Interactions between dopamine and glutamate in the prefrontal cortex are essential for cognitive functions such as working memory. Modulation of N-methyl-D-aspartic acid (NMDA) receptor functions by dopamine D1 receptor is believed to play a critical role in these functions. The aim of the work reported here is to explore the signaling pathway underlying D1 receptor-mediated trafficking of NMDA receptors in cultured rat prefrontal cortical neurons.Activation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits. This effect was completely blocked by small interfering RNA knockdown of Fyn, but not Src. Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression. D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.Dopamine D1 receptor-mediated increase of NMDA receptors is thus Fyn kinase dependent. Targeting this signaling pathway may be useful in treating drug addiction and schizophrenia. The prefrontal cortex (PFC) plays a well-established role in working memory function ,2 and dyRecent evidence indicates that NMDA receptors are not static, as traditionally believed; instead they can move into and out of synapses -20. IndeTo characterize the expression pattern of D1 and NMDA receptors in cultured PFC neurons, we performed double immunofluorescent staining of both D1 and NR2B receptors in low-density cultures at 14 DIV. As shown in Figure Although NMDA receptors are largely composed of NR1 (obligatory subunit), NR2A and NR2B, we focused on the effect of D1 class-mediated regulation in NR2B trafficking to test our hypothesis. This is because NR2B is the major tyrosine-phosphorylated subunit in the post-synaptic density ,30,31 an2 in normal medium for another 15 min to allow the receptor trafficking. When the PFC neurons were treated with the D1 agonist SKF-81297 alone, expression of NR2B subunits in cultured PFC neurons was significantly enhanced compared with control, as shown in Figure To study D1 receptor activation-mediated NR2B expression in cultured PFC neurons, the cultures were treated for 10 min with DMSO (0.1%) as vehicle control, the selective dopamine D1 receptor agonist SKF-81297 (10 μM), or the dopamine D1 receptor antagonist SCH-23390 (15 μM) followed by SKF-81297 (10 μM) for 10 min. The cultures were then restored to 37°C/5% CO2 for 48 h to allow for detectable knockdown. The efficiencies of the transfections were assessed with the neurons that stained with antibodies of either anti-Fyn or Src. We found that both Fyn and Src kinase expressions were significantly decreased in the siRNA-transfected neurons. As shown in Figure To test our hypothesis regarding the roles of Fyn and Src in D1-NR2B interaction, we used Fyn- and Src-specific small interfering RNAs (siRNAs) to transfect PFC neurons at 11 DIV. First, we determined the efficiency of Fyn and Src siRNA transfection in cultured PFC neurons. Four hours after siRNA application, the neurons were washed out and then maintained in neurobasal medium  at 37°C/5% CORecent studies have emphasized the importance of Src family kinases in regulation of NMDA receptors in the CNS, possibly in enhancing NMDA receptor function ,26. Howe2 in normal medium for another 15 min. As shown in Figure We next examined the specific roles of Src and Fyn in D1 receptor-mediated modulation of NMDA receptor trafficking. In our recent study , we founPrevious studies in other brain regions have demonstrated that activation of Src family kinases leads to phosphorylation of NR2B which appears to be important for surface expression of this subunit ,32. TherFinally, we used a surface biotinylation assay to examine whether Fyn and Src affect NR2B trafficking under this condition. We found that treatment of high-density PFC cultured neurons with D1 receptor agonist SKF-81297 significantly increased the ratio of surface/total NR2B expression compared with the control group  might stabilize NMDA receptors on the cell surface, thereby increasing NMDA receptor responses. Accordingly, Src family kinase activation might inhibit NMDA receptor endocytosis ,52. We oOur RNAi efficiency is similar to that of others as reported in neurons . The reaOur findings demonstrate that dopamine receptors may regulate synaptic plasticity by modulating NMDA receptor synaptic expression through a Fyn-dependent signaling pathway. Considering that NMDA receptor activity alterations are involved in the clinical features of schizophrenia and drug abuse, our study not only provides insight into the roles of Src family kinases in NMDA receptor trafficking but also offers the possibility of generating new pharmacologic reagents targeting Fyn kinase signaling pathway. This intervention could be promising for psychiatric disorders involving D1-NMDA interaction, such as schizophrenia and drug addiction.The chemicals used for treatment of PFC neurons were purchased from the following sources: selective D1 agonist SKF-81297, D1 antagonist SCH-23390, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich . Antibodies used for immunofluorescence and Western blotting included mouse anti-NR2B , rabbit phospho-Y1472 NR2B , rabbit anti-PSD95 , rat anti-D1 , rabbit anti-Fyn , mouse anti-Src , and mouse antitubulin . The primary antibody dilutions listed above were for Western blotting. For immunofluorescence staining, dilutions were all 1:800. The following reagents were obtained from Jackson ImmunoResearch : Cy3-conjugated donkey antimouse secondary antibody (1:800), FITC-conjugated donkey anti-mouse secondary antibody (1: 800), Cy3-conjugated donkey anti-rabbit secondary antibody (1:800), FITC-conjugated donkey anti-rabbit secondary antibody (1:800), peroxidase-conjugated goat anti-mouse secondary antibody (1:1000), and peroxidase-conjugated goat anti-rabbit secondary antibody (1:1000).5 cells per well on poly-l-lysine coated glass coverslips in 6-well plates  and kept in Neurobasal medium supplemented with B27, L-glutamine, and 5% FBS at 37°C/5% CO2 for 4 h. Cells were then maintained in Neurobasal medium supplemented with B27, and L-glutamine without FBS at 37°C/5% CO2. Cells were fed 2 times per week, with one third of the media changed each time. Cultured PFC neurons were used between 14 and 18 days in vitro (DIV) for the studies described.Timed pregnant rats and adult rats (3 months old) were purchased from Marshall Farms  and the animal procedures were in strict accordance with the National Institute of Health (NIH) animal use guideline. The experimental protocols were approved by the Institutional Animal Care and Use Committee at Drexel University College of Medicine. Primary neuron cultures were prepared from embryonic day 20 rat PFC. Briefly, PFC tissues were isolated and dissociated with trypsin  at 37°C, filtered by cell filter , rinsed with Neurobasal medium  supplemented with B27and L-glutamine and 5% fetal bovine serum (FBS). Cultures were plated at 2 × 10Src and Fyn siRNAs were purchased from Millipore, including pKD-Fyn-v6  and pKD-Src-v2 .Src siRNA sequence:Sense strand:5'-TGGTGGCCTACTACTCCAAACTTCAAGAGAGTTTGGAGTAGTAGGCCACCATTTTTG-3'Antisense strand:5'-AATTCAAAAATGGTGGCCTACTACTCCAAACTCTCTTGAAGTTTGGAGTAGTAGGCCACCA-3'Fyn siRNA sequence:Sense strand:5'-ACATCGTCACCGAGTATGTGATTCAAGAGATCATATACTCGGTGACGATGTTTTTTG-3'Antisense strand:5'-AATTCAAAAAACATCGTCACCGAGTATATGATCTCTTGAATCACATACTCGGTGACGATGT-3'2 for 48 to 72 h to allow for detectable knockdown prior to the treatment with other pharmacologic reagents. The efficiencies of siRNA transfection were assessed by both immunostaining and Western blotting with antibodies against Fyn or Src, whereas the antibody specificities were demonstrated by re-probing the Src and Fyn, respectively.The siRNAs were used to transfect PFC neurons (750 ng per well) with GeneSilencer siRNA transfection reagent from Genlantis  when cultures reached 70% confluence. Neurons in the control conditions were transfected with the same volume of transfection reagent without siRNA. After 4 h, neurons were washed with serum-free Neurobasal medium, and neurons were maintained in Neurobasal medium supplemented with B27 and L-glutamine at 37°C/5% CO2 for another 15 min to allow the trafficking of NMDA receptors. For control, the same volume of DMSO (0.1%) was added as vehicle and the cultured neurons were subjected to the same conditions. Precautions were taken to protect samples from light exposure and oxidation. The treated PFC neurons were used for both immunofluorescent staining and Western blotting.To assess the roles of Fyn and Src in D1 receptor-mediated modulation of NMDA receptor, PFC neurons with or without transfection were treated with the selective D1 receptor agonist SKF-81297 (10 μM) for 10 min or pretreated with selective D1 receptor antagonist SCH-23390 (15 μM) followed by SKF-81297 (10 μM) for 10 min. The cultured neurons were then restored to normal medium at 37°C/5% COPFC neurons were fixed by methanol at -20 °C for 5 min and rinsed with 0.2% Triton X-100  in phosphate-buffered saline (PBS) to permeabilize the plasma membrane. Coverslips were blocked with 10% bovine serum albumin in PBS for 1 h at room temperature. Double immunofluorescent stainings were conducted with antibodies against NMDA receptors (NR2B), D1 receptor, Fyn, Src, or postsynaptic marker PSD95, followed by appropriate secondary antibodies. Images were acquired using a Zeiss Axiovert 200 M inverted microscope with Axiovision software . All analysis and quantifications were performed using the NIH Image J software. The dendritic segments in neurons (40 μm) were randomly selected for puncta analysis. The average intensity of fluorescence staining and the punctate number of NR2B/Fyn/Src were quantified using the NIH Image J software and were blindly confirmed by other researchers in the laboratory. Results were presented as the mean number of total puncta or mean intensity of fluorescence ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey