Deleted In Colorectal Cancer Expression Research Articles

Amphetamine in adolescence disrupts the development of medial prefrontal cortex dopamine connectivity in a DCC-dependent manner.

Initiation of drug use during adolescence is a strong predictor of both the incidence and severity of addiction throughout the lifetime. Intriguingly, adolescence is a period of dynamic refinement in the organization of neuronal connectivity, in particular medial prefrontal cortex (mPFC) dopamine circuitry. The guidance cue receptor, DCC (deleted in colorectal cancer), is highly expressed by dopamine neurons and orchestrates their innervation to the mPFC during adolescence. Furthermore, we have shown that amphetamine in adolescence regulates DCC expression in dopamine neurons. Drugs in adolescence may therefore induce their enduring behavioral effects via DCC-mediated disruption in mPFC dopamine development. In this study, we investigated the impact of repeated exposure to amphetamine during adolescence on both the development of mPFC dopamine connectivity and on salience attribution to drug context in adulthood. We compare these effects to those induced by adult exposure to an identical amphetamine regimen. Finally, we determine whether DCC signaling within dopamine neurons is necessary for these events. Exposure to amphetamine in adolescence, but not in adulthood, leads to an increase in the span of dopamine innervation to the mPFC, but a reduction of presynaptic sites present on these axons. Amphetamine treatment in adolescence, but not in adulthood, also produces an increase in salience attribution to a previously drug-paired context in adulthood. Remarkably, DCC signaling within dopamine neurons is required for both of these effects. Drugs of abuse in adolescence may therefore induce their detrimental behavioral consequences by disrupting mesocortical dopamine development through alterations in the DCC signaling cascade.

Progressive disorganization of paranodal junctions and compact myelin due to loss of DCC expression by oligodendrocytes.

Paranodal axoglial junctions are critical for maintaining the segregation of axonal domains along myelinated axons; however, the proteins required to organize and maintain this structure are not fully understood. Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC) are proteins enriched at paranodes that are expressed by neurons and oligodendrocytes. To identify the specific function of DCC expressed by oligodendrocytes in vivo, we selectively eliminated DCC from mature myelinating oligodendrocytes using an inducible cre regulated by the proteolipid protein promoter. We demonstrate that DCC deletion results in progressive disruption of the organization of axonal domains, myelin ultrastructure, and myelin protein composition. Conditional DCC knock-out mice develop balance and coordination deficits and exhibit decreased conduction velocity. We conclude that DCC expression by oligodendrocytes is required for the maintenance and stability of myelin in vivo, which is essential for proper signal conduction in the CNS.

Haloperidol treatment downregulates DCC expression in the ventral tegmental area

A core feature in the pathophysiology of schizophrenia is abnormal development and function of mesocorticolimbic dopamine (DA) circuitry. We have previously shown that variations in the function of the netrin-1 receptor, deleted in colorectal cancer (DCC), result in changes to the development, organization and ongoing plasticity of DA circuitry. In rodents, repeated exposure to the indirect DA-agonist, amphetamine upregulates DCC expression in the ventral tegmental area (VTA), but not in DA terminal regions. This elevation in DCC expression is associated with increased vulnerability to developing and maintaining sensitized mesolimbic DA function. Antipsychotic medications remain the best treatment option for managing the symptoms in schizophrenia. The peak effects of these medications are gradual, suggesting that a therapeutic component of antipsychotic treatment involves structural reorganization. Here we assessed whether repeated exposure to typical and atypical antipsychotics could also regulate DCC. Adult mice were orally administered haloperidol, clozapine, or risperidone via their drinking water for 4 weeks. Levels of DCC were measured by Western blot analysis of tissue punches of the VTA, medial prefrontal cortex, nucleus accumbens, and dorsal striatum. Haloperidol decreased DCC levels by approximately 50% in the VTA, but not in DA targets. Furthermore, haloperidol did not alter UNC-5 homologue levels, another family of netrin-1 receptors, confirming that its effects target DCC-mediated netrin-1 signaling specifically. The atypical antipsychotics did not alter DCC expression. These results suggest that typical antipsychotics induce selective functional reorganization in the VTA via DCC-mediated netrin-1 signaling.

