Monocarboxylate transporter 1 is up‐regulated in basal‐like breast carcinoma

Pinheiro C, Albergaria A, Paredes J, Sousa B, Dufloth R, Vieira D, Schmitt F & Baltazar F
(2010) Histopathology 56, 860–867
Monocarboxylate transporter 1 is up‐regulated in basal‐like breast carcinoma Aims: Monocarboxylate transporters (MCTs) have been considered promising targets for cancer therapy, since they facilitate lactate efflux in glycolytic tumours. However, their role in solid tumours is still poorly understood. Thus, the present work aimed to contribute to understanding the involvement of MCT1 and MCT4 in breast cancer progression as well as MCT regulation by CD147. Methods and results: The expression of the membrane transporters MCT1 and MCT4 was analysed in a series of breast carcinomas (249 cases) and their clinicopathological significance investigated. Additionally, we analysed the significance of CD147 co‐expression, as an important regulator of MCT expression and activity. MCT1 was significantly increased in breast carcinomas when compared with normal breast tissue and, importantly, both MCT1 and CD147 were associated with poor prognostic variables such as basal‐like subtype and high grade tumours. Conclusions: These results provide evidence for a prognostic value of MCT1 in breast carcinoma and support the exploitation of the complex MCT1/CD147 as a promising target for cancer therapy, especially in basal‐like breast carcinoma.     

Family‐based association study of 5‐HTTLPR,TPH, MAO‐A,and DRD4 polymorphisms in mood disorders

Variants of the functional polymorphism in the serotonin transporter (upstream regulatory region: 5‐HTTLPR), the tryptophan hydroxylase (TPH), the monoamine oxidase A (MAO‐A), and the dopamine receptor D4 (DRD4) genes have all been associated with mood disorders. The aim of this study was to test those hypotheses by using a family‐based association approach. Both diagnoses and psychopathology were used for phenotype definitions. A total of 134 nuclear families with mood disorders, with probands affected by bipolar (n = 103) or major depressive (n = 58) disorders, were included in the study. All subjects were typed for the above‐mentioned gene variants using polymerase chain reaction (PCR) technique. No significant transmission disequilibrium was found in the overall sample for any polymorphism. A separate analysis of bipolar subjects only, or the use of continuous psychopathologic traits as affectation status did not influence the observed results. Our study did not support the involvement of 5‐HTTLPR, TPH, MAO‐A, or DRD4 polymorphisms in mood disorders. © 2002 Wiley‐Liss, Inc.     

Induced arginine transport via cationic amino acid transporter‐1 is necessary for human T‐cell proliferation

Availability of the semiessential amino acid arginine is fundamental for the efficient function of human T lymphocytes. Tumor‐associated arginine deprivation, mainly induced by myeloid‐derived suppressor cells, is a central mechanism of tumor immune escape from T‐cell‐mediated antitumor immune responses. We thus assumed that transmembranous transport of arginine must be crucial for T‐cell function and studied which transporters are responsible for arginine influx into primary human T lymphocytes. Here, we show that activation via CD3 and CD28 induces arginine transport into primary human T cells. Both naïve and memory CD4⁺ T cells as well as CD8⁺ T cells specifically upregulated the human cationic amino acid transporter‐1 (hCAT‐1), with an enhanced and persistent expression under arginine starvation. When hCAT‐1 induction was suppressed via siRNA transfection, arginine uptake, and cellular proliferation were impaired. In summary, our results demonstrate that hCAT‐1 is a key component of efficient T‐cell activation and a novel potential target structure to modulate adaptive immune responses in tumor immunity or inflammation.     

Dendritic cells phenotype fitting under hypoxia or lipopolysaccharide; adenosine 5′‐triphosphate‐binding cassette transporters far beyond an efflux pump