multiple-comparison tests for several experimental groups. Images were prepared for printing with Adobe Photoshop  and Canvas .The biotinylation was performed as described with minor modification . After tCrude synaptosome P2) was prepared from rat frontal cortex as previously described ,59 with  was prepProteins were isolated from adult rat PFC or high-density prefrontal neuronal cultures with RIPA buffer. After centrifugation at 14,000 rpm at 4 °C for 15 min, the supernatants were resolved by electrophoresis on 7.5% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked in 5% nonfat milk in TBS for 1 h and were incubated with the following antibodies overnight at 4 °C: mouse anti-NR2B, rabbi anti-PSD95, rat anti-D1, rabbit anti-Fyn, mouse anti-Src, and mouse antitubulin. After incubation with appropriate horseradish peroxidase-conjugated secondary antibodies, antigens were identified by enhanced chemiluminescence reagents . Blots wThe authors declare that they have no competing interests.JLH carried out the biochemical experiments, data analysis, and wrote the manuscript. GL did cell cultures, carried out immunostaining, participated in biochemical experiments and data analysis. YCL participated in immunostaining and data analysis. WJG helped the manuscript preparation and prepared the manuscript for publication. YQH conceived and designed the study and revised the manuscript. All authors read and approved the final manuscript.D1-mediated Increase of NMDA Receptors May Require Protein Synthesis and Depends on PKA Signaling. The file includes graphs which show the effects of protein synthesis inhibitor anisomycin and PKA inhibitor KT5720 and PKC inhibitor Go6983 on D1-mediated increase of NR2B cluster number and fluorescence.Click here for file


The profiles of 19 microsatellite markers gave a 100% positive predictive value and an 80.8% negative predictive value for hepatocellular carcinoma. In conclusion, we have determined a profile of microsatellite markers appropriate for differential diagnosis of primary liver cancer. The discovery may permit a high-throughput screening of hepatocellular carcinoma at an early stage of disease.Frequent loss of heterozygosity of microsatellites markers on specific chromosomal region have been reported in various types of primary human cancer. The same loss of heterozygosity has also been identified in the matched plasma/serum DNA. Using 109 microsatellite markers representing 24 chromosomal arms, we have examined the loss of heterozygosity in 21 cases of hepatocellular carcinoma, six of cholangiocarcinoma, and 27 cases of chronic hepatitis or cirrhosis. All cases of the hepatocellular carcinoma showed deletion from two to 10 chromosomal arms, while deletion of chromosomes from two to eight regions was detected in five of six cholangiocarcinoma patients. One or more loss of heterozygosity in the paired serum DNA could be detected in 16 of 25 (76.2%) hepatocellular carcinoma patients. In contrast, no alterations in serum DNA test could be found in cholangiocarcinoma patients. Five of seven (71.4%) hepatocellular carcinoma patients with alpha-fetoprotein levels less than 20 ng mlBritish Journal of Cancer (2002) 87, 1449–1453. doi:10.1038/sj.bjc.6600649www.bjcancer.com© 2002 Cancer Research UK Primary liver cancer ranks fifth in frequency among all malignancies in the world with an estimated number of 437 000 new cases in 1990. The vast majority of primary liver cancer is hepatocellular carcinoma (HCC), a malignant tumour derived from hepatocytes. HCC is increasing in many countries, particularly in areas where hepatitis C virus infection is more common than hepatitis B virus infection . The choThe prognosis of HCC or CC is usually poor, with 3-year survival rates estimated at 44.9% and 34% after hepatic resection, respectively  and hepatic ultrasonography are widely used as surveillance modalities for high-risk patients. Elevated levels of AFP, with normal range set at 20 ng mln=21) and CC (n=6). The results suggest that microsatellite serum DNA analysis may be a valuable, non-invasive method for the early detection of primary liver cancer.Recent advances in tumour genetics reveal that the genesis and progression of tumours follow an accumulation of multiple genetic alterations, including inactivation of tumour suppressor genes and/or activation of proto-oncogenes. Frequent loss of heterozygosity (LOH) of microsatellites markers on specific chromosomal region have been reported in various types of human cancer. In addition, nucleic acid-based molecular techniques have successfully identified the same LOH in the paired plasma/serum DNA from patients with diverse types of epithelial cancer, such as lung , head ann=20) or cirrhosis (n=7) as controls. Both peripheral blood lymphocytes and serum samples were obtained with written informed consent. Briefly, DNA was extracted from peripheral blood lymphocytes, tumour and matched serum samples by digestion with 1% SDS/proteinase K  overnight followed by phenol-chloroform extraction and ethanol precipitation. Each tumour was reviewed for histologic grading according to the Edmondson classification (1958). Clinical staging was determined according to the tumour-node-metastasis staging protocol of the American Joint Committee on Cancer (1997) with surveys of the clinical details, image studies, and pathologic data. The clinical characteristics, including AFP, hepatitis B surface antigen (HBsAg) and hepatitis C virus antibody (anti-HCV), were obtained from medical records.Primary tumour from 21 cases of HCC and six cases of CC were collected from the Department of Surgery, National Cheng Kung University Hospital, Tainan, Taiwan between September 1998 and March 2000 . The marker pairs selected for HCC  included 1p , 3p (D3S1317), 4p (D4S394), 4q , 5q (D5S409), 6q (D6S264), 8p , 8q , 9p , 10q (D10S109), 11p , 11q , 12p (D12S93), 13q , 14q , 16p , 16q , and 17p  based on previous reports  and T4 kinase   in a total of 10 μl. PCR for each primer set was performed for 35 cycles consisting of denaturation at 95°C for 30 s, annealing at 50–60°C for 1 min, and extension at 70°C for 1 min . The reaction products were separated on 7% urea- formamide-polyacrylamide gels  and exposed for 12–24 h. For informative tumours, LOH was scored if the tumour allele demonstrated a greater than 30% reduction in intensity compared to the corresponding normal control visually as previously described  positive test: presence of LOH in the primary tumour and in the matched serum DNA (true positive); (B) positive test: absence of LOH in tumour but presence of LOH in patient serum ; (C) negative test: presence of LOH in tumour but absence in paired serum samples ; and (D) negative test: absence of LOH either in tumour or in paired serum samples (true negative). Decision diagnostic criteria were obtained from the results A, B, C, and D. Test sensitivity is equal to A/A+C. Test specificity is equal to D/D+B. Test accuracy is equal to: A+D/A+B+C+D. Predictive positive result or positivity prediction is equal to A/A+B. Predictive negative result or negativity prediction is equal to D/D+C.−1 (P=0.02), and 13q with satellite lesion (P=0.04) (data not shown). There was a trend toward positive association of 8p or 13q loss with advanced tumour staging (P=0.06), and 8p loss with AFP greater than 20 ng ml−1 (P=0.06). LOH on 14q and 16q occurred more frequently in hepatitis C virus-negative patients . Otherwise, patterns of allelic loss did not relate to histological grading or tumour size . In addition, novel alleles indicative of microsatellite instability were found in three patients  at chromosome 2, 13 and 16, respectively (data not shown).All of the HCC cases showed deletion from two to 10 chromosomal arms . The higP>0.1, respectively). If the cut-off values were set at AFP 400 ng ml−1, negative serum LOH test was observed in two of 10 (20%) patients with abnormal AFP level (cases 1 and 15), and in three of 11 (27.3%) patients having AFP level within normal limits . In contrast, five of seven (71.4%) patients  having AFP less than 20 ng ml−1 had positive LOH test. The results imply that microsatellite DNA analysis may have potential as a non-invasive diagnostic test for the early detection of HCC, especially for those who having AFP below reference range. However, none of three cases showing microsatellite instability in primary tumours had the same alterations detected in the serum DNA (data not shown).As for tumour diagnosis, a total of 16 of 21 (76.2%) HCC patients were found to have LOH in serum DNA from one to four microsatellite markers . The preWith regard to cholangiocarcinoma, five of six patients belong to advanced stage of tumour . DeletioAltogether, the data of this pilot study indicate that microsatellite analysis of serum DNA could successfully detect around three-quarters (76.2%) of HCC by screening the following profile of microsatellite markers, i.e. D1S160, D1S163, D1S170, D4S1545, D5S409, D7S495, D8S200, D8S555, D9S171, D9S747, D11S1344, D13S227, D14S51, D16S289, D16S402, D16S419, D16S422, D16S498, D16S516. The positive predictive value was 100%, and negative predictive value was 80.8%.Currently, only ultrasound-guided needle biopsy yields diagnostic material and allows a definitive differential diagnosis between HCC and CC   may secrete little or no AFP into the circulation  is feasible by screening the serum samples with a profile of 19 microsatellite markers. However, a recent study reported that around 50% of the cirrhotic nodules are monoclonal and already have chromosomal aberrations (In conclusion, we have determined a profile of molecular markers appropriate for differential diagnosis of primary liver cancer in the serum. The discovery may permit a high-throughput screening of hepatocellular carcinoma at an early, and potentially resectable, stage of disease.