Adolescence: a time of transition for the phenotype of dcc heterozygous mice

Stark differences exist between adult (>PND 70) and juvenile (∼PND 21-34) rodents in how DCC (deleted in colorectal cancer) receptors and sensitization to amphetamine interact. In adults, repeated amphetamine upregulates DCC receptor expression selectively in the ventral tegmental area (VTA), an effect that is critical for sensitization. In contrast, amphetamine administered to juveniles downregulates VTA DCC expression. Moreover, whereas adult dcc heterozygous mice fail to sensitize when repeatedly treated with amphetamine, drug treatment during the juvenile period actually abolishes this adult "protective" phenotype. We set out to determine whether adolescence (PND ∼35-55) is a period during which: (1) amphetamine-induced alterations in VTA DCC expression switch from downregulation to upregulation; (2) the "protective" phenotype of adult dcc heterozygotes against sensitization becomes evident; and (3) the adult "protective" phenotype of dcc heterozygotes can still be abolished by repeated amphetamine treatment. Repeated amphetamine did not significantly alter VTA DCC expression in adolescent rodents when assessed 1 week later. Both wild-type and dcc heterozygous mice exhibited sensitization at this time. Remarkably, wild-type mice, but not dcc heterozygotes, exhibited sensitization when tested during adulthood. Adolescence is a time of transition for dcc heterozygotes as related to sensitization. Our results support the hypothesis that DCC may be a key factor in determining age-dependent individual differences in vulnerability to sensitization. Given that exposure to drugs of abuse during adolescence can have profound consequences for adulthood, the resilience of adult dcc heterozygous mice against adolescent exposure to amphetamine is particularly salient.

DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain

The transmembrane protein deleted in colorectal cancer (DCC) and its ligand, netrin-1, regulate synaptogenesis during development, but their function in the mature central nervous system is unknown. Given that DCC promotes cell-cell adhesion, is expressed by neurons, and activates proteins that signal at synapses, we hypothesized that DCC expression by neurons regulates synaptic function and plasticity in the adult brain. We report that DCC is enriched in dendritic spines of pyramidal neurons in wild-type mice, and we demonstrate that selective deletion of DCC from neurons in the adult forebrain results in the loss of long-term potentiation (LTP), intact long-term depression, shorter dendritic spines, and impaired spatial and recognition memory. LTP induction requires Src activation of NMDA receptor (NMDAR) function. DCC deletion severely reduced Src activation. We demonstrate that enhancing NMDAR function or activating Src rescues LTP in the absence of DCC. We conclude that DCC activation of Src is required for NMDAR-dependent LTP and certain forms of learning and memory.

Netrin-1 rescues neuron loss by attenuating secondary apoptosis in ipsilateral thalamic nucleus following focal cerebral infarction in hypertensive rats

