![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA; 2 Department of Biochemistry and Center for Molecular Neuroscience, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA; 3 Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 BE Amsterdam, The Netherlands; 4 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA; 5 Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA; 6 The Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts 02141, USA; 7 Department of Medicine and Cellular and Structural Biology, San Antonio Cancer Institute, University of Texas Health Science Center, San Antonio, Texas 78229, USA; 8 Laboratoire de Biochimie and Hormonologie, Centre de Biologie et Pathologie, CHRU de Lille 59037, Lille cedex, France; 9 Department of Neurology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA; 10 Division of Oncology, Children’s Hospital of Philadelphia, Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; 11 Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA
 |
Abstract |
|---|
 Top Abstract ResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
[Keywords: Apoptosis; kinesin; neuroblastoma; pheochromocytoma; prolyl hydroxylase]]
Received January 7, 2008; revised version accepted February 14, 2008.
). Deregulation of this process can cause disease (Zhu and Parada 2002). Paragangliomas and neuroblastomas are tumors of the sympathetic nervous system (paragangliomas arising in the adrenal medulla are called pheochromocytomas). During normal embryological development, neuronal progenitor cells, including cells capable of forming the sympathetic nervous system, compete with one another for growth factors such as nerve growth factor (NGF), with the losers undergoing apoptosis. Germline VHL, NF-1, c-Ret, and Succinate Dehydrogenase Subunits B and D (SDHB and SDHD) mutations have been linked to the development of paragangliomas (Nakamura and Kaelin 2006). We reported recently that the products of these genes define a pathway that is activated upon NGF withdrawal, leading to apoptosis mediated by the EglN3 prolyl hydroxylase (Lee et al. 2005). This suggests that at least some paragangliomas arise due to failure to properly cull neuronal progenitor cells during development. |
Results |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
).
|
p53 is a critical regulator of apoptosis and has been implicated in developmental cell death of sympathetic neurons (Aloyz et al. 1998). Isogenic HCT116 cells that are p53+/+ or p53–/– were both killed by EglN3 (Supplemental Fig. 2A). Likewise, EglN3-induced cell death in neural crest-derived cells (SK-Mel28 melanoma cells) was not prevented by an effective p53 shRNA or by SV40 T antigen, which blocks p53 function (Supplemental Fig. 2B–D). Therefore, EglN3-induced apoptosis does not require p53.
To begin to understand how EglN3 induces apoptosis, we screened for shRNAs that can prevent EglN3-induced death. In pilot experiments, we determined the Ad-EglN3 titer required to kill all of the SK-Mel28 melanoma cells in subconfluent cultures (Fig. 1C; data not shown). Next, SK-Mel28 cells were infected with a previously described retroviral shRNA library (Berns et al. 2004) (or with empty retrovirus) prior to Ad-EglN3 infection (Fig. 2A). No survivors emerged among the control cells pretreated with the empty virus. In cells pretreated with the shRNA library, however, 12 surviving colonies emerged and were expanded for further analysis. Three died when rechallenged with Ad-EglN3 and were therefore considered false-positives, while nine remained resistant (Fig. 2B). The shRNA inserts from these latter colonies were isolated, sequenced, and retested for their ability to protect naive SK-Mel28 cells from EglN3-induced apoptosis. Sequence analysis of one of the two shRNAs that scored positively in this assay predicted that it targeted the β splice variant of KIF1B, a member of the kinesin 3 family (Nagai et al. 2000; Yang et al. 2001; Zhao et al. 2001). Down-regulation of endogenous KIF1Bβ, but not the alternative splice variant KIF1B
, was confirmed by Western blot analysis (Fig. 2C). KIF1B and KIF1Bβ share an N-terminal motor domain but contain different C-terminal cargo domains. Protection against EglN3-induced cell death was conferred by two additional, independent, KIF1Bβ shRNAs, arguing that modulation of KIF1Bβ, rather than an off-target effect, was responsible for this protection (data not shown). Since KIF1B maps to 1p36 (Nagai et al. 2000; Yang et al. 2001; Zhao et al. 2001), which is frequently deleted in multiple tumor types including nervous system tumors (Schwab et al. 1996), we hypothesized that it might function as a tumor suppressor gene through regulation of apoptosis in neuronal, and perhaps other, tissues.
