappears in HF diet-fed mice where there is increased cholesterol and triglycerides across all genotypes. These data demonstrate that knockout of Tpcn2 may increase insulin action in male, but not female, mice. In addition, both male and female KO mice are protected against diet-induced weight gain, but this protection is likely independent from glucose tolerance, insulin sensitivity, and plasma lipid levels.