Tail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown.TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1.Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle. Th. Th51]. The endogenous localisation of SLMAP in COS7 cells was examined using anti-SLMAP antibodies Figure  and comp1/L   Figure . We tran) Figure . When we) Figure . We co-t) Figure . Thus it10/L+A11/L (TA2-2L)  Figure . When we) Figure . The Myc) Figure . We co-t) Figure . The resThe TA2-4L mutant has a transmembrane region that is only slightly less hydrophobic than the wild type TA1 Figure  They wer1/L+R27/L mutant which has the positively charged amino acids replaced with leucine does have an increase in hydrophobicity compared with wild type TA2 but only in the regions flanking the transmembrane domain  membrane proteins are important in several cellular processes including neurotransmitter release, vesicle transport, membrane fusion and signal transduction -8. All tThe information required for the targeting of SLMAP to intracellular membranes was not contained in specific amino acids that flank the predicted transmembrane domain but was dictated by the overall hydrophobic profile of the TA. We found that SLMAP1 could target either the ER alone or the ER and the mitochondria in Cos7 cells depending on which of the two alternative tail anchors (TA1 or TA2) were being expressed. The disruption of the cytoskeleton with nocodazole showed that the distribution patterns of SLMAP1 are dependent on an intact microtubule network. This is wholly consistent with changes in distribution noted for other ER resident proteins, and the known role of the cytoskeleton in anchoring intracellular membranes in place ,53. It iTail anchors comprise a single membrane spanning helix of 17 to 21 amino acid residues at the C-terminal end of the protein ,10. Inveet al., [The two different tail anchors in SLMAP are generated by alternative splicing and both appear to target the protein to subcellular locations. Both TA1 and TA2 target SLMAP1 to the ER. TA2 also targets SLMAP1 the mitochondria as evidenced by co-localization with Tom20. We found that this differential targeting was due to the lower hydrophobic content of the trans-membrane helix in TA2 when compared to the alternatively spliced TA1. Immunohistochemistry indicates that TA1 directs SLMAP1 predominantly to the ER and not the mitochondria probably due to the more hydrophobic nature of the transmembrane domain. We were able to prevent TA2 from targeting the mitochondria by the substitution of moderately hydrophobic amino acids with the highly hydrophobic leucine residues implying that the important targeting information in TA2 is the relative hydrophobicity of the transmembrane region. In this regard Bielharz 1/L R27/L mutants despite the hydrophobicity of the flanking regions being increased to a level above that of the TA1 isoform. Recently it has been shown that artificial TAs require a moderately hydrophobic transmembrane region to target the MOM [We found no evidence to suggest that the positively charged amino acids which flank the membrane spanning helix are responsible for the targeting of TA2 to the mitochondria per-se. This is in contrast to the view that basic amino acids flanking the TA are responsible for MOM targeting ,14,24,25 the MOM . ConsistThe presence of the two alternatively spliced exons encoding tail anchors which target SLMAP1 to different locations suggests SLMAP1 may perform multiple subcellular functions. Given the coiled-coil nature of the majority of the SLMAP protein, it may be that it tethers proteins into place on both the ER and perhaps the MOM. Alternatively, it may be involved in tethering of the two membrane systems together through homo/hetero-dimerisation. Differential levels of expression of SLMAP with the different TAs, suggest that SLMAP may serve varying subcellular roles in tissue specific manner ,30,31. IWhile the mechanism of TA protein membrane insertion remains to be defined, it appears that both the SRP  and a novel mechanism utilizing ATP appear to be involved ,57. WhicOur data here supports the view that the sub-cellular targeting of SLMAP is determined by the overall hydrophobicity of its alternative tail-anchor (TA). Further, the basic amino acid residues in the regions flanking the transmembrane helix contribute to the overall hydrophobic profile of the TA and its targeting rather then the individual residues being a targeting signal. Further more, the potential association of SLMAP with the MOM implies additional roles for this molecule in mitochondrial function.Membrane helix predictions were performed using the HMMTOP program [Hydropathy calculations were performed using the ProtScale tool  with the Eisenberg algorithm using a window size of 9 and the relative weight for the window edges was 100%. [ program ,50. Hydras 100%. .Site directed mutagenesis was performed using the Stratagene "Quikchange" kit. Template DNA concentration was 30 μg per reaction. The K1/I mutant (AAA/ATA) was created using 5' (GGA AAT AAT ATA CCC TGG CCC) and 3' (GGG CCA GGG TAT ATT ATT TCC). The R27/I mutant (AGA/ATA) was created using 5' (CCA GGT CTG GCC ATA GCT TCT CCG TG) and 3' (CA CGG AGA AGC TAT GGC CAG ACC TGG). The K1/I+R27/I mutant was created by performing SDM on the K1/I mutant using the R27/I primers (5' (CCA GGT CTG GCC ATA GCT TCT CCG TG) and 3' (CA CGG AGA AGC TAT GGC CAG ACC TGG)). The P4/L mutant (CCC/CTC) was created using 5' (ATA CCC TGG CTC TGG ATG CCC) and 3' (GGG CAT CCA GAG CCA GGG TAT). The A10/L + A11/L mutant (GCT/CTT + GCC/CTC) was created using 5' (ATG CCC ATG TTG CTT CTC CTG GTT GCG GTG) and 3' (CAC CGC AAC CAG GAG AAG CAA CAT GGG CAT). The T16/L + A17/L mutant (ACA/CTA + GCT/CTC) was created using 5' (CTG GTT GCG GTG CTA CTC ATC GTG CTG TAT) and 3' (ATA CAG CAC GAT GAG TAG CAC CGC AAC CAG). The A10/L+A11/L+T16/L+ A17/L mutant was created by SDM on the A10/L+A11/L construct using the T16/L + A17/L creation primers (5' (CTG GTT GCG GTG CTA CTC ATC GTG CTG TAT) and 3' (ATA CAG CAC GAT GAG TAG CAC CGC AAC CAG)).Since the targeting of the tail anchored proteins resides at the C-terminal regions in general we used GFP and 6Myc-tagged expression constructs of the naturally occurring SLMAP1-TA1, SLMAP1-TA2, as previously described ,31,32. SCell Culture and Immunohistochemistry. Transfections were performed as previously described [COS7 African green monkey kidney and C2C12 cells were maintained at 37°C in Dulbecco modified essential media (DMEM) supplemented with 10% heat inactivated fetal bovine serum and antibiotics. Transient transfection experiments were performed using the fugene™  transfection reagent according to the manufacturers' specifications. Disruptions of microtubules were induced in COS7 cells with 10 μM nocodazole (Sigma-Aldrich) for two hours at 37°C. Filamentous actin was disrupted in COS7 cells with 1 μM cytochalain D (Sigma-Aldrich) for 2 hours at 37°C. escribed , and visescribed . For endet al., [2PO4/K2HPO4 pH 6.5 and 5 mM MgCl2). The homogenate was centrifuged at 1500 × g for 10 minutes to remove unbroken cells, debris and nuclei. Postnuclear supernatant (PNS) was loaded onto a sucrose step gradient  and centrifuged at 100,000 × g for one hour. Fractions collected from the gradient included S1 (0.25–0.5 M interface), A1 (0.5 M layer), S2 (0.5–0.86 M interface), B1 (0.86 M layer), S3 (0.86–1.3 M interface), C1 (1.3 M layer), and pellet (P). S2 fraction was adjusted to 1.15 M sucrose, and then overlaid with 1.0 M sucrose, 0.86 M sucrose and 0.25 M sucrose. This sucrose gradient was centrifuged at 76,000 × g for 3 hours. Fractions collected included: SGF1 (0.26–0.86 M interface), SGF2 (0.86–1.0 M interface), C2 (1.0 M layer), SGF3 (1.0–1.15 M interface), and SGFL (1.15 M layer). Protein content of each fraction was determined via the BCA protein assay (Pierce). Equal amounts (10 μg) of each fraction were loaded onto a 10% SDS polyacrylamide gel. ER-Golgi protein transport assays The ts045-VSVG-GFP expression plasmid was donated by Dr. Lippincott-Schwartz [Stacked Golgi fractions (SGF) and endoplasmic reticulum fractions were isolated from rabbit liver according to the method described by Taylor et al., . All proSchwartz . The plaJB conducted the experiments on the site directed mutagenesis and the hydrophobicity predictions while RG carried out the trafficking and biochemical studies and MS analyzed sequences and made constructs. BST conceptualized the experiments and drafted the manuscript together with JB, RG and MS. All authors read and approved the final manuscript. JTB and RMG contributed equally to this work.