This study examines adenosine 5′‐triphosphate‐binding cassette (ABC) transporters as a potential therapeutic target in dendritic cell (DC) modulation under hypoxia and lipopolysaccharide (LPS). Functional capacity of dendritic cells (DCs) (mixed lymphocyte reaction: MLR) and maturation of iDCs were evaluated in the presence or absence of specific ABC‐transporter inhibitors. Monocyte‐derived DCs were cultured in the presence of interleukin (IL)‐4/granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Their maturation under hypoxia or LPS conditions was evaluated by assessing the expression of maturation phenotypes using flow cytometry. The effect of ABC transporters on DC maturation was determined using specific inhibitors for multi‐drug resistance (MDR1) and multi‐drug resistance proteins (MRPs). Depending on their maturation status to elicit T cell alloresponses, the functional capacity of DCs was studied by MLR. Mature DCs showed higher P‐glycoprotein (Pgp) expression with confocal microscopy. Up‐regulation of maturation markers was observed in hypoxia and LPS‐DC, defining two different DC subpopulation profiles, plasmacytoid versus conventional‐like, respectively, and different cytokine release T helper type 2 (Th2) versus Th1, depending on the stimuli. Furthermore, hypoxia‐DCs induced more B lymphocyte proliferation than control‐iDC (56% versus 9%), while LPS‐DCs induced more CD8‐lymphocyte proliferation (67% versus 16%). ABC transporter‐inhibitors strongly abrogated DC maturation [half maximal inhibitory concentration (IC₅₀): P‐glycoprotein inhibition using valspodar (PSC833) 5 μM, CAS 115104‐28‐4 (MK571) 50 μM and probenecid 2·5 μM], induced significantly less lymphocyte proliferation and reduced cytokine release compared with stimulated‐DCs without inhibitors. We conclude that diverse stimuli, hypoxia or LPS induce different profiles in the maturation and functionality of DC. Pgp appears to play a role in these DC events. Thus, ABC‐transporters emerge as potential targets in immunosuppressive therapies interfering with DCs maturation, thereby abrogating innate immune response when it is activated after ischaemia.     

Regulation of apical transporter of L‐DOPA in human intestinal Caco‐2 cells

The present study examined the nature of the apical inward L ‐3,4‐dihydroxyphenylalanine (L ‐DOPA) transporter in human intestinal epithelial Caco‐2 cells, and whether protein kinases modulate the activity of this transporter. The apical inward transfer of L ‐DOPA was promoted through an energy‐dependent and sodium‐insensitive transporter (K_m=33 μM ; V_max=2932 pmol/mg protein/6 min). This transporter was insensitive to N‐(methylamino)‐isobutyric acid, but competitively inhibited by 2‐aminobicyclo(2,2,1)‐heptane‐2‐carboxylic acid (BCH; IC₅₀=83 μM ). The organic cation inhibitor decynium 24 failed to affect the accumulation of L ‐DOPA, whereas the organic anion inhibitor 4,4′‐diisothiocynatostilbene‐2,2′‐disulphonic acid (DIDS) competitively inhibited L ‐DOPA uptake (IC₅₀=83 μM ). However, the apical‐to‐basal and basal‐to‐apical transepithelial transport and the cell accumulation of [³H]‐PAH was close to that of [¹⁴C]‐sorbitol and insensitive to DIDS (300 μM ). Modulators of protein kinase A (PKA) [cyclic adenosine monophosphate (cAMP), forskolin, H‐89 and cholera toxin], protein kinase G (PKG) [cyclic guanosine monophosphate (GMP), zaprinast, LY 83583 and sodium nitroprusside] and protein kinase C (PKC) (phorbol 12,13‐dibutirate and chelerythrine) failed to affect the accumulation of L ‐DOPA. The Ca²⁺/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L ‐DOPA uptake (IC_50s of 53 and 252 μM , respectively), but the rise of intracellular Ca²⁺ by A23187 (1 μM ) and thapsigargin (1 μM ) played no role on L ‐DOPA uptake. It is concluded that Caco‐2 cells take up L ‐DOPA over the apical cell border through the sodium‐independent and pH‐sensitive L‐type amino acid transporter.     