|
(Fig. 3A). Conversely, knockdown of human EglN3 in HeLa cervical carcinoma cells with two independent siRNAs decreased KIF1Bβ levels (Fig. 3B; Supplemental Fig. 3). Notably, an siRNA against EglN1, which regulates the HIF transcription factor (Berra et al. 2003), did not affect KIF1Bβ, consistent with the earlier conclusion that regulation of apoptosis by EglN3 is HIF-independent (Lee et al. 2005). zKIF1B levels were also markedly attenuated in zebrafish treated with a morpholino oligonucleotide directed against zEglN3 (Fig. 3C).
|
). Pheochromocytomas are derived from sympathetic neuronal progenitor cells, and PC12 cells have been used extensively as a model to study the effects of NGF on neuronal differentiation and survival. Consistent with previous reports (Lipscomb et al. 1999; Lee et al. 2005), NGF withdrawal from PC12 cells caused the accumulation of EglN3, which coincided with the onset of apoptosis (Fig. 3D; data not shown). Importantly, KIF1Bβ was induced with similar kinetics. Similarly, NGF withdrawal induced both EglN3 and KIF1Bβ in primary rat sympathetic neurons (Supplemental Fig. 4). Moreover, the induction of KIF1Bβ by NGF withdrawal was prevented by DMOG in primary rat sympathetic neurons and did not occur in EglN3–/– primary mouse sympathetic neurons (Fig. 3E,F). Likewise, KIFBβ was not induced by Ara-C in EglN3–/– CGN (Fig. 3G). Collectively, these results indicate that EglN3 hydroxylase activity is necessary and sufficient for KIF1Bβ induction. Regulation of KIF1Bβ by EglN3 appears to be post-transcriptional (Supplemental Fig. 5). Whether KIF1Bβ is hydroxylated by EglN3 remains to be determined.
Introduction of KIF1Bβ into PC12 cells was, like EglN3 itself, sufficient to induce apoptosis, although apoptosis occurred more rapidly with KIF1Bβ (1–2 d vs. 3 d) (Fig. 4A; data not shown), which is consistent with KIF1Bβ acting downstream from EglN3. The percentage of apoptotic cells at any time point did not exceed 20%, however, because KIF1Bβ, like NGF withdrawal itself, killed asynchronously (Lee et al. 2005). KIF1Bβ also induced apoptosis in primary rat sympathetic neurons (Fig. 5B). Conversely, multiple KIF1Bβ shRNAs prevented apoptosis of primary rat sympathetic neurons following NGF withdrawal (Fig. 4B). Therefore, KIF1Bβ is both necessary and sufficient for apoptosis in this setting.
|
; White et al. 2005). KIF1Bβ mRNA levels are decreased in neuroblastoma tumors with 1p36 deletions and are also decreased in advanced-stage disease (Caren et al. 2005; Wang et al. 2006; A. Nakagawara, pers. comm.). In contrast, KIF1Bβ protein levels appear to be highly variable across neuroblastoma lines and do not strictly correlate with the presence or absence of 1p deletions, possibly due to adaptation in culture as well as alternative mechanisms of KIF1Bβ down-regulation (data not shown). The neuroblastoma cell line NB-1 has an 
500-kb homozygous deletion at 1p36 that spans KIF1B and five other known genes (Ohira et al. 2000; Yang et al. 2001; Krona et al. 2003). In contrast to SK-N-SH and CHP212 cells, NB-1 cells were resistant to EglN3-induced apoptosis but were, like other neuroblastoma lines, killed by wild-type KIF1Bβ (Fig. 4C–E). Notably, restoring the function of the other five 1p genes deleted in NB-1 cells has no effect (A. Nakagawara, pers. comm.). Similarly, LAN6 neuroblastoma cells and H460 lung carcinoma cells, both of which also produce low levels of KIF1Bβ, were resistant to EglN3 (Fig. 4C,D). Interestingly, H460 cells exhibit neuroendocrine features (Lee et al. 1992). Killing by KIF1Bβ in neuroblastoma cells appears to be independent of its kinesin motor function, since KIF1Bβ(600–1770), which lacks the KIF1Bβ motor domain, still induced apoptosis (Figs. 4E, 5A,B).