Previously we performed a linkage scan of 638 African American affected sibling pairs (ASP) with type 2 diabetes (T2D) enriched for end-stage renal disease (ESRD). Ordered subset linkage analysis (OSA) revealed a linkage peak on chromosome 7p in the subset of families with earlier age of T2D diagnosis.GCK1, IL6, IGFBP1 and IGFBP3) for association with age of T2D diagnosis, age of ESRD diagnosis, duration of T2D to onset of ESRD, body mass index (BMI) in African American cases and T2D-ESRD in an African American case-control cohort. OSA of fine mapping markers supported linkage at 28 cM on 7p (near D7S3051) in early-onset T2D families . SNPs in candidate genes and 70 ancestry-informative markers (AIMs) were evaluated in 577 African American T2D-ESRD cases and 596 African American controls.We fine mapped this region by genotyping 11 additional polymorphic markers in the same ASP and investigated a total of 68 single nucleotide polymorphisms (SNPs) in functional candidate genes (GCK1 gene (recessive T2D age-adjusted P = 0.0006). Nominal associations were observed with GCK1 SNPs and T2D age of diagnosis (BMI-adjusted P = 0.014 to 0.032). Also, one IGFBP1 and four IGFBP3 SNPs showed nominal genotypic association with T2D-ESRD (P = 0.002-0.049). After correcting for multiple tests, only rs730497 remanined significant.The most significant association was observed between ESRD age of diagnosis and SNP rs730497, located in intron 1 of the GCK1 gene appears to play a role in early ESRD onset in African Americans.Variant rs730947 in the  A genome wide linkage scan was performed on 638 African American affected sibling pairs (ASPs) with type 2 diabetes (T2D) from 247 families; 166 families contained at least one ASP concordant for diabetic end-stage renal disease (T2D-ESRD) [GCK1), interleukin-6 (IL6), insulin growth factor binding protein 1 (IGFBP1) and insulin growth factor binding protein 3 (IGFBP3).Fine mapping of this region using the same African American families was undertaken in order to localize the peak and to determine the primary phenotype of association. There are several plausible diabetes and nephropathy candidate genes in the resulting region of interest, including glucokinase isoform 1 (GCK have been identified in subjects with maturity-onset diabetes of the Young (MODY) [n repeat element located approximately 10 kb 3' to the coding region has also shown association with T2D across several diverse ethnic populations [Mutations of g (MODY) ,4. Howevg (MODY) -7. A dinulations -10.IL6 promoter region have been shown to affect promoter strength [The cytokine interleukin 6 (IL-6) is an essential regulator of the acute phase response associated with T2D and diabetic nephropathy ,12. Varistrength .IGFBP3 was associated with levels of HbA1c [in vivo and in vitro studies have demonstrated that IGFBP-3 is a potent insulin antagonist [The insulin like growth factors 1 and 2 circulate tightly bound to IGFBP-1 and IGFBP-3. A cross-sectional study of T2D cases and controls found that polymorphisms in of HbA1c . Also, itagonist .We fine mapped the region of interest using additional microsatellite markers, and investigated associations with SNPs in four plausible candidate genes with age of T2D diagnosis, age of ESRD diagnosis, duration of T2D to onset of ESRD, BMI and T2D-ESRD in an African American cohort.This study was conducted under Institutional Review Board approval from Wake Forest University School of Medicine and adhered to the tenets of the Declaration of Helsinki. All participants provided written informed consent. Clinical characteristics and recruitment of African American patients have been described previously ,2. DNA sIdentification, clinical characteristics, and recruitment of African Americans and European American patients and controls have been described previously . BrieflyFor admixture analyses, 39 unrelated European American controls were recruited using the same criteria as African American controls. DNA was also obtained from 44 YRI from the National Institute of General Medical Sciences Human Variation Collection .http://research.marshfieldclinic.org/genetics/ and the Genome Database http://www.gdb.org/gdb/. The polymerase chain reaction (PCR) was used to incorporate fluorescently labeled primers from Integrated DNA Technologies, Inc.  and Applied Biosystems  into PCR products, which were then electrophoresed on an ABI PRISM 3700 DNA Analyzer and results analyzed with Genescan Analysis Software . Electropherograms were imported into GeneMapper  for binning and allele calling. Familial relationships and Mendelian inconsistencies were checked using Prest [Eleven fine mapping microsatellites at an average spacing of 4 cM were selected from the Marshfield Center of Medical Genetics database ng Prest  and PedCng Prest . InconsiP value (ΔP).A series of OSA  were comIL6, GCK1, IGFBP1 and IGFBP3) that lie under the intersection of the optimal linkage peaks for early age of T2D, lower BMI and longer duration to ESRD was selected ; interleukin-6 ; insulin growth factor binding protein 1  and insulin growth factor binding protein 3 .Plausible functional candidate genes . Six SNPs were genotyped using iPlex methodology on a MassARRAY system  : IL6 rs1Seventy biallelic Admixture Informative Markers (AIMs), selected to maximize European and African allele frequency differences and sample all non-acrocentric arms of the autosomal genome, were genotyped by Illumina Inc.'s Custom Genotyping Service  in 577 African American cases, 596 African American controls, 44 YRI and 39 European Americans. Primer sequences are available on request. Genotyping success rates for AIMS were >97.4% in African American cases and >95.6% in African American controls.2 tests for categorical variables, and one-way ANOVA test for continuous variables. Hardy-Weinberg equilibrium (HWE) was assessed using the χ2 goodness-of-fit statistic at a significance level of ≤ 0.01. Haplotype block structure was established using Haploview 3.2 [et al. [Characteristics were compared and tested for significant differences using χview 3.2 , using t [et al. .a priori genetic tests of association  are reported. Due to a lack of validity of the large sample χ2 test statistic, only the dominant model was considered for SNPs with ten or fewer individuals that were homozygous for the minor allele.Tests of association under the three Quantitative trait  association analysis for all SNPs was performed using a series of analysis of variance (ANOVA) tests implemented in QSNPGWA .a priori genotypic models, and conducted using Stata 10 .SNPs that showed nominal evidence for association with age of T2D onset were adjusted by BMI. Additionally, ESRD age, duration of T2D to ESRD onset and BMI were adjusted for age of T2D onset. All analyses were adjusted using linear regression on Unadjusted odds ratios (ORs) and 95% confidence intervals (CI) in the case-control population were computed using SNPGWA to test all SNPs for genotypic association with T2D-ESRD . SNPs thIndividual ancestral proportions were calculated using an expectation-maximization (EM) algorithm (FRAPPE)  under a PS power and sample size program specifying a two-sided test at an alpha level of ≤ 0.05, to detect an OR of 1.30 was utilized to approximate study power .P ≤ 0.05 level. To correct for multiple tests at the gene level the conservative Bonferonni method was used, with a P-value ≤ 0.0007 considered significant evidence for association when assuming independence based on LD.We report nominally significant associations at the P = 0.002) with a mean age at diagnosis 29.5 ± 3.0 years vs. mean age at diagnosis of the unlinked families 44.5 ± 7.3. Although the max. LOD score dropped  the LOD-1 interval increased from 11.5-38.2 cM to 11.5-49.5 cM. There was no significant difference (P = 0.56) between mean BMI in the linked early-onset families (31.5 kg/m2 ± 7.62) versus the unlinked (31.8 kg/m2 ± 7.51) pedigrees. The evidence for linkage in subsets of T2D-ESRD families with lower BMI  and longer duration of diabetes before onset of ESRD  was no longer significant .Characteristics of the study population used for tests of association with positional candidate genes are shown in Table P ≤ 0.01) were observed for IL6 rs1474347 in controls (P = 0.002) and IGFBP1 rs1496495 in cases (P = 0.001). Although inconsistent with HWE proportions, these SNPs were retained for exploratory analyses, however there was no evidence for association of these SNPs with any of the traits investigated.All SNPs in candidate genes were examined for departures from HWE assumptions in cases and controls separately. Deviations from HWE  and the 11 genotyped IGFBP3 SNPs fell within two blocks of LD (data not shown).The 24 GCK1 SNPs nominally associated with T2D age at diagnosis and five GCK1 SNPs associated with age at ESRD onset . The homozygote recessive genotype ("AA") for IL6 SNP rs2069849, located in exon 5, was nominally associated with age at ESRD onset (T2D age-adjusted P = 0.013). SNPs in IGFBP1 and IGFBP3 were not associated with T2D age at diagnosis or ESRD onset  with shorter duration of T2D to ESRD onset . Results are further summarized in Table Association results for duration of T2D to ESRD onset are presented in Additional file IGFBP1 and IGFBP3 SNPs were not associated when adjusted for age of T2D diagnosis . Single SNP association results for BMI are presented thoroughly in Additional file Association results for BMI are shown in Table P ≤ 0.05) for T2D-ESRD are shown in Additional file IGFBP3: two promoter SNPs: rs903889 , rs924140  and two SNPs located in intron 3; rs10255707  and rs3110697 . Nominal evidence of association was also detected with IGFBP1 rs9658233 located in the 3' UTR region. GCK1 and IL6 SNPs showed no evidence of association with T2D-ESRD  in non-diabetic European Americans from the Women's Genome Health Study (WGHS) and participants from the Boston metropolitan area. The MAF of the rs730497 A "risk"allele was similar in African American cases (0.20) and previously reported European Americans from WGHS (0.17) [1c and nephropathy [A" allele and ESRD progression is unknown, however, marker rs730497 is located 4.8 kb from exon 1 and 30.7 kb from exon 2. The slightly more common G allele appears to be ancestral since it is present in the chimpanzee sequence.Recently, Paré et al.  reportedS (0.17) . Severalhropathy . The relGCK1 was also observed with T2D age at diagnosis (Table GCK1 SNPs were found to be associated with T2D risk (recessive model: OR ranging from 1.36-1.87) in a case-control population from Finland [et al. [GCK1 SNPs genotyped captured all 32 HapMap GCK1 SNPs with MAF > 5% with r2 values > 0.80.Nominal associations with MODY gene  Finland . Bonnyca [et al.  observed [et al.  and the  [et al. , and Sou [et al. , were noIL6 SNP rs2069849, located in exon 5, was nominally associated with earlier age of ESRD onset. This variant causes a synonymous coding