Ronald E. Cannon

Arsenic Exposure In utero Exacerbates Skin Cancer Response in Adulthood with Contemporaneous Distortion of Tumor Stem Cell Dynamics

Arsenic is a carcinogen with transplacental activity that can affect human skin stem cell population dynamics in vitro by blocking exit into differentiation pathways. Keratinocyte stem cells (KSC) are probably a key target in skin carcinogenesis. Thus, we tested the effects of fetal arsenic exposure in Tg.AC mice, a strain sensitive to skin carcinogenesis via activation of the v-Ha-ras transgene likely in KSCs. After fetal arsenic treatment, offspring received topical 12-O-tetradecanoyl phorbol-13-acetate (TPA) through adulthood. Arsenic alone had no effect, whereas TPA alone induced papillomas and squamous cell carcinomas (SCC). However, fetal arsenic treatment before TPA increased SCC multiplicity 3-fold more than TPA alone, and these SCCs were much more aggressive (invasive, etc.). Tumor v-Ha-ras levels were 3-fold higher with arsenic plus TPA than TPA alone, and v-Ha-ras was overexpressed early on in arsenic-treated fetal skin. CD34, considered a marker for both KSCs and skin cancer stem cells, and Rac1, a key gene stimulating KSC self-renewal, were greatly increased in tumors produced by arsenic plus TPA exposure versus TPA alone, and both were elevated in arsenic-treated fetal skin. Greatly increased numbers of CD34-positive probable cancer stem cells and marked overexpression of RAC1 protein occurred in tumors induced by arsenic plus TPA compared with TPA alone. Thus, fetal arsenic exposure, although by itself oncogenically inactive in skin, facilitated cancer response in association with distorted skin tumor stem cell signaling and population dynamics, implicating stem cells as a target of arsenic in the fetal basis of skin cancer in adulthood.

Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain

P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to the delivery of small-molecule drugs across the blood-brain barrier and into the CNS. Here we test a unique signaling-based strategy to overcome this obstacle. We used a confocal microscopy-based assay with isolated rat brain capillaries to map a signaling pathway that within minutes abolishes P-glycoprotein transport activity without altering transporter protein expression or tight junction permeability. This pathway encompasses elements of proinflammatory- (TNF-α) and sphingolipid-based signaling. Critical to this pathway was signaling through sphingosine-1-phosphate receptor 1 (S1PR1). In brain capillaries, S1P acted through S1PR1 to rapidly and reversibly reduce P-glycoprotein transport activity. Sphingosine reduced transport by a sphingosine kinase-dependent mechanism. Importantly, fingolimod (FTY720), a S1P analog recently approved for treatment of multiple sclerosis, also rapidly reduced P-glycoprotein activity; similar effects were found with the active, phosphorylated metabolite (FTY720P). We validated these findings in vivo using in situ brain perfusion in rats. Administration of S1P, FTY720, or FTY729P increased brain uptake of three radiolabeled P-glycoprotein substrates, 3H-verapamil (threefold increase), 3H-loperamide (fivefold increase), and 3H-paclitaxel (fivefold increase); blocking S1PR1 abolished this effect. Tight junctional permeability, measured as brain 14C-sucrose accumulation, was not altered. Therefore, targeting signaling through S1PR1 at the blood-brain barrier with the sphingolipid-based drugs, FTY720 or FTY720P, can rapidly and reversibly reduce basal P-glycoprotein activity and thus improve delivery of small-molecule therapeutics to the brain.

Nrf2 Upregulates ATP Binding Cassette Transporter Expression and Activity at the Blood–Brain and Blood–Spinal Cord Barriers

Activation of nuclear factor E2-related factor-2 (Nrf2), a sensor of oxidative stress, is neuroprotective in animal models of cerebral ischemia, traumatic brain injury, subarachnoid hemorrhage, and spinal cord injury. We show here that Nrf2 activation with sulforaphane (SFN) in vivo or in vitro increases expression and transport activity of three ATP-driven drug efflux pumps at the blood–brain barrier [P-glycoprotein, ATP binding cassette b1 (Abcb1); multidrug resistance-associated protein-2 (Mrp2), Abcc2; and breast cancer resistance protein (Bcrp), Abcg2]. Dosing rats with SFN increased protein expression of all three transporters in brain capillaries and decreased by 50% brain accumulation of the P-glycoprotein substrate verapamil. Exposing rat or mouse brain capillaries to SFN increased P-glycoprotein, Bcrp, and Mrp2 transport activity and protein expression; SFN increased P-glycoprotein activity in mouse spinal cord capillaries. Inhibiting transcription or translation abolished upregulation of P-glycoprotein activity. No such effects were seen in brain capillaries from Nrf2-null mice, indicating Nrf2 dependence. Nrf2 signaled indirectly to increase transporter activity/expression. The p53 inhibitor pifithrin abolished the SFN-induced increase in transporter activity/expression, and the p53-activator nutlin-3 increased P-glycoprotein activity. SFN did not alter P-glycoprotein transport activity in brain and spinal cord capillaries from p53-null mice. Inhibitors of p38 MAPK and nuclear factor κB (NF-κB) blocked the effects of SFN and nutlin-3 on P-glycoprotein activity. These results implicate Nrf2, p53, and NF-κB in the upregulation of P-glycoprotein, Bcrp, and Mrp2 at blood–CNS barriers. They imply that the barriers are tightened selectively (efflux transporter upregulation) by oxidative stress, providing increased neuroprotection, but also reduced penetration of many therapeutic drugs.