Neurological deficit following cerebral infarction correlates with not only primary injury, but also secondary neuronal apoptosis in remote loci connected to the infarction. Netrin-1 is crucial for axonal guidance by interacting with its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5H (UNC5H). DCC and UNC5H are also dependence receptors inducing cell apoptosis when unbound by netrin-1. The present study is to investigate the role of netrin-1 and its receptors in ipsilateral ventroposterior thalamic nucleus (VPN) injury secondary to stroke in hypertensive rats. Renovascular hypertensive Sprague–Dawley rats underwent middle cerebral artery occlusion (MCAO). Continuous intracerebroventricular infusion of netrin-1 (600ng/d for 7days) or vehicle (IgG/Fc) was given 24h after MCAO. Neurological function was evaluated by postural reflex 8 and 14days after MCAO. Then, immunoreactivity was determined in the ipsilateral VPN for NeuN, glial fibrillary acidic protein, netrin-1 and its receptors (DCC and UNC5H2), apoptosis was detected with Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) assay, and the expressions of caspase-3, netrin-1, DCC, and UNC5H2 were quantified by western blot analysis. MCAO resulted in the impaired postural reflex after 8 and 14days, with decreased NeuN marked neurons and increased TUNEL-positive cells, as well as an up-regulation in the levels of cleaved caspase-3 and UNC5H2 protein in the ipsilateral VPN, without significant change in DCC or netrin-1 expression. By exogenous netrin-1 infusion, the number of neurons was increased in the ipsilateral VPN, and both TUNEL-positive cell number and caspase-3 protein level were reduced, while UNC5H2 expression remained unaffected, simultaneously, the impairment of postural reflex was improved. Taken together, the present study indicates that exogenous netrin-1 could rescue neuron loss by attenuating secondary apoptosis in the ipsilateral VPN after focal cerebral infarction, possibly via its receptor UNC5H2, suggesting that relative insufficiency of endogenous netrin-1 be an underlying mechanism of secondary injury in the VPN post stroke.

Increased Expression of Netrin-1 and Its Deleted in Colorectal Cancer Receptor in Human Diseased Lumbar Intervertebral Disc Compared With Autopsy Control

The expression of netrin-1 and its deleted in colorectal cancer (DCC) receptor was investigated in human lumbar discs using immunohistochemistry. To investigate the expression of netrin-1 and DCC receptor in human diseased and healthy lumbar intervertebral discs (IVDs) and to clarify the correlation between netrin-1 expression and the degree of neurovascular ingrowth. Previous studies have shown neurovascular ingrowth into the inner regions of degenerated IVD and suggested that the ingrowth may contribute to discogenic low back pain. Netrin-1 is an axon guidance molecule that regulates axons seeking their final targets and has been identified as involved in various pathological conditions, so is its DCC receptor. However, the role of netrin-1 in diseased IVDs remains unknown. Thirty-five diseased IVD specimens were collected from 34 patients with different lumbar diseases during posterior lumbar interbody fusion. Eight normal discs were obtained at autopsy as control. Using polyclonal or monoclonal antibody, the disc slides were immmunostained to detect the expression and distribution of netrin-1, the DCC, the neuronal marker (neurofilament), and the vascular endothelial cell marker (CD34). Netrin-1 and DCC immunopositive cells distributed substantially from the annulus fibrosus to the nucleus pulposus (NP), and the immunopositivity was detected in the disc cells, endothelial cells and granulation tissue cells in the diseased discs. The percentage of netrin-1 positive disc cells of the NP was more than that of the annulus fibrosus. The expression of netrin-1 and DCC was weak in the normal discs. A significant positive correlation between the percentage of netrin-1 immunopositive disc cells and neurovascular scores was found. The increased expression of netrin-1 and DCC in diseased IVDs compared with controls suggested that they might play an important role in the process of neurovascular ingrowth.

Association between schizophrenia and genetic variation in DCC: A case–control study

Schizophrenia is a highly heritable neurodevelopmental disorder associated with alterations in synaptic connectivity. Deleted in colorectal cancer (DCC), a receptor for the guidance cue netrin-1, plays a pivotal role in organizing neuronal circuitry by guiding growing axons and dendrites to their correct targets and by influencing synaptic connectivity. Results from experiments we previously conducted in dcc-heterozygous mice show that DCC plays a critical role in the developmental organization of the mesocorticolimbic dopamine (DA) circuitry. Furthermore we have shown that reduced expression of DCC during development and/or throughout life confers resilience to the development of schizophrenia-like DA and behavioural abnormalities. Importantly, this “protective” phenotype only emerges after puberty. Here we assess whether DCC may contribute to the risk of schizophrenia. We examined single nucleotide polymorphisms (SNPs) located in the DCC gene for association with schizophrenia using a case–control sample consisting of 556 unrelated schizophrenic patients and 208 healthy controls. We found one SNP, rs2270954, to be nominally associated with schizophrenia; patients were less likely to be heterozygous at this locus and more likely to be homozygous for the minor allele (χ2=9.84, df=2, nominal p=0.0071). Intriguingly, this SNP is located within the 3′ untranslated region, an area known to contain a number of regulatory sequences that determine the stability and translation efficacy of mRNA. These results, together with our previous findings from studies in rodents, point at DCC as a promising novel candidate gene that may contribute to the genetic basis behind individual differences in susceptibility to schizophrenia.

