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. [Cancer Res 2008;68(20):8278-85]
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, C-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.ABC transporters | chemotherapy | brain endothelium | ABCB1 | breast cancer related protein | multidrug resistance-associated protein
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 B (NF-B) blocked the effects of SFN and nutlin-3 on P-glycoprotein activity. These results implicate Nrf2, p53, and NF-B 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.
Schizophrenia and bipolar disorder are leading causes of morbidity across all populations, with heritability estimates of approximately 80% indicating a substantial genetic component. Population genetics and genome-wide association studies suggest an overlap of genetic risk factors between these illnesses but it is unclear how this genetic component is divided between common gene polymorphisms, rare genomic copy number variants, and rare gene sequence mutations. We report evidence that the lipid transporter gene ABCA13 is a susceptibility factor for both schizophrenia and bipolar disorder. After the initial discovery of its disruption by a chromosome abnormality in a person with schizophrenia, we resequenced ABCA13 exons in 100 cases with schizophrenia and 100 controls. Multiple rare coding variants were identified including one nonsense and nine missense mutations and compound heterozygosity/homozygosity in six cases. Variants were genotyped in additional schizophrenia, bipolar, depression (n > 1600), and control (n > 950) cohorts and the frequency of all rare variants combined was greater than controls in schizophrenia (OR = 1.93, p = 0.0057) and bipolar disorder (OR = 2.71, p = 0.00007). The population attributable risk of these mutations was 2.2% for schizophrenia and 4.0% for bipolar disorder. In a study of 21 families of mutation carriers, we genotyped affected and unaffected relatives and found significant linkage (LOD = 4.3) of rare variants with a phenotype including schizophrenia, bipolar disorder, and major depression. These data identify a candidate gene, highlight the genetic overlap between schizophrenia, bipolar disorder, and depression, and suggest that rare coding variants may contribute significantly to risk of these disorders.
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.
MDR1/P-gp induction 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 (4-fold). 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 three day exposure to 100 nM 1,25(OH)2D3 increased MDR1 mRNA expression (40%) and P-gp protein (3-fold); 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 Alzheimer’s disease.
The Tg.AC transgenic mouse, which harbors an activated v-Ha-ras coding region that is fused to an embryonic zeta 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.
This study identifies genes expressed early in 12-Otetradecanoylphorbol-13-acetate (TPA)-induced skin carcinogenesis in genetically initiated Tg⅐AC v-Ha-ras transgenic mice. Keratinocyte progenitor cells from TPA-treated Tg⅐AC mice were isolated with fluorescence-activated cell sorting and expression was analyzed using cDNA microarray technology. Eleven genes were identified whose expression changed significantly in response to carcinogen treatment. Deleted in split hand/split foot 1 (Dss1) is a gene associated with a heterogeneous limb developmental disorder called split hand/split foot malformation. cDNA microarray expression analysis showed that the mouse homologue of Dss1 is induced by TPA. Dss1 overexpression was detected by Northern blot analysis in early TPA-treated hyperplastic skins and in JB6 Cl 41-5a epidermal cells. Interestingly, Dss1 expression was also shown to be elevated in skin papillomas relative to normal skins, and further increased in squamous cell malignancies. Functional studies by ectopically constitutive expression of Dss1 in JB6 Cl 41-5a preneoplastic cells strongly increased focus formation and proliferation of these cells and enhanced efficiency of neoplastic transformation of the cells in soft agar. These results strongly suggest that Dss1 is a TPA-inducible gene that may play an important role in the early stages of skin carcinogenesis.Skin carcinogenesis is a complex multistage process that progresses through distinct stages of initiation, promotion, progression, and malignancy (1-3). The Tg⅐AC mouse is a genetically modified (transgenic) form of the FVB/N mouse strain that carries a genomic copy of the v-Ha-ras gene fused to a fetal -globin gene promoter (4). Tg⅐AC mice have already entered the initiation stage of cancer development and have a higher sensitivity to many types of environmentally inducible cancer than wild type mice. Tg⅐AC mice develop hyperplasia in skin keratinocytes after exposure to tumor promoters such as TPA 1 (4), full thickness wounding (5), ultraviolet radiation (6), or carcinogens such as 7,12-dimethylbenz[a]anthracene (7). These hyperplasias eventually develop into benign papillomas, some of which become malignant tumors such as squamous cell carcinomas or spindle cell tumors (7). The in vivo Tg⅐AC mouse model is a valuable tool to study the early stages of skin carcinogenesis.The epidermis is a stratified, rapidly renewing tissue in which terminally differentiated cells are continuously lost from the skin surface and replaced by an intricate and highly regulated proliferative process. Skin cells are regenerated through the proliferative capacity of keratinocyte stem cells (KSCs) and transit amplifying (TA) cells in the basal layer. KSCs are a minor subpopulation of relatively quiescent cells that have broad proliferative potential and an unlimited capacity for self-renewal (8, 9). It has been proposed that carcinogens generate mutations in the population of stem cells which are transformed them into initiated preneoplastic cells (10). It has also been r...
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