Two cDNAs encode two nearly identical Cu/Zn superoxide dismutase proteins in maize

SummarySOD-4, a cytosolic form of superoxide dismutase in maize, originally was defined as a single band of activity by zymogram analysis. The protein was purified to “homogeneity” as shown by a single band on native or denaturing polyacrylamide gels and a single spot on two dimensional gels. The N-terminal amino acid sequence for the first 20 residues was determined for the purified SOD-4 protein. All residues were clearly determined except for residue twelve, where both glutamic and aspartic acids were found. A maize λgt11 cDNA library was constructed from scutellar poly(A)+RNA. Two cDNAs were isolated, restriction mapped, and their DNA sequences determined. The amino acid sequence deduced from both cDNAs matched perfectly the N-terminal sequence of the purified protein except for the residue at position 12. Significantly, at the twelfth codon, one cDNA was found to code for glutamic acid and the other cDNA had a codon for aspartic acid. Both cDNAs contained similar but not identical 5′ and 3′ untranslated sequences. Both cDNAs contained polyadenylation signals and tails. cDNA isolations, RNA, and genomic DNA blots confirm the existence and expression of two genes that produce indistinguishable SOD-4 proteins.

Retrotransposition-Competent Human LINE-1 Induces Apoptosis in Cancer Cells With Intact p53

Retrotransposition of human LINE-1 (L1) element, a major representative non-LTR retrotransposon in the human genome, is known to be a source of insertional mutagenesis. However, nothing is known about effects of L1 retrotransposition on cell growth and differentiation. To investigate the potential for such biological effects and the impact that human L1 retrotransposition has upon cancer cell growth, we examined a panel of human L1 transformed cell lines following a complete retrotransposition process. The results demonstrated that transposition of L1 leads to the activation of the p53-mediated apoptotic pathway in human cancer cells that possess a wild-type p53. In addition, we found that inactivation of p53 in cells, where L1 was undergoing retrotransposition, inhibited the induction of apoptosis. This suggests an association between active retrotransposition and a competent p53 response in which induction of apoptosis is a major outcome. These data are consistent with a model in which human retrotransposition is sensed by the cell as a “genetic damaging event” and that massive retrotransposition triggers signaling pathways resulting in apoptosis.

Kinetics of wound-induced v-Ha-ras transgene expression and papilloma development in transgenic Tg.AC mice

The Tg.AC transgenic mouse, which harbors an activated v‐Ha‐ras coding region that is fused to an embryonic ζ globin transcriptional control region and a 3′ simian virus 40 polyadenylation sequence, rapidly develops epidermal papillomas in response to topical application of chemical carcinogens or tumor promoters or to full‐thickness wounding of the dorsal skin. In this report, we investigated the localization and temporal induction of v‐Ha‐ras transgene expression after full‐thickness wounding of Tg.AC mouse skin. Surgically inflicted full‐thickness incisions 3 cm long yielded four to six papillomas per Tg.AC mouse by 5 wk after wounding. Similar wounding of the FVB/N isogenic host strain did not produce tumors, which implicates a causal role for the v‐Ha‐ras transgene. Reverse transcription–polymerase chain reaction assays detected the v‐Ha‐ras transgene transcript in total RNA samples isolated from wound‐associated tissue 3 and 4 wk after wounding. Tissues 1–2 wk after wounding and all non‐wound–associated tissues were negative for transgene expression. In situ hybridization experiments using transgene‐specific 35S‐labeled antisense RNA probes localized transgene expression to the basal epidermal cells in wound‐induced papillomas. Adjacent normal and hyperplastic skin tissues were negative for transgene expression by this assay. This work supports the hypothesis that the wound repair response leads to the transcriptional activation and continued expression of the v‐Ha‐ras transgene in specific cells in the skin, which alters normal epithelial differentiation and ultimately results in neoplastic growth. Mol. Carcinog. 20:108–114, 1997.

1α,25-Dihydroxyvitamin D3-liganded vitamin D receptor increases expression and transport activity of P-glycoprotein in isolated rat brain capillaries and human and rat brain microvessel endothelial cells.