A functional SNP in the 3′‐UTR of TAP2 gene interacts with microRNA hsa‐miR‐1270 to suppress the gene expression

The transporter associated with antigen processing 2 (TAP2) is involved in the development of multidrug resistance and the etiology of immunological diseases. In this study, we investigated whether the expression of TAP2 can be perturbed by single nucleotide polymorphisms (SNPs) located in 3′‐untranslated region (3′‐UTR) of the gene via interactions with microRNAs. Using a series of in silico assays, we selected the candidate microRNAs (miRNAs) with the potential to interact with functional SNPs of TAP2. The SNP rs241456—located in the 3′‐UTR of TAP2—resides in a potential binding site for hsa‐miR‐1270 and hsa‐miR‐620. HEK 293 cells, from a human kidney cell line, were used to characterize the extent of binding of miRNAs to each polymorphic allele of the SNP by a luciferase reporter gene assay. RNA electrophoretic mobility shift assays were used to evaluate the interaction between the miRNAs and each allele sequence of the SNP. We found that hsa‐miR‐1270 inhibited luciferase activity by binding to the T allele of the SNP in an allele‐specific manner. A negative correlation was also found between the expression of hsa‐miR‐1270 and the T allele of the SNP in kidney tissues. Our findings support the hypothesis that hsa‐miR‐1270 suppresses the production of TAP2 by binding to this SNP in the 3′‐UTR of this gene. Environ. Mol. Mutagen. 59:134–143, 2018. © 2017 Wiley Periodicals, Inc.     

Stress‐Induced Upregulation of SLC19A3 is Impaired in Biotin‐Thiamine‐Responsive Basal Ganglia Disease

Biotin‐thiamine‐responsive basal ganglia disease (BTBGD) is a potentially treatable disorder caused by mutations in the SLC19A3 gene, encoding the human thiamine transporter 2. Manifestation of BTBGD as acute encephalopathy triggered by a febrile infection has been frequently reported, but the underlying mechanisms are not clear. We investigated a family with two brothers being compound heterozygous for the SLC19A3 mutations p.W94R and p.Q393*fs. Post‐mortem analysis of the brain of one brother showed a mixture of acute, subacute and chronic changes with cystic and necrotic lesions and hemorrhage in the putamen, and hemorrhagic lesions in the caudate nucleus and cortical layers. SLC19A3 expression was substantially reduced in the cortex, basal ganglia and cerebellum compared with an age‐matched control. Importantly, exposure of fibroblasts to stress factors such as acidosis or hypoxia markedly upregulated SLC19A3 in control cells, but failed to elevate SLC19A3 expression in the patient's fibroblasts. These results demonstrate ubiquitously reduced thiamine transporter function in the cerebral gray matter, and neuropathological alterations similar to Wernicke's disease in BTBGD. They also suggest that episodes of encephalopathy are caused by a substantially reduced capacity of mutant neuronal cells to increase SLC19A3 expression, necessary to adapt to stress conditions.     

Temporal Changes of Astrocyte Activation and Glutamate Transporter‐1 Expression in the Spinal Cord After Spinal Nerve Ligation‐Induced Neuropathic Pain

Astrocyte activation is involved in the neuropathic pain. As a glutamate scavenger, the glutamate transporter‐1 (GLT‐1) is exclusively expressed on the astrocytes and probably correlates with astrocyte activation. In the present study, we attempted to clarify the temporal changing courses of astrocyte activation and GLT‐1 expression, as well as their correlations induced by a neuropathic pain model, namely, spinal nerve ligation (SNL) in which rapidly appearing (<3 days) and persistent (>21 days) mechanical allodynia and thermal hyperalgesia were presented. Immunofluorescent staining showed that GLT‐1 was expressed exclusively in most (not all) of the astrocytes, even when the GLT‐1 expression reached its peak. The expression of GLT‐1 displayed an interesting biphasic change, with an initial up‐regulation followed by a down‐regulation after SNL. Our results also demonstrated that SNL induced a marked and long‐term (>21 days) activation of astrocytes in the ipsilateral spinal dorsal horn. These results suggest that astrocyte activation, the change of GLT‐1 expression and the potential relationship between them might play key roles in the induction and/or maintenance of neuropathic pain. The present results provide novel clues in understanding the mechanisms underlying the involvement of astrocytes and GLT‐1 in the neuropathic pain. Anat Rec, 291:513–518, 2008. © 2008 Wiley‐Liss, Inc.     