|
; data not shown). In addition, we repeatedly identified common polymorphic alleles that lead to a Y1087C or V1554M substitution (data not shown).
|
Next, primary rat sympathetic neurons were electroporated with plasmids encoding wild-type KIF1Bβ or these variants. The induction of apoptosis by all of the putative disease-causing variants (S34L, E646V, T827I, P1217S, S1481N, and E1628K) was clearly impaired relative to wild-type KIF1Bβ or the polymorphic variants Y1087C and V1554M (Fig. 5B,C; Supplemental Fig. 7). Comparable levels of protein production were confirmed by immunofluorescence and immunoblot analysis (Fig. 5C; Supplemental Fig. 7). These data argue that putative disease-causing variants are pathogenic rather than the result of benign polymorphisms or passenger mutations.
|
Discussion |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
; Haag et al. 1981; Stoler and Bouck 1985). Our data suggest that one such tumor suppressor gene is KIF1Bβ, and that this gene is relevant to certain tumors of neuronal origin. Nonetheless, we and others observed that the remaining KIF1Bβ allele in 1p deleted tumors and cell lines is often wild-type, contrary to the Knudson Two-Hit scenario (Ohira et al. 2000; Yang et al. 2001; A. Nakagawara, pers. comm.; data not shown). Moreover, two of the variants we identified (S34L and S1481N) were not associated with the loss of the remaining wild-type allele. Perhaps KIF1Bβ haploinsufficiency is adequate for tumorigenesis in some contexts, especially when combined with the loss of other contiguous 1p genes such as CHD5 (Bagchi et al. 2007). In this regard, we noted substantial protection against apoptosis with an shRNA that decreased KIF1Bβ levels by 50%, and the existence of multiple neuroblastoma and pheochromocytoma suppressor genes on 1p has been suggested before (Takeda et al. 1994; Cheng et al. 1995; Ichimiya et al. 1999; Benn et al. 2000; Opocher et al. 2003; Wang et al. 2006). At the same time, complete loss of KIF1Bβ promotes (rather than inhibits) neuronal apoptosis (Zhao et al. 2001), as does nearly complete elimination of KIF1Bβ using shRNAs (Supplemental Fig. 8). Tumor development was, however, associated with loss of the remaining wild-type allele for the two germline neuroblastoma mutations E646V and P1217S and the pheochromocytoma mutation E1628K. We note that these mutations are not completely null, based on our apoptotic assay, and therefore loss of the remaining wild-type allele might still confer a survival advantage. The NB-1 line appears to be unusual insofar as it has a homozygous, rather than heterozygous, KIF1Bβ deletion. Among several possibilities, these cells might harbor additional mutations that allow them to tolerate total loss of KIF1Bβ function.
Our findings have potential implications with respect to the pathogenesis of certain neural crest-derived tumors such as pheochromocytomas and neuroblastomas. Many cases of pheochromocytoma without a positive family history are nonetheless due to previously unsuspected germline mutations involving VHL, c-Ret, NF1, SDHB, or SDHD. We reported earlier that these genes, together with EglN3, define a pathway (Fig. 4I) that is responsible for the elimination of excess neuroblasts during normal embryological development when growth factors such as NGF become limiting. It is noteworthy that neuroblastomas frequently express an NGF receptor (Brodeur 1994), and both NF1 and VHL mutations have been linked to this form of cancer also (Johnson et al. 1993; The et al. 1993; H. Greulich and M. Meyerson, unpubl.). In this report, we placed KIF1Bβ downstream from EglN3 and identified loss-of-function germline KIF1Bβ mutations in some pheochromocytomas and neuroblastomas. Moreover, we obtained functional data consistent with the idea that partial loss of KIF1Bβ, such as might occur with the loss of one KIF1B allele, would protect neuroblasts from apoptosis in response to stimuli such as NGF withdrawal. We therefore suggest that some neuroblastomas, like pheochromocytomas, result from germline alterations that directly or indirectly compromise KIF1Bβ function and allow certain neuronal progenitor cells to escape