Induction of the multidrug resistance protein 1 (MDR1)/P‐glycoprotein (P‐gp) by the vitamin D receptor (VDR) was investigated in isolated rat brain capillaries and rat (RBE4) and human (hCMEC/D3) brain microvessel endothelial cell lines. Incubation of isolated rat brain capillaries with 10 nM of the VDR ligand, 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3] for 4 h increased P‐gp protein expression fourfold. Incubation with 1,25(OH)2D3 for 4 or 24 h increased P‐gp transport activity (specific luminal accumulation of NBD‐CSA, the fluorescent P‐gp substrate) by 25–30%. In RBE4 cells, Mdr1b mRNA was induced in a concentration‐dependent manner by exposure to 1,25(OH)2D3. Concomitantly, P‐gp protein expression increased 2.5‐fold and was accompanied by a 20–35% reduction in cellular accumulation of the P‐gp substrates, rhodamine 6G (R6G), and HiLyte Fluor 488‐labeled human amyloid beta 1‐42 (hAβ42). In hCMEC/D3 cells, a 3 day exposure to 100 nM 1,25(OH)2D3 increased MDR1 mRNA expression (40%) and P‐gp protein (threefold); cellular accumulation of R6G and hAβ42 was reduced by 30%. Thus, VDR activation up‐regulates Mdr1/MDR1 and P‐gp protein in isolated rat brain capillaries and rodent and human brain microvascular endothelia, implicating a role for VDR in increasing the brain clearance of P‐gp substrates, including hAβ42, a plaque‐forming precursor in Alzheimers disease.

Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation.

Deleted in Split hand/Split foot 1 (DSS1) was previously identified as a novel 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible gene with possible involvement in early event of mouse skin carcinogenesis. The mechanisms by which human DSS1 (HsDSS1) exerts its biological effects via regulation of the ubiquitin-proteasome system (UPS) are currently unknown. Here, we demonstrated that HsDSS1 regulates the human proteasome by associating with it in the cytosol and nucleus via the RPN3/S3 subunit of the 19S regulatory particle (RP). Molecular anatomy of HsDSS1 revealed an RPN3/S3-interacting motif (R3IM), located at amino acid residues 15 to 21 of the NH(2) terminus. Importantly, negative charges of the R3IM motif were demonstrated to be required for proteasome interaction and binding to poly-ubiquitinated substrates. Indeed, the R3IM motif of HsDSS1 protein alone was sufficient to replace the ability of intact HsDSS1 protein to pull down proteasome complexes and protein substrates with high-molecular mass ubiquitin conjugates. Interestingly, this interaction is highly conserved throughout evolution from humans to nematodes. Functional study, lowering the levels of the endogenous HsDSS1 using siRNA, indicates that the R3IM/proteasome complex binds and targets p53 for ubiquitin-mediated degradation via gankyrin-MDM2/HDM2 pathway. Most significantly, this work indicates that the R3IM motif of HsDSS1, in conjunction with the complexes of 19S RP and 20S core particle (CP), regulates proteasome interaction through RPN3/S3 molecule, and utilizes a specific subset of poly-ubiquitinated p53 as a substrate.

Induction of transgene expression in Tg.AC (v-Ha-ras) transgenic mice concomitant with DNA hypomethylation

Tg.AC transgenic mice have a transgene composed of a ζ‐globin transcriptional control region, a v‐Ha‐ras coding region, and a simian virus 40 3′ polyadenylation signal sequence. Induced ectopic expression of the transgene by chemical treatment or full‐skin‐thickness wounding leads to the development of skin papillomas. Reverse transcription–polymerase chain reaction assays and protein blotting indicated that the transgene was expressed 16–28 d after full‐skin‐thickness surgical wounding. Normal unwounded skin did not express the transgene. DNA blotting indicated that the position of the transgene remained stable during wound‐induced tumorigenesis. Concomitant with the v‐Ha‐ras mRNA and protein expression was the hypomethylation of specific MspI/HpaII sites within the transgene. These results are consistent with the hypothesis that hypomethylation is required for the induced and sustained expression of the Tg.AC v‐Ha‐ras transgene in spontaneous and induced tumors in Tg.AC mice. Mol. Carcinog. 21:244–250, 1998.

Mrp1 is essential for sphingolipid signaling to p-glycoprotein in mouse blood-brain and blood-spinal cord barriers.

At the blood–brain and blood–spinal cord barriers, P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to central nervous system (CNS) pharmacotherapy. Recently, we showed that signaling through tumor necrosis factor-α (TNF-α), sphingolipids, and sphingosine-1-phosphate receptor 1 (S1PR1) rapidly and reversibly reduced basal P-glycoprotein transport activity in the rat blood–brain barrier. The present study extends those findings to the mouse blood–brain and blood–spinal cord barriers and, importantly, identifies multidrug resistance-associated protein 1 (Mrp1, Abcc1) as the transporter that mediates S1P efflux from brain and spinal cord endothelial cells. In brain and spinal cord capillaries isolated from wild-type mice, TNF-α, sphingosine, S1P, the S1PR agonist fingolimod (FTY720), and its active, phosphorylated metabolite, FTY720P, reduced P-glycoprotein transport activity; these effects were abolished by a specific S1PR1 antagonist. In brain and spinal cord capillaries isolated from Mrp1-null mice, neither TNF-α nor sphingosine nor FTY720 reduced P-glycoprotein transport activity. However, S1P and FTY720P had the same S1PR1-dependent effects on transport activity as in capillaries from wild-type mice. Thus, deletion of Mrp1 alone terminated endogenous signaling to S1PR1. These results identify Mrp1 as the transporter essential for S1P efflux from the endothelial cells and thus for inside-out S1P signaling to P-glycoprotein at the blood–brain and blood–spinal cord barriers.