L‐type amino acid transporter‐1 and CD98 expression in bone and soft tissue tumors

L‐type amino acid transporter‐1 (LAT1) is expressed in many cancers. We examined LAT1 and CD98 expression immunohistochemically in surgically resected specimens of various bone and soft tissue tumors. Out of 226 cases, 79 (35%) were LAT1⁺ and 95 (42%) were CD98⁺. In bone tumors, LAT1 was highly expressed in osteoblastoma (89%), chondrosarcoma (50%), and osteosarcoma (60%); in soft tissue tumors, LAT1 was highly expressed in rhabdomyosarcoma (80%), synovial sarcoma (63%), Ewing's sarcoma (60%), epithelioid sarcoma (100%) and angiosarcoma (100%). In malignant soft tissue tumors, LAT1 expression was associated with higher histological grade. High CD98 expression was seen in many bone tumors of intermediate and high malignancy. Among soft tissue tumors, CD98 was expressed in tendon sheath giant cell tumor and malignant peripheral nerve sheath tumor (57%), Ewing's sarcoma (50%) and undifferentiated sarcoma (64%). Some of the malignant soft tissue tumors expressed both LAT1 and CD98. This study showed that LAT1 and CD98 was expressed in many malignant and intermediate bone tumors, and some malignant soft tissue tumors.     

Immunohistological expression of HIF‐1α, GLUT‐1, Bcl‐2 and Ki‐67 in consecutive biopsies during chemoradiotherapy in patients with rectal cancer

The aim of this study was to describe the dynamics of HIF‐1α, GLUT‐1, Bcl‐2 and Ki‐67 during chemoradiotherapy (CRT) of rectal cancer, and to investigate the fluctuation of these biomarkers in relation to pathological response to CRT. The study included 86 patients with rectal adenocarcinoma receiving preoperative CRT (>50.4 Gy and Uracil/Tegafur). Immunohistological expressions of HIF‐1α, GLUT‐1, Bcl‐2 and Ki‐67 were investigated in biopsies taken before treatment, after 2, 4 and 6 weeks of CRT and in specimens from the operation. Decreasing expressions of HIF‐1α, Bcl‐2 and Ki‐67 were observed during CRT, whereas GLUT‐1 overall was unchanged. No significant changes of the markers were observed in the interval between CRT and surgery. A significant association was observed between the presence of residual carcinoma after 6 weeks of treatment and pathological response to CRT, but no association was seen between the fluctuations of any of the markers and response to CRT. This unique material containing specimens before, after and during CRT for rectal cancer demonstrated biological dynamics in HIF‐1α, Bcl‐2 and Ki‐67, but not GLUT‐1, expression during CRT, and a significant association was seen between the presence of residual carcinoma after 6 weeks of treatment and pathological response to CRT.     

Cocaine‐induced locomotor sensitization in rats correlates with nucleus accumbens activity on manganese‐enhanced MRI

A long‐standing goal of substance abuse research has been to link drug‐induced behavioral outcomes with the activity of specific brain regions to understand the neurobiology of addiction behaviors and to search for drug‐able targets. Here, we tested the hypothesis that cocaine produces locomotor (behavioral) sensitization that correlates with increased calcium channel‐mediated neuroactivity in brain regions linked with drug addiction, such as the nucleus accumbens (NAC), anterior striatum (AST) and hippocampus, as measured using manganese‐enhanced MRI (MEMRI). Rats were treated with cocaine for 5 days, followed by a 2‐day drug‐free period. The following day, locomotor sensitization was quantified as a metric of cocaine‐induced neuroplasticity in the presence of manganese. Immediately following behavioral testing, rats were examined for changes in calcium channel‐mediated neuronal activity in the NAC, AST, hippocampus and temporalis muscle, which was associated with behavioral sensitization using MEMRI. Cocaine significantly increased locomotor activity and produced behavioral sensitization compared with saline treatment of control rats. A significant increase in MEMRI signal intensity was determined in the NAC, but not AST or hippocampus, of cocaine‐treated rats compared with saline‐treated control rats. Cocaine did not increase signal intensity in the temporalis muscle. Notably, in support of our hypothesis, behavior was significantly and positively correlated with MEMRI signal intensity in the NAC. As neuronal uptake of manganese is regulated by calcium channels, these results indicate that MEMRI is a powerful research tool to study neuronal activity in freely behaving animals and to guide new calcium channel‐based therapies for the treatment of cocaine abuse and dependence. Copyright © 2015 John Wiley & Sons, Ltd.     