change for phenylalanine at position 201 (F201), which may alter mRNA stability and translation. Promoter polymorphisms rs1800795 and rs1800796 have shown association with T2D and diabetic nephropathy respectively [ectively -42. We iectively , did notIGFBP1 (rs9658233) and four IGFBP3 SNPs . The major "T" allele for SNP rs9658233, located in the downstream region of IGFBP1, appears to confer risk with T2D-ESRD, with OR between 1.25-1.35  of the African American controls (mean 93.6 mg/dl). Five of these individuals had glucose levels greater than 126 mg/dl. Four of these measures were non-fasting and the fifth had unknown dietary status, suggesting the overall misclassification rate of controls is likely to be below 2%. While this has not impacted our ability to detect an association, this may have reduced our power to detect more subtle influences of additional variants, and underestimate effect sizes.IGFBP1 rs9658233 and IGFBP3  remained nominally significant. Multiple comparisons were corrected by conservative Bonferroni method (P ≤ 0.00005) however, when assuming independence based on LD, one would consider a P-value ≤ 0.0007 as evidence for association, only SNP rs730497 remained significant (T2D age-adjusted P = 0.0006).While adjustments for admixture reduced the strength for the observed associations with T2D-ESRD  [IGF2BP3), warrant investigation.We have confirmed linkage to early-onset T2D on chromosome 7p, and examined common variants across positional candidate genes  (GRB10) ,49 and IGCK1 play a role with ESRD risk. Further studies in other populations and functional studies may be warranted.Our findings support that intron 1 of the The authors declare that they have no competing interests.TSL carried out the genotyping of additional polymorphic markers, prepared linkage data for analysis, performed association analysis, and drafted the manuscript. CDL developed software programs SNPGWA and QSNPGWA, and directed LL in conducting linkage analyses. KLK performed the admixture analysis. JCM assisted with statistical analysis. CJG assisted in preparing linkage data for analysis. SSR, BIF and DWB participated in the design of the study and participant recruitment. MMS conceived of the study, participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/22/prepubSupplementary tables. Supplemental tables S1-S6.Click here for file


Functional polymorphisms within the CYP1B1 genotyping was performed on blood samples of 60 CRPC patients treated with docetaxel, using TaqMan probes-based assays. Association between CYP1B1-142C>G (leading to the 48ArgGly transition), 4326C>G , and 4390A>G (453AsnSer) polymorphisms and treatment response, progression-free-survival (PFS) and overall-survival (OS) was estimated using Pearson χ2 test, Kaplan-Meier curves and Log-rank test.CYP1B1-432ValVal genotype experienced a significantly lower response-rate (P = 0.014), shorter progression-free-survival (P = 0.032) and overall-survival (P < 0.001). Multivariate analyses and correction for multiple comparisons confirmed its prognostic significance for OS. No significant associations were found among other polymorphisms and both response and clinical outcome.Patients carrying the CYP1B1-4326C>G  polymorphism emerged as possible predictive marker of response and clinical outcome to docetaxel in CRPC patients and may represent a potential new tool for treatment optimization. Larger prospective trials are warranted to validate these findings, which might be applied to the future practice of CRPC treatment. Prostate cancer is the most common malignancy in men and the second leading cause of cancer death among males in the Western World . ApproxiTwo multicenter phase III randomized clinical trials, TAX 327 and SWOG 9916, showed a survival advantage in Castration-Resistant Prostate Cancer (CRPC) patients treated with taxane-based chemotherapy ,5. In boDespite the relative success in the treatment of these patients, high variability in the clinical response to docetaxel has been observed . This vaThere is evidence from preclinical and clinical studies that the cellular response to docetaxel is linked to expression and/or activity of cytochrome-P450 1B1 (CYP1B1). Although docetaxel is not directly metabolized by CYP1B1 ,8, CYP1BCYP1B1 gene expression [CYP1B1 activity is regulated by several functional non-synonymous single-nucleotide-polymorphisms (SNPs). The 4326C>G SNP (rs1056836), leading to the 432LeuVal (432LV) amino-acid transition, is associated with increased catalytic activity of CYP1B1 ,13, whilpression ,14, withpression .CYP1B1*3-allele) has been associated with enhanced synthesis of 4-OHE2, increasing the intracellular ratio with the minor CYP1B1 metabolite 2-OHE2 [CYP1B1*3 had a significantly shorter OS after docetaxel-based therapies [Interestingly, the homozygous variant of the 4326C>G SNP . Patients of age > 18, performance status 0-3  with histologically confirmed diagnosis of prostate cancer, were eligible for the study. Other eligibility criteria were hormone refractory disease, ANC-count ≥ 1.5 × 102 on day-1 every 21 days or docetaxel 30 mg/m2 weekly for five of every six weeks plus prednisone 10 mg os daily.All patients received docetaxel given intravenously over 1-hour at a dose of 75 mg/mThe primary endpoint of this analysis was the correlation of the candidate polymorphisms with antitumour response as determined by the effect of treatment on PSA concentration, whereas secondary endpoints included correlation with progression-free-survival (PFS) and overall survival (OS). Serum PSA and physical examination were evaluated every three weeks while CT imaging and bone scan were performed at baseline and every three cycles. A response was defined as a reduction from baselines of at least 50% that was maintained for at least three weeks, whereas PSA progression was defined as an increase from the nadir of either at least 25% for men with no PSA response or at least 50% for all others. Stable disease was defined as an increase from the nadir less than 25% for men with no PSA response or less than 50% for all others. PFS was defined from the date of randomization to the date of PSA progression as an increase ≥ 25% in PSA level from baseline or ≥ 50% from the lowest value achieved; the PSA increase should be at least 5 ng/ml confirmed by three measurements at 3 week intervals. Other criteria of progressive disease were the appearance of a new lesion or a ≥ 25% increase using standard bidimensional measurements, in accordance with WHO guidelines, of previously measured disease.Genomic DNA was extracted from blood samples (5 ml) drawn before drug administration, using the QIAmp-DNA-mini-Kit . DNA yields and integrity were checked by optical density at 260 nm, while testing for contamination was performed by measuring absorbance at 280 nm and calculating the 260/280 ratio.CYP1B1 polymorphisms at 48ArgGly, 432LeuVal and 453AsnSer were studied with Taqman®-probes-based assays using the ABIPRISM-7900HT instrument equipped with the Sequence-Detection-System version 2.0 software . Forward (F) and reverse (R) primers and probes (P) were obtained from Assay-on-Design-SNPs products, using the File-Builder software version 2.0 (Applied Biosystems), i.e. for 142C>G: (F) 5'-GCT GCT GAG GCA ACG GA-3'; (R) 5'-CAG TGG CCA CGC AAA CG-3'; (VIC-P) 5'-CAG CTC CGG TCC GC-3' (FAM-P); 5'-AGC TCG GGT CCG C-3' for 4326C>G: (F) 5'-CAG CTC GAT TCT TGG ACA AGG A-3'; (R) 5'-TGC CCA CTG AAA AAA TCA TCA CTC T-3'; (VIC-P) 5'-CCT CAT CAA CAA GGA C-3'; (FAM-P) 5'-CTC ATC AGC AAG GAC-3'; and for 4390A>G: (F) 5'- TTT GTC AAC CAG TGG TCT GTG AAT-3'; (R) 5'-GGA TCA AAG TTC TCC GGG TTA GG-3'; (VIC-P) 5'-CAT GAC CCA CTG AAG TG-3'; (FAM-P) 5'-ATG ACC CAG TGA AGT G-3'.®, and 0.625 μl of the assay mix (F and R primers and the specific probes), in a total volume of 25 μl. After thermal cycling , the instrument determined the allelic content of each sample by reading the generated fluorescence, as described [PCR reactions were performed using 20 ng of DNA diluted in 11.875 μl DNAse/RNAse-free water, 12.5 μl of TaqMan Universal-PCR-Master-Mix, with AmpliTaq-Goldescribed .CYP1B1 SNPs with response to docetaxel, while the secondary endpoints included the correlation with PFS, and OS.This study was aimed at evaluating the correlation of selected 2 test. Analyses included information on genotypes, ECOG-performance status , age (≤ median vs. > median), pre-treatment PSA (≤ median vs. > median), visceral metastatic disease (yes vs. no), anaemia , and schedule (3 week vs. weekly administration). PFS was calculated from the start of chemotherapy to the date of clinical and/or biochemical and/or radiological evidence of progression or death, whichever occurred first. OS was calculated from the day of treatment start to the endpoint (death or last follow-up). The Kaplan-Meier method was used to plot PFS and OS and the Log-rank test to compare curves. Known baseline prognostic variables were also included in multivariate analyses, using Cox's proportional hazards model to identify factors of independent significance. A step-down procedure was used based on the likelihood ratio test, and hazard ratios were calculated to estimate the magnitude and the direction of the effect.Demographic and clinical information on response were compared across genotype, using Pearson-χSPSS/PC+17 software . Statistical significance was set at P < 0.05. However, in the univariate analyses for clinical outcome according to the 3 studied polymorphisms, a Bonferroni correction required a P < 0.05/3 = 0.016 for statistical significance.Data were analyzed using A total of 60 consecutive patients were enrolled in this study. Clinical characteristics are listed in Table CYP1B1 SNPs. For the 4326C>G CYP1B1, the frequencies of CC, GC and GG genotypes were 20%, 42% and 38%, respectively. The wild-type CYP1B1 4390AA had a frequency of 62%, while the heterozygous 4390AG and homozygous 4390GG variants had a frequency of 30% and 8%, respectively. Regarding CYP1B1 142CG, the CC variant was found in 13% of cases, while the heterozygous CG and the GG variants were observed in 35% and 52% of cases, respectively.All the samples were evaluable for SNP- analyzer-software . Genotype frequencies of 4326C>G allele were comparable  with those reported in a study in a Caucasian population of prostate cancer patients [All polymorphisms followed Hardy-Weinberg's equilibrium as calculated with the patients , while tpatients ,8. PatieThe overall response rate (CR+PR = RR) of the patients was 48.3%. Age, ECOG-PS, docetaxel-schedule, pre-treatment PSA and visceral metastasis were not associated with response , whereas anaemia was significantly associated with response  have Hb ≥ 13.0 g/dl, P = 0.004).None of the clinical characteristics was associated with PFS, while the occurrence of visceral metastasis and anaemia were associated with significantly shorter OS. In particular, median OS was 15.8  months