A genome-wide association study identifies SNP in DCC is associated with gallbladder cancer in the Japanese population

Gallbladder cancer (GC) is a relatively uncommon cancer with higher incidence in certain areas including Japan. Because of the difficulty in diagnosis, prognosis of GC is very poor. To identify genetic determinants of GC, we conducted a genome-wide association study (GWAS) in 41 GC patients and 866 controls. Association between each single-nucleotide polymorphism (SNP) with GC susceptibility was evaluated by multivariate logistic regression analysis conditioned on age and gender of subjects. SNPs that showed suggestive association (P<1 × 10(-4)) with GC were further examined in 30 cases and 898 controls. SNP rs7504990 in the DCC (deleted in colorectal cancer, 18q21.3) that encodes a netrin 1 receptor achieved a combined P-value of 7.46 × 10(-8) (OR=6.95; 95% CI=3.43-14.08). Subsequent imputation analysis identified multiple SNPs with similarly strong associations in an adjacent genomic region, where loss of heterozygosity was reported in GC and other cancers. Reduced expression of DCC was indicated to be associated with the poorly differentiated histological type, increased proliferation and metastasis through loss of adhesiveness. However, due to the limited sample size investigated here, further replication study and functional analysis would be necessary to further confirm the result of the association.

Deleted in colorectal carcinoma suppresses metastasis in p53-deficient mammary tumours

Since its discovery in the early 1990s the deleted in colorectal cancer (DCC) gene, located on chromosome 18q21, has been proposed as a tumour suppressor gene as its loss is implicated in the majority of advanced colorectal and many other cancers. DCC belongs to the family of netrin 1 receptors, which function as dependence receptors as they control survival or apoptosis depending on ligand binding. However, the role of DCC as a tumour suppressor remains controversial because of the rarity of DCC-specific mutations and the presence of other tumour suppressor genes in the same chromosomal region. Here we show that in a mouse model of mammary carcinoma based on somatic inactivation of p53, additional loss of DCC promotes metastasis formation without affecting the primary tumour phenotype. Furthermore, we demonstrate that in cell cultures derived from p53-deficient mouse mammary tumours DCC expression controls netrin-1-dependent cell survival, providing a mechanistic basis for the enhanced metastatic capacity of tumour cells lacking DCC. Consistent with this idea, in vivo tumour-cell survival is enhanced by DCC loss. Together, our data support the function of DCC as a context-dependent tumour suppressor that limits survival of disseminated tumour cells.

Juvenile exposure to methylphenidate reduces cocaine reward and alters netrin-1 receptor expression in adulthood

The guidance cue netrin-1 acting on mesocorticolimbic dopamine (DA) neurons through its receptor DCC (deleted in colorectal cancer) has been implicated in the neuronal plasticity induced by psychostimulant drugs. We examined in C57/BL6 mice the effects of repeated juvenile methylphenidate (MPH) exposure on cocaine-reward sensitivity in adulthood and determined whether early MPH treatment alters adult expression of DCC in mesocorticolimbic DA regions. By using place conditioning, we show that adult mice exposed to MPH during the juvenile period are less sensitive to cocaine-reward compared to saline-controls. Furthermore, by means of immunoblotting, we demonstrate that early MPH treatment attenuates adult DCC expression in the ventral tegmental area (VTA) selectively. These results support previous evidence that developmental MPH treatment diminishes cocaine-reward in adulthood and are the first to suggest that DCC in the VTA may participate in this enduring effect.