The Role of the Dopamine Transporter in Dopamine‐Induced DNA Damage

The neurotransmitter dopamine causes DNA damage, oxidative stress and is involved in the pathology of neurological diseases. To elucidate this potential link we investigated the mechanism of dopamine‐induced DNA damage. We studied the role of the dopamine transporter (DAT) in MDCK and MDCK‐DAT cells, containing the human DAT gene. After treatment with dopamine, only MDCK‐DAT cells showed elevated chromosomal damage and dopamine uptake. Although stimulation of dopamine type 2 receptor (D₂R) with quinpirole in the absence of dopamine did not induce genotoxicity in rat neuronal PC12 cells, interference with D₂R signaling by inhibition of G‐proteins, phosphoinositide 3 kinase and extracellular signal‐regulated kinases reduced dopamine‐induced genotoxicity and affected the ability of DAT to take up dopamine. Furthermore, the D₂R antagonist sulpiride inhibited the dopamine‐induced migration of DAT from cytosol to cell membrane. To determine whether oxidation of dopamine by monoamine oxidase (MAO) is relevant in its genotoxicity, we inhibited MAO, which reduced the formation of micronuclei and of the oxidative DNA adduct 8‐oxodG. Overall, dopamine exerted its genotoxicity in vitro upon transport into the cells and oxidation by MAO. D₂R signaling was involved in the genotoxicity of dopamine by affecting activation and cell surface expression of DAT and hence modulating dopamine uptake.     

The norepinephrine transporter gene and attention‐deficit hyperactivity disorder

The adrenergic system plays a known role in attentional systems and a suspected causal role in attention‐deficit hyperactivity disorder (ADHD), based on evidence from pharmacological interventions and animal models. The efficacy of the highly selective noradrenergic reuptake inhibitor, tomoxetine, in treating ADHD symptoms supports the system's role in ADHD and points to the norephinephrine transporter as a candidate gene. This study tested the gene for the norepinephrine transporter (NET1) as a susceptibility factor in ADHD using three polymorphisms located in exon 9, intron 9, and intron 13. We examined the inheritance of these polymorphisms in a sample of 122 families with a total of 155 children with ADHD identified through an ADHD proband. Use of the transmission disequilibrium test failed to show significant evidence for biased transmission of any of the alleles or the haplotypes of these polymorphisms. We further investigated this gene by screening the probands for five known amino acid variants to determine if they contributed to the ADHD phenotype but observed only one (Thr99Ile) in our sample. Since the frequency of this variant (1.8%) was similar to that previously reported in a control sample (2.2%), it is unlikely that this variant is related to the ADHD phenotype. Our results do not support the NET1 gene as a major genetic susceptibility factor in ADHD. © 2002 Wiley‐Liss, Inc.     

<Emphasis Type="Italic">KNQ1</Emphasis>, a<Emphasis Type="Italic"> Kluyveromyces lactis</Emphasis> gene encoding a drug efflux permease

Several transport systems play an important role in conferring multiple drug resistance, presumably due to their catalysis of the energy-dependent extrusion of a large number of structurally and functionally unrelated compounds out of the cells. In the present work, the gene named KNQ1 (encoding Kluyveromyces lactis membrane permease) was cloned by functional complementation of the cycloheximide-hypersensitivity phenotype of the Saccharomyces cerevisiae mutant strain lacking a functional PDR5 gene. The isolated gene exhibited 48.9% identity with the S. cerevisiae ATR1 gene conferring resistance to aminotriazole and 4-nitroquinoline-N-oxide and encoded a protein of 553 amino acids. When present in multicopy, it efficiently complemented the phenotype associated with the pdr5 or pdr1pdr3 mutations in S. cerevisiae. Overexpression of the KNQ1 gene in K. lactis wild-type strains led to resistance against several cytotoxic compounds, like 4-nitroquinoline-N-oxide, 3-aminotriazole, bifonazole and ketoconazole. The gene was assigned to K. lactis chromosome III and its expression was found to be responsive to oxidative stress induced by hydrogen peroxide. Based on the phenotype of homologous and heterologous transformants, we propose that the gene encodes a membrane-associated component of the machinery responsible for decreasing the concentration of several toxic compounds in the cytoplasm of yeast cells.Communicated by K. Breunig