in patients with Hb < 13.0 g/dl vs. 28.3  months in patients with Hb ≥ 13.0 g/dl (P = 0.006). Similarly, patients with visceral metastasis had a median OS of 13.9  months, while patients without visceral metastasis had a median OS of 23.2  months (P = 0.001).Finally, responding patients had a significantly longer OS  vs. 15.4  months, P < 0.001) and PFS  vs. 4.1  months, P = 0.009) than patients with SD or PD.CYP1B1 polymorphisms at position 4390 and 142 .When grouping patients between those with response (CR and PR) and without response (SD and PD), and between those with genotype leading to increased CYP1B1 activity or expression (4326GG or 142GG) and with genotype leading to reduced CYP1B1 expression (4390AG or 4390GG), no significant correlations with response were observed for the CYP1B1 4326GG and 412GG genotype (N = 9) also experienced a significant lower rate of response (P = 0.039), with respect to patients (N = 51) harbouring the other genotypes. Indeed, 8 out of 9 (88.9%) patients with the CYP1B1 4326GG and G412GG genotype experienced PD or SD, while 28 out of 51 (54.9%) patients harbouring the other genotypes achieved CR or PR. Furthermore, the patients carrying both the CYP1B1 4326GG and the 4390AG or 4390GG genotype (N = 13) experienced a significant lower prevalence of response with respect to patients (N = 47) harbouring the other genotypes (P = 0.003). Indeed, 12 out of 13 patients with the CYP1B1 4326GG and the 4390AG or 4390GG genotype experienced PD or SD, whereas 28 out of the 47 patients carrying the other genotyes achieved CR or PR. In contrast, no differences were observed grouping patients with the CYP1B1 4390AG or 4390GG and the 412GG genotypePatients harbouring both the CYP1B1 4326GG genotype was associated with shorter PFS and OS . The median age of the 23 patients harbouring the CYP1B1 4326GG was 69 years (range = 58-80). Nine (39%) of these patients had visceral metastasis, while 19 (83%) had anaemia. No significant correlations were observed between OS and PFS in patients grouped according to the other studied polymorphisms , while no correlations were observed with PFS.However, the  OS Fig. . In part OS Fig. . In ordeCYP1B1 4326C>G polymorphism). In this model, limited by the small sample size, we found that response and visceral metastasis were always associated with OS, while pre-treatment PSA and anaemia were not independent predictors of OS (Table CYP1B1 4326GG genotype resulted a significant predictor of OS (P = 0.003). In a second multivariate we also evaluated the grouped CYP1B1 4326GG and 412GG as well as the grouped CYP1B1 4326GG and 4390AG or 4390GG genotypes, and both these grouped genotypes showed a significant association with OS .To evaluate the risk of death we carried out a Cox regression analysis entering known prognostic baseline factors  and the polymorphisms significantly associated with OS in the univariate model  patients, none of whom had an objective response to paclitaxel [A major mechanism of resistance to taxanes may be represented by the mutation/overexpression of tubulin. In particular, clitaxel , but we MDR1 mRNA expression in leukocytes than subjects with the TT genotype [Other studies have evaluated polymorphisms of the multidrug resistance (MDR1) gene, whose product P-glycoprotein functions as an ATP-dependent exporter of several drugs, including paclitaxel and docetaxel, from cells. Subjects homozygous for C in position 3435 (3435C>T) had higher genotype . This SNgenotype ,23.MDR1 linked alleles had significantly improved overall survival, but no data were available on response [In CRPC patients receiving docetaxel alone, individuals carrying a diplotype consisting of the 1236C-2677G-3435C response .Assessing germline genetic polymorphisms as either predictive or prognostic markers is very appealing, especially in the CRPC patients. In these patients primary tumours, as well as bone metastasis, are not resected so that the avaibility of tumour material can be problematic. SNPs are inherited genetic variants harboured by all the cells and their analysis can be easily performed in blood and is easier to adopt in the routine clinical setting than tumour expression arrays, which need biopsies of patient's tumours, laser-microdissection and sophisticated infrastructure.CYP1B1, which play an important role in cancer risk and progression as well as in the metabolism of cancers modulated by sex hormones. In particular, estrogen exposure has been implicated in the disease aetiology of prostate cancer, and CYP1B1 is up-regulated in prostate cancer [CYP1B1 polymorphisms may be involved in cancer risk, alone or in combination with other factors [Therefore, in the present study we evaluated functional polymorphisms of e cancer . Previou factors . In part factors , where tCYP1B1 polymorphisms in order to establish their possible relationships with clinical outcome in CRPC patients treated with docetaxel.However, this study focused on the analysis of CYP1B1 4326C>G polymorphism. Patients carrying the CYP1B1 4326CC or the 4326CG genotype experienced positive clinical response in a significantly higher rate than CYP1B1 4326GG patients.The primary end-point of this study was the correlation with response and significant response differences were observed according to CYP1B1 4326GG and the 412GG polymorphisms [CYP1B1 4326GG and 412GG genotype. These patients experienced a significantly lower rate of response, with respect to patients harbouring the other genotypes.Since previous studies reported a linkage-disequilibrium between the CYP1B1 4326GG and the 4390AG or 4390GG genotype also experienced a significantly lower response rate with respect to all the patients harbouring the other genotypes. However, the patients carrying the CYP1B1 412GG and the 4390AG or the 4390GG genotype had similar response rate with respect to patients harbouring the other genotypes, suggesting that the 4326GG genotype might be the best pharmacogenetic marker of lower prevalence of response to docetaxel in CRPC patients.Of note, the patients carrying both the CYP1B1 4326GG genotype was associated with significantly shorter PFS and OS, whereas no correlations were observed between both the grouped CYP1B1 4326GG and 412GG and the grouped CYP1B1 4326GG and 4390AG or 4390GG genotypes with PFS.Furthermore, the CYP1B1 C4326C genotype remained an independent predictive parameter of death risk.Given the small number (N = 60) of patients enrolled in the study, in order to evaluate whether other prognostic factors could potentially explain their short survival, we performed corrections for multiple comparison and checked carefully several known baseline demographic, pathological and biochemical characteristics that predicted PSA decline and survival in previous studies in CRPC -29. The CYP1B1 4326GG genotype and the observed clinical outcome to docetaxel are not entirely clear. In view of previous studies demonstrating no correlation between docetaxel pharmacokinetics and CYP1B1 genotype [in vitro studies indicating that docetaxel is not directly metabolized by CYP1B1 [The underlying molecular mechanisms for the association between genotype , as welly CYP1B1 , this asy CYP1B1 ,32, and y CYP1B1 , showingy CYP1B1 .CYP1B1 polymorphism in a recent study in ovarian cancer patients treated with carboplatin and taxane regimens in the Scottish Randomised Trial in Ovarian Cancer phase III trial [However in the study by Sissung et al.  no assocII trial . These cCYP1B1-4326GG polymorphism, while its predictive role in docetaxel resistance should be evaluated in a prospective randomized study including a non-docetaxel containing arm.Given the high variant allele frequencies of these polymorphisms, the statistical power to detect associations with these SNPs and response is greatly improved. Furthermore, we used very conservative statistical methods and correction for multiple comparisons to evaluate these relationships and reduce the chances of spurious findings. However, as most previous studies in this cancer setting, the present research is limited by the small sample and residual confounding could have resulted in our findings. Larger prospective studies are needed to validate these preliminary results, after adjustment for known prognostic factors. Furthermore, our data can only suggest a prognostic role for the CYP1B1-4236GG polymorphism is linked to the clinical response to docetaxel, and may represent a potential new tool for treatment optimization. These results should prompt to perform multicenter prospective clinical trials, to validate CYP1B1 polymorphisms as possible biomarkers of docetaxel activity in CRPC patients.The data from the present investigational study provide the first evidence that the The authors declare that they have no competing interests.IP and EG participated in the design of the study, performed the experiments and the statistical analysis and drafted the manuscript. AC and FC participated in the analysis of the clinical and experimental data. CO participated in the acquisition and statistical analysis of clinical data. CC and SR were involved in the enrollement of the patients. RD, TSM, DKP, SR and WF participated in the conception, design, and coordination of the study. IP, EG, AC and TSM revised the manuscript critically. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/511/prepub