Autocrine Netrin Function Inhibits Glioma Cell Motility and Promotes Focal Adhesion Formation

Deregulation of mechanisms that control cell motility plays a key role in tumor progression by promoting tumor cell dissemination. Secreted netrins and their receptors, Deleted in Colorectal Cancer (DCC), neogenin, and the UNC5 homologues, regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Netrin and netrin receptor expression have previously been shown to be disrupted in invasive tumors, including glioblastoma. We determined that the human glioblastoma cell lines U87, U343, and U373 all express neogenin, UNC5 homologues, and netrin-1 or netrin-3, but only U87 cells express DCC. Using transfilter migration assays, we demonstrate DCC-dependent chemoattractant migration of U87 cells up a gradient of netrin-1. In contrast, U343 and U373 cells, which do not express DCC, were neither attracted nor repelled. Ectopic expression of DCC by U343 and U373 cells resulted in these cells becoming competent to respond to a gradient of netrin-1 as a chemoattractant, and also slowed their rate of spontaneous migration. Here, in addition to netrins' well-characterized chemotropic activity, we demonstrate an autocrine function for netrin-1 and netrin-3 in U87 and U373 cells that slows migration. We provide evidence that netrins promote the maturation of focal complexes, structures associated with cell movement, into focal adhesions. Consistent with this, netrin, DCC, and UNC5 homologues were associated with focal adhesions, but not focal complexes. Disrupting netrin or DCC function did not alter cell proliferation or survival. Our findings provide evidence that DCC can slow cell migration, and that neogenin and UNC5 homologues are not sufficient to substitute for DCC function in these cells. Furthermore, we identify a role for netrins as autocrine inhibitors of cell motility that promote focal adhesion formation. These findings suggest that disruption of netrin signalling may disable a mechanism that normally restrains inappropriate cell migration.

Effects of treadmill exercise on the expression of netrin-1 and its receptors in rat brain after cerebral ischemia

Recent evidence suggests that exercise improves functional outcome in animal models of cerebral ischemia. Since netrin-1 and its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5B (Unc5B), act as important regulators in neural and vascular activities, we sought to determine whether netrin-1 and DCC and Unc5B are involved in the neuroprotective effects of exercise on rats with induced cerebral ischemia. A total of 108 rats were randomly distributed into three groups: sham-operated group (n = 12), middle cerebral artery occlusion (MCAO) group (n = 48), MCAO+treadmill exercise group (n = 48). Behavioral testing indicated that treadmill exercise could significantly improve neurologic deficits of rats with cerebral ischemia at day 14 and 28 after MCAO (n = 12, P<0.05 and P<0.01), but there was no significant difference at day 4 and 7. Quantitative reverse transcription polymerase chain reaction (qPCR) and Western blot analysis revealed that treadmill exercise enhanced netrin-1 and DCC expression, while it suppressed Unc5B expression in rat peri-ischemic brain area, especially at day 14 and 28 after MCAO (n = 4, P<0.05 or P<0.01). Immunofluorescence analysis showed that in the peri-ischemic area, netrin-1 was expressed in neuronal perikarya, DCC, however, was expressed in neural processes and peri-vascular astrocytes, while Unc5B was expressed mostly in neuronal perikarya and some processes. These results suggest that netrin-1 and its receptors DCC and Unc5B may engage in exercise-induced neural circuit remodeling in the peri-ischemic area, and exercise may promote survival of neurons in this area by regulating netrin-1-Unc5B signaling. Additionally, netrin-1 may also play a role in brain-blood barrier via DCC-immunoreactive peri-vascular astrocytes. In conclusion, we demonstrate that treadmill exercise has beneficial effects that may be attributed, at least in part, to the involvement of netrin-1 and its receptors DCC and Unc5B in the neuronal and vascular activities in brain-ischemic rats.

Abolition of the behavioral phenotype of adult netrin-1 receptor deficient mice by exposure to amphetamine during the juvenile period

Netrin-1 guidance cues contribute to amphetamine-induced plasticity of the adult mesocorticolimbic dopamine system in rodents. The netrin-1 receptor, deleted in colorectal cancer (DCC), is upregulated by repeated amphetamine treatment selectively in the ventral tegmental area (VTA) of adult rats and wild-type mice. Furthermore, adult dcc heterozygous mice fail to show amphetamine-induced increases in VTA DCC expression and do not develop sensitization to this drug. The effects of netrin-1 receptor signaling on mesocorticolimbic dopamine system function change across development. However, the effects of AMPH on DCC receptor regulation and behavioral sensitization before puberty have not been determined. Here we examined whether (1) repeated amphetamine treatment would also alter DCC expression in juvenile rats and wild-type mice, and (2) dcc heterozygotes treated with amphetamine during the juvenile period (PND 22-32) would develop behavioral sensitization to this drug. Repeated amphetamine downregulates DCC expression selectively in the VTA of juvenile rodents. Moreover, the behavioral phenotype of adult dcc heterozygous mice is not present before puberty and is abolished by amphetamine treatment during the juvenile period. Remarkably, adult dcc heterozygotes pretreated with amphetamine as juveniles no longer exhibit reduced DCC expression in the VTA compared to wild-type controls. Our results indicate that netrin-1 receptor signaling may be a key factor in determining individual differences in vulnerability to the behaviorally sensitizing effects of amphetamine at different ages. Moreover, they suggest that the juvenile period marks a window of vulnerability during which exposure to stimulant drugs can reverse the behavioral phenotype of adult dcc heterozygous mice.

Peri-pubertal emergence of UNC-5 homologue expression by dopamine neurons in rodents.

Puberty is a critical period in mesocorticolimbic dopamine (DA) system development, particularly for the medial prefrontal cortex (mPFC) projection which achieves maturity in early adulthood. The guidance cue netrin-1 organizes neuronal networks by attracting or repelling cellular processes through DCC (deleted in colorectal cancer) and UNC-5 homologue (UNC5H) receptors, respectively. We have shown that variations in netrin-1 receptor levels lead to selective reorganization of mPFC DA circuitry, and changes in DA-related behaviors, in transgenic mice and in rats. Significantly, these effects are only observed after puberty, suggesting that netrin-1 mediated effects on DA systems vary across development. Here we report on the normal expression of DCC and UNC5H in the ventral tegmental area (VTA) by DA neurons from embryonic life to adulthood, in both mice and rats. We show a dramatic and enduring pubertal change in the ratio of DCC:UNC5H receptors, reflecting a shift toward predominant UNC5H function. This shift in DCC:UNC5H ratio coincides with the pubertal emergence of UNC5H expression by VTA DA neurons. Although the distribution of DCC and UNC5H by VTA DA neurons changes during puberty, the pattern of netrin-1 immunoreactivity in these cells does not. Together, our findings suggest that DCC:UNC5H ratios in DA neurons at critical periods may have important consequences for the organization and function of mesocorticolimbic DA systems.

Spatio-temporal deleted in colorectal cancer (DCC) and netrin-1 expression in human foetal brain development