These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic.The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007–2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes . The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity  Most of the currently circulating pandemic H1N1/2009 (“swine”) influenza viruses are susceptible to the anti-influenza drug oseltamivir. Many countries have stockpiled oseltamivir for pandemic preparedness, and to date only a small proportion of the H1N1/2009 viruses isolated have been oseltamivir-resistant. However, if these viruses can be readily transmitted, oseltamivir resistance may spread. We evaluated the transmissibility of a pair of pandemic H1N1/2009 influenza viruses in ferrets. One virus was oseltamivir-sensitive and the other carried the oseltamivir resistance-associated H275Y NA mutation. We also investigated the viruses' susceptibility to NA inhibitors (the drug class to which oseltamivir belongs), their NA enzyme kinetics, and their replication efficiency in cultured cells. Under identical conditions, the resistant H1N1/2009 virus was not transmitted by respiratory droplets but was efficiently transmitted by direct contact, while the sensitive H1N1/2009 virus was efficiently transmitted by both routes. A novel swine-origin H1N1 influenza virus emerged in Mexico in April 2009 and rapidly spread worldwide, causing the first influenza pandemic of the 21st century The NA-inhibitor resistance-associated mutations in influenza viruses are drug-specific and NA subtype-specific During the H1N1/2009 influenza pandemic, to date, almost all tested viruses have remained susceptible to oseltamivir and zanamivir in vitro and in a ferret model a pair of pandemic H1N1/2009 clinical isolates. The pandemic A/Denmark/524/09 (A/DM/524/09) and A/Denmark/528/09 (A/DM/528/09) viruses were isolated from a small cluster of patients with H1N1/2009 virus infection The increasing concern about oseltamivir-resistant H1N1/2009 viruses prompted us to evaluate transmissibility and growth fitness of one oseltamivir-resistant variant. The infectivity and transmissibility  of several NA inhibitor-resistant influenza viruses have previously been studied in experimental animal models Sequence analysis of the NA genes revealed that A/DM/524/09 virus encoded a conserved H residue at amino acid position 275, whereas A/DM/528/09 virus had an H275Y amino acid mutation caused by a single T-to-C nucleotide substitution at codon 275 . Pairwis50: 5.0 nM), but the A/DM/528/09 carrying the H275Y NA mutation had IC50 values approximately 200 times that of the wild-type virus (50 of zanamivir was comparable for both viruses and was uniformly low (mean IC50≤1.3 nM)  and zanamivir. The wild-type A/DM/524/09 virus was susceptible to oseltamivir carboxylate (mean ICpe virus . The IC5≤1.3 nM) . These rTo understand the impact of the H275Y mutation on the NA enzymatic properties of the H1N1/2009 viruses, we determined the NA enzyme kinetics of both viruses. Km is an estimate of the dissociation equilibrium for substrate binding to enzyme and the reciprocal of Km approximates the affinity of substrate binding, while Vmax reflects the enzyme's catalytic activity. The NA of resistant A/DM/528/09 virus had a slightly higher Km and lower Vmax than the NA of the sensitive A/DM/524/09 virus . The H27in vitro, we characterized virus plaque morphology and growth kinetics in both MDCK and MDCK-SIAT1 cells. The latter have increased surface expression of human-like α2,6-linked terminal sialic acids To determine whether the H275Y NA mutation affects virus growth in vitro, we performed single- and multiple-cycle growth studies of both viruses in MDCK and MDCK-SIAT1 cells. In single-cycle growth in the two cell lines, the two viruses reached comparable levels 6 hours post-infection, but the initial growth of the oseltamivir-resistant virus was significantly delayed in comparison to its sensitive counterpart (P<0.05) (10TCID50/ml lower (P<0.05). Likewise, in multiple-cycle growth, the two viruses reached comparable yields 24 hours post-infection, but the resistant virus showed a significant growth delay during the first 12 hours post-infection (P<0.05); this delay was more conspicuous in MDCK-SIAT1 cells than in MDCK cells  : at 4 hoCK cells , probablThe transmissibility of pandemic H1N1/2009 viruses was studied in a ferret model. Two naïve ferrets were housed at day 2 post-inoculation (p.i.) in the same cage with one inoculated ferret (direct contact), and two naïve ferrets were placed in an adjacent cage separated from the donor's cage by two layers of wire mesh (respiratory droplet exposure). Transmission of H1N1 virus was assessed by detection of infection in recipient ferrets . Virus samples in nasal washes at day 4 p.i. or post-contact (p.c.) were sequenced to detect the presence of the H275Y NA mutation.The donor ferret inoculated with oseltamivir-sensitive A/DM/524/09 virus shed virus until day 6 p.i. , Table 2The donor ferret inoculated with oseltamivir-resistant A/DM/528/09 virus shed virus until day 8 p.i. , with a We verified the sequence stability of the NA at position 275 in each virus after replication and transmission in ferrets. Direct sequencing of the NA genes from nasal wash samples revealed no sequence change at this position in either virus (data not shown). Therefore, no spontaneous H275Y NA mutation emerged in the wild-type virus and the H275Y mutation remained stable in the mutant after transmission to a new host.Because both the oseltamivir-sensitive and the oseltamivir-resistant H1N1/2009 viruses were efficiently transmitted by direct contact, hosts could potentially be exposed to both types of virus. To compare the relative growth capability and transmissibility of the sensitive and resistant H1N1/2009 viruses within the host, we co-inoculated a ferret with a 1∶1 ratio of the sensitive A/DM/524/09 and resistant A/DM/528/09 viruses. The pattern of virus shedding and the clinical signs were similar to those in ferrets inoculated with either A/DM/524/09 or A/DM/528/09 virus . By usinin vitro may at least in part explain its impaired transmission among ferrets.This study is the first, to our knowledge, to demonstrate the inefficient respiratory droplet transmission of an oseltamivir-resistant H275Y mutant of H1N1/2009 in ferrets, which are an established animal model of the pathogenesis and transmission of human influenza viruses. The oseltamivir-resistant mutant virus retained efficient transmission only by direct contact, whereas the oseltamivir-sensitive pandemic virus was efficiently transmitted by both routes. These results show that the transmissibility of the oseltamivir-resistant H1N1/2009 influenza virus had been altered. We suggest that the lower fitness of oseltamivir-resistant variant within the host along with its reduced NA enzyme efficiency and delayed growth of the H275Y mutant virus in vitro and in vivoThere are limited experimental data about the routes of transmission of oseltamivir-resistant influenza viruses. The two natural routes of influenza virus transmission, direct contact with fomites and respiratory droplets , were approved by the St. Jude Animal Care and Use Committee, and complied with the policies of the National Institutes of Health and the Animal Welfare Act.2. All strains of virus underwent a limited number of passages in MDCK cells to maintain their original properties. MDCK cells transfected with cDNA encoding human 2,6-sialyltransferase (MDCK-SIAT1 cells) were maintained as described previously A/Denmark/524/09 (H1N1) influenza virus (A/DM/524/09) and an oseltamivir-resistant A/Denmark/528/09 (H1N1) virus (A/DM/528/09) were provided by Statens Serum Institute, Copenhagen, Denmark. The resistant virus was isolated from the tthroat swab of a patient who had influenza-like symptoms and received post-exposure oseltamivir prophylaxis (75 mg once daily) 3R, 4R, 5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]-1-cyclohexene-1-carboxylic acid) and zanamivir (4-guanidino-Neu5Ac2en) were provided by Hoffmann-La Roche, Ltd. . The compounds were dissolved in distilled water and aliquots were stored at −20°C until the time of use.The NA inhibitors oseltamivir carboxylate ([50) was determined in MDCK cells. The cells were infected with serial log dilutions of the stock viruses, incubated for 1 h at 37°C, washed, and overlaid with infection medium (MEM with 0.3% BSA and 1 µg/ml TPCK-trypsin). Infection of cells was determined by hemagglutination assay (HI) after incubation for 3 d at 37°C, and TCID50 was calculated by the Reed-Muench method The 50% tissue culture infectious dose (TCIDSingle-step growth curves were generated for influenza viruses in MDCK cells or MDCK-SIAT1 cells. Confluent cell monolayers were infected with viruses at a multiplicity of infection (MOI) of ∼2.0 PFU/cell. After incubation, the cells were washed with 0.9% aqueous NaCl solution (pH 2.2) to remove free infectious virus particles and then were washed twice with phosphate-buffered saline (PBS) to adjust the pH. Supernatants were collected 2, 4, 6, 8, 10 and 12 h p.i. and stored at −70°C for titration. To generate multi-step growth curves, MDCK cells or MDCK-SIAT1 cells were infected with viruses at a MOI of 0.001 PFU/cell. Supernatants were collected 12, 24, 36, 48, 60 and 72 h p.i. and stored at −70°C for titration in the same cell line.Confluent MDCK or MDCK-SIAT cells were incubated for 1 h at 37°C with 10-fold serial dilutions of virus in 1 ml of infection medium. The cells were then washed and overlaid with freshly prepared MEM containing 0.3% BSA, 0.9% bacto-agar, and 1 µg/ml TPCK trypsin. The plaques were visualized after incubation at 37°C for 3 d by staining with 0.1% crystal violet solution containing 10% formaldehyde.50) was determined by plotting the percent inhibition of NA activity as a function of compound concentration calculated in the GraphPad Prism 4 software from the inhibitor-response curve. The NA inhibitor–sensitive A/Fukui/20/04 (H3N2) influenza virus was included in every plate for comparison.A modified fluorometric assay using the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) (Sigma-Aldrich) was used to determine viral NA activity 6.0 PFU/ml. We measured NA enzyme kinetics at pH 6.5 with 33 mM 2-(N-Morpholino) ethanesulfonic acid hydrate , 4 mM CaCl2, and MUNANA with a final substrate concentration of 0 to 400 µM. The reaction was conducted at 37°C in a total volume of 50 µl, and the fluorescence of released 4-methylumbelliferone was measured every 60 sec for 60 min in a Synergy 2 multi-mode microplate reader (BioTek) using excitation and emission wavelengths of 360 and 460 nm, respectively. The Km and Vmax were calculated by fitting the data to the appropriate Michaelis-Menten equations by using nonlinear regression in the GraphPad Prism 4 software. The A/PR/8/34 (H1N1) influenza virus was included for comparison in all assays.All H1N1 viruses were standardized to an equivalent dose of 106 TCID50 of A/DM/524/09, A/DM/528/09 virus in 1.0 ml sterile PBS. One donor ferret was inoculated with 106 TCID50 of a mixture of A/DM/524/09 and A/DM/528/09 viruses (1∶1 infectivity ratio). After the donor ferrets were confirmed to shed virus on day 2 p.i. by the Directigen Flu A+B quick test , each was then housed in the same cage with 2 naïve direct-contact ferrets. Two additional recipient ferrets were placed in an adjacent cage isolated from the donor's cage by a two layers of wire mesh (∼5 cm apart) that prevented physical contact but allowed the passage of respiratory droplets. A Borazine gun  was used to ensure non-directional air flow inside the isolator. The donor and recipient ferrets were housed together since day 2 p.i until day 21 p.i. Ferret weight and temperature were recorded daily for 21 days. Body temperature was measured by subcutaneous implantable temperature transponders .Young adult ferrets (4–5 months of age) were obtained from the ferret breeding program at St. Jude Children's Research Hospital. All ferrets were seronegative for influenza A H1N1 and H3N2 viruses and for influenza B viruses. Ferrets were housed in the isolators in ABSL3+ facilities and monitored for 3–5 days to establish baseline body temperature and overall health. Donor ferrets were initially housed separately from contact ferrets. The donor ferrets were lightly anesthetized with isoflurane and inoculated with 1050 titers were determined in MDCK cells. Inflammatory cell counts were determined as described previously