Deleted in colorectal cancer (DCC) and its ligand netrin-1 are known as axonal guidance factors, being involved in angiogenesis, migration and survival of precursor cells in the embryonic mammalian central nervous system (CNS). So far, little is known about the distribution of those molecules in human CNS development. We investigated 22 human foetal brain specimens (12th and 28th week of gestation) for DCC and netrin-1 expression by means of immunohistochemistry, immunofluorescence and confocal laser microscopy. Statistical analysis was performed by applying a semi-quantitative score, including staining intensity and frequency and correlation with foetal age. DCC and netrin-1 were differentially expressed throughout the developing human foetal telencephalic and cerebellar cortical layers. Netrin-1 exhibited the highest levels in telencephalic germinal layers, whereas the strongest DCC immunoreactivity was seen in the developing cortical plate. Netrin-1 and DCC were predominantly present on cerebellar external granule layer cells. Distinct co-expression was seen in maturing foetal brainstem nuclei, cerebellar external granular layer and the choroid plexus. In contrast, endothelial cells showed strong netrin-1 expression with subsidiary DCC immunoreactivity. Pontine and telencephalic axonal fibre tracts also demonstrated strong netrin-1 expression. We show that DCC and netrin-1 are ubiquitously expressed in the human foetal brain; however, both exhibit a distinct spatio-temporal expression pattern. Together with the data from animal experiments, our findings might indicate also an important role for DCC and netrin-1 in human foetal CNS development.

Netrin‐1 receptor in the ventral tegmental area is required for sensitization to amphetamine

Fundamental to neural organization during development, the netrin-1 guidance cue and its receptor, deleted in colorectal cancer (DCC), continue to be expressed in the adult brain. We have shown recently that adult dcc heterozygous mice do not develop sensitization to the stimulant drug of abuse amphetamine (AMPH) and that repeated exposure to AMPH upregulates DCC expression in adult rats. This upregulation is selective to the ventral tegmental area (VTA), a site critical for the initiation of behavioral plasticity induced by stimulant drugs, and is glutamate-dependent. Here we demonstrate that the lack of AMPH-induced sensitization in dcc heterozygotes is associated with a failure of AMPH to upregulate DCC receptor expression in the VTA. Further, we show that, in wild-type mice, repeated AMPH induces increases in VTA expression of the dendritic spine-associated protein, spinophilin. Significantly, however, this effect is not observed in dcc heterozygotes. In parallel experiments conducted in adult rats, we show that VTA DCC receptor activation, at the time of AMPH pretreatment, is critical for sensitization to AMPH. Together, these results demonstrate that the DCC netrin-1 receptor, a protein traditionally known for its role in organizing brain development, plays a critical function in adult brain plasticity, possibly via orchestration of neuronal circuitry reorganization. We propose VTA DCC receptor signaling as a novel mechanism in the series of glutamate-dependent cellular processes that lead to enduring plasticity by drugs of abuse.

Alterations of DCC Gene in B-Cell Malignancies.

Abstract 4428Many genetic alterations have been detected in various tumors to date, and these are concerned with oncogenesis closely. Oligonucleotide array analysis has contributed to detect genomic gains and losses implicated in the development of human malignant tumors, identifying either oncogenes or tumor suppressor genes. In our recent data, oligonucleotide array analysis showed recurrent genomic copy number variations at 18q21-22 in malignant lymphoma and multiple myeloma cell lines. There are a lot of genes related to oncogenesis at 18q21-22, such as SMAD4, DCC, MBD2, TCF4, TXNL1, WDR7, FECH, NARS, MALT1, and BCL2. We studied expression of these genes by reverse-transcriptase polymerase chain reaction (RT-PCR). Although most genes examined expressed in almost all cell lines examined, DCC (deleted in colorectal cancer) could be detected in a small number of malignant lymphoma cell lines.DCC, which encodes a receptor for netrin-1, would control apoptosis and be concerned in oncogenesis. Loss of one DCC allele or decreased DCC expression occur in many solid tumors, suggesting that DCC inactivation constitutes a critical event in the development of these tumors. Similarly, the inactivation of DCC can have a role in the development of hematologic malignancies, including leukemia and malignant lymphoma. We analyzed DCC expression in 15 multiple myeloma and 9 malignant lymphoma cell lines, by RT-PCR using various primer pairs. However, no products could be detected using any primer pairs containing DCC exon 1 primer in three multiple myeloma cell lines, although other primer pairs could amplify products, suggesting the DCC transcripts lacking exon 1 in these cell lines. This result made us predict the abnormal form of DCC. Therefore, we tried to clone the DCC transcripts fused to unknown sequences instead of exon 1, using bubble PCR method for cDNA in these three cell lines. Some products were obtained and sequenceanalysis revealed that the bubble PCR products contained exon 2 of DCC fused to unknown sequences. BLAST search revealed that these unknown sequences were part of intron 1 of DCC, resulting in the lack of start codon in exon 1. Putative abnormal DCC protein lacks a part of N-terminus, suggesting that structural abnormalities of DCC play some roles in oncogenesis of hematologic malignancies. Although the previous studies described the abnormalities of DCC derived from allelic loss or decreased expression, structural abnormalities of DCC we found in this study might be another critical mechanism for DCC abnormalities even if the normal expression could be detected by RT-PCR which could amplify only limited portion of the gene. Disclosures:No relevant conflicts of interest to declare.