Nasal washes were collected from donors and recipients on days 1, 2, 4, 6, 8, 10, 12, and 14 p.i. by flushing both nostrils with 1.0 ml PBS, and TCIDSerum samples were collected from ferrets 3 weeks after virus inoculation, treated with receptor-destroying enzyme, heat-inactivated at 56°C for 30 min, and tested by HI assay with 0.5% packed chicken red blood cells (CRBC) as described previously Viral RNA was isolated from ferret nasal washes by using the RNeasy Mini kit . Samples were reverse-transcribed and analyzed by PCR using primers specific for the NA gene segment, as described previously 5′-AGAACACAAGAGTCTGAATGTG-3′ and 5′-CCATTTGCTCCATTAGACGATACT-3′. Single nucleotide primer extension was performed using a SNaPshot kit (ABI) per the manufacturer's protocol. The reaction consisted of 2.5 µl SNaPshot Reaction Mix, 3 µl RT-PCR product, and 0.2 µmol/L of the extension primer in a 5 µl final reaction volume. The extension primer, 5′-CAGTCGAAATGAATGCCCCTAATTAT-3′, was synthesized by IDTDNA and used to detect the first nucleotide of NA 275 codon. After the SNaPshot reaction, a unit of shrimp alkaline phosphatase (USB) was added to remove 5′ phosphoryl groups of unincorporated dideoxynucleotide substrates as directed by manufacturer's protocol. One µl of the SNaPshot products was mixed with deionized formamide and LIZ120 (ABI) size standard and was injected into the ABI 3730xl capillary electrophoresis instrument (ABI) per the manufacturer's protocol. Data were analyzed by using ABI GeneMapper software. Serially diluted DNA template (35 ng/µl to 0.02 ng/µl) from each genotype was used for signal standardization. Spike-in samples were generated by using 11 different ratios of wild-type and mutant DNA fragments, e.g. 100% wild-type, 90% wild-type, etc. A good correlation was achieved between the spike-in ratios and ratios of fluorescence intensity values (R2 = 0.9877) (data not shown).The relative quantification of SNP assay was performed as described previously t-test or analysis of variance (ANOVA) was used for all comparisons.The unpaired


Fbx4-αBcrystallin E3 ubiquitin ligase. Although cyclin D1 overexpression occurs in numerous malignancies, overexpression of cyclin D1 alone is insufficient to drive transformation. In contrast, cyclin D1 mutants refractory to phosphorylation-dependent nuclear export and degradation are acutely transforming. This raises the question of whether overexpression of cyclin D1 is a significant contributor to tumorigenesis or an effect of neoplastic transformation. Significantly, recent work strongly supports a model wherein nuclear accumulation of cyclin D1-dependent kinase during S-phase is a critical event with regard to transformation. The identification of mutations within SCFFbx4-αBcrystallin ligase in primary tumors provides mechanistic insight into cyclin D1 accumulation in human cancer. Furthermore, analysis of mouse models expressing cyclin D1 mutants refractory to degradation indicate that nuclear cyclin D1/CDK4 kinase triggers DNA re-replication and genomic instability. Collectively, these new findings provide a mechanism whereby aberrations in post-translational regulation of cyclin D1 establish a cellular environment conducive to mutations that favor neoplastic growth.Mitogenic induction of cyclin D1, the allosteric regulator of CDK4/6, is a key regulatory event contributing to G1 phase progression. Following the G1/S transition, cyclin D1 activation is antagonized by GSK3β-dependent threonine-286 (Thr-286) phosphorylation, triggering nuclear export and subsequent cytoplasmic degradation mediated by the SCF The t cascade -10. This cascade -13. Foll cascade .in vitro and in vivo [While cyclin D1 overexpression occurs frequently in human cancer and is considered a causative factor in many tumor types, simple over-expression of wild type cyclin D1 is insufficient to drive neoplastic transformation . In cont in vivo ,16 imply in vivo ,18. HoweCCND1 gene changing proline-287 (Pro-287) to a serine or threonine residue in endometrioid endometrial carcinoma correlates with overexpression of cyclin D1 in the nucleus of neoplastic cells. Additionally, a 12-base pair in frame deletion corresponding to deletion of amino acids 289–292 was reported with overexpression of cyclin D1 [Cyclin D1 overexpression occurs in carcinomas of the breast, esophagus, head and neck, and lung; in a majority of these cases, alterations in gene expression cannot account for its overexpression. Perturbations in cyclin D1 degradation have been suggested as the primary contributor in a large percentage of cases. Indeed mutations that interfere with Thr-286 phosphorylation occur, but are rare. Such mutations have been noted in endometrial and esophageal cancer ,18. For yclin D1 . Signifiyclin D1 .CCND1) in a panel of 90 patient esophageal carcinomas revealed mutation of Thr-286 to arginine and a deletion of C-terminal residues 266–295. Additionally, screening of human tumor-derived esophageal carcinoma cell lines also identified a Pro-287 to alanine mutation in three of these lines [In accordance with endometrial cancer studies, recently identified cyclin D1 mutations in esophageal cancer and tumor-derived cell lines also disrupt Thr-286 phosphorylation . Sequencse lines .in vitro, much like the phosphorylation-deficient T286A mutant [Alternative splicing may also contribute to cancer specific accumulation of cyclin D1 proteins that cannot undergo cytoplasmic degradation . Tumor-sA mutant ,21. It iFbx4-αB crystallin [Cell cycle progression is driven by alternating phases of cyclin expression and destruction. Degradation is coordinated by substrate ubiquitylation and destruction via the 26S proteasome ,23. Polyystallin . Substraystallin .Given the low frequency of mutations within cyclin D1 that directly impact is turnover, a logical prediction was the occurrence of inactivating mutations in its E3 ligase. The first clue to cyclin D1 ligase involvement came from analysis of several breast cancer cell lines exhibiting increased cyclin D1 half-life without mutations disrupting phosphorylation. These analyses revealed that MCF-7 and MDA-MB-231 cells lack αB crystallin expression as a consequence of chromosome deletions. In addition, tumor microarray analysis of esophageal carcinomas revealed a reduction in both αB crystallin and Fbx4 mRNA levels in tumor tissues . CollectFbx4 sequence in primary esophageal carcinoma samples identified hemizygous, missense mutations in 14 percent of the tumors; no mutations occurred in αB crystallin or CCND1 genes in tumors expressing mutated Fbx4. A high percentage of mutations reside within a putative Fbx4 dimerization domain, while others target Ser-12 in the N-terminus. Additionally, one mutation identified within the F box domain results in production of a dominant negative protein incapable of recruiting Skp1 and Cul1[Significantly, recent assessment of the  and Cul1. The res and Cul1-30.Further analysis of the cyclin D1 ligase revealed that GSK3β, the kinase responsible for cyclin D1 Thr-286 phosphorylation, also catalyzes phosphorylation of Fbx4 Ser-12. Fbx4 phosphorylation correlates with low cyclin D1 expression during G2/M and early G1, with a marked decrease during cell cycle entry due to growth factor-dependent GSK3β inhibition . Fbx4 phin vitro, revealing that such mutations are indeed transforming [The tumorigenic potential of Fbx4 mutations disrupting phosphorylation and dimerization was assessed sforming . These fin vitro and B-cell lymphomagenesis in mice [Aberrant nuclear accumulation of cyclin D1 during S-phase drives cell transformation  in mice ,16. Rece in mice . Cdt1 is in mice ,33. The  in mice ,35. In c in mice ,37. Ulti in mice .Fbx4-αBcrystallin activation generate genomic instability, ultimately driving tumor formation (Figure Active cyclin D1/CDK4 complexes influence transcriptional regulation of the Cul4 proteins; however, the precise mechanism of regulation remains to be elucidated. Strikingly, impaired cyclin D1 ligase function results in nuclear accumulation of active cyclin D1/CDK4 complexes, accumulation of Cdt1, and triggers cellular transformation analogous to cyclin D1 T286A . Taken tGenomic integrity is monitored by cell cycle checkpoints that promote cell cycle arrest or apoptosis upon detection of damaged DNA . ChronicProvided with this new data suggesting that cyclin D1-dependent kinase contributes to neoplasia at least in part through perturbations in DNA replication and loss of genomic integrity, can we utilize this information for increased therapeutic modalities? One scenario might be to take advantage of the fact that tumors harbouring mutations in cyclin D1 or Fbx4 have a compromised DNA damage checkpoint due to loss of p53. In theory, treatment of normal cells with chemotherapeutic agents that generate DNA crosslinks should trigger an intra S-phase checkpoint, thereby providing an opportunity for repair. Expression of stabilized cyclin D1 should promote maintenance of Cdt1 and MCM complexes and in so doing promote continued origin firing without allowing for repair of damaged DNA, ultimately resulting in mitotic catastrophe. Further work is required to investigate how alterations in cyclin D1 proteolysis might influence cellular responses to DNA damaging therapeutics.The alternative is the development of drugs that directly target the kinase subunits that cyclin D1 regulates. If continued activation of CDK4/6 is required for tumor proliferation and survival, such drugs may have significant clinical use. Consistent with this notion of oncogene addiction, treatment of mammary epithelial cells derived from murine tumors harbouring a MMTV-T286A transgene with the CDK4/6 inhibitor PD0332991  triggereFbx4-αB crystallin in human cancers implicate ligase function in cyclin D1 overexpression and subsequent nuclear accumulation of active cyclin D1/CDK4 complexes. Importantly, GSK3β functions as the master switch, turning on cyclin D1 destruction at the G1/S transition by regulating both ligase activation and cyclin D1 phosphorylation. Given the phenotypic outcome of accumulated nuclear cyclin D1/CDK4 complexes, further mechanistic investigation is required for development of novel therapeutic strategies to promote tumor cell death in cancers overexpressing cyclin D1.Phosphorylation-dependent nuclear export and subsequent degradation of cyclin D1 is essential to maintain cellular homeostasis. Disruption of this regulatory pathway has been extensively shown to promote neoplastic transformation; however, the precise mechanism of this event has been elusive. Significantly, recent work revealed that nuclear accumulation of active cyclin D1/CDK4 complexes generates genomic instability through a mechanism of Cdt1 stabilization and DNA re-replication. Furthermore, mutations targeting SCFThe authors declare that they have no competing interests.LLP and JAD contributed to the discussion and preparation of this manuscript.