DCC promoter hypermethylation in esophageal squamous cell carcinoma

Deleted in Colorectal Cancer (DCC) is a putative tumor suppressor gene, whose loss has been implicated in colorectal tumorigenesis. Decreased or loss of DCC expression has been demonstrated in a number of human cancers, including esophageal cancer. In this study, we analyzed esophageal squamous cell carcinoma (ESCC) cell lines and primary ESCCs as well as normal esophageal tissues for DCC methylation by bisulfite sequencing, methylation-specific PCR (MSP) and/or quantitative methylation-specific PCR (qMSP). When a qMSP cut-off value for positivity was set to 1.0, DCC methylation was detected in 10 of 12 ESCC cell lines tested, 74% of primary ESCCs (n = 70), 0% of corresponding normal esophageal tissues (n = 20) and 0% of normal esophagus from healthy individuals (n = 19). DCC expression was undetectable in the majority of ESCC cell lines, and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reactivated gene expression. DCC overexpression suppressed colony formation in ESCC cell lines, suggesting that DCC may function as a tumor suppressor gene in the esophagus. However, DCC methylation was not associated with any clinical or pathologic parameters measured. We have demonstrated that DCC methylation is a frequent and cancer-specific event in primary ESCCs, suggesting that DCC and associated pathways may represent a new diagnostical therapeutic target.

Regulation of netrin-1 receptors by amphetamine in the adult brain

Netrin-1 is a guidance cue molecule fundamental to the organization of neuronal connectivity during development. Netrin-1 and its receptors, deleted in colorectal cancer (DCC) and UNC-5 homologues (UNC-5), continue to be expressed in the adult brain, although neither their function nor the kinds of events that activate their expression are known. Two lines of evidence suggest a role for netrin-1 in amphetamine-induced dopamine plasticity in the adult. First, DCC is highly expressed by adult dopamine neurons. Second, adult mice with reduced DCC levels do not develop amphetamine-induced behavioral sensitization. To explore the role of netrin-1 in amphetamine-induced plasticity, we examined the effects of sensitizing treatment regimens of amphetamine on DCC and/or UNC-5 protein expression in the adult rat. These treatments produced striking and enduring increases in DCC and UNC-5 expression in the cell body, but not terminal regions, of the mesocorticolimbic dopamine system. Notably, neuroadaptations in the cell body region of mesocorticolimbic dopamine neurons underlie the development of sensitization to the effects of amphetamine. Furthermore, these localized amphetamine-induced changes were prevented by co-treatment with an N-methyl- d-aspartate receptor antagonist, a treatment known to block the development of amphetamine-induced sensitization of behavioral activation, dopamine release and motivated behavior. Using immunohistochemistry, we showed that both DCC and UNC-5 receptors are highly expressed by adult mesocorticolimbic dopamine neurons. These results provide the first evidence that repeated exposure to a stimulant drug such as amphetamine affects netrin-1 receptor expression in the adult brain. Taken together, our findings suggest that changes in netrin-1 receptor expression may play a role in the lasting effects of exposure to amphetamine and other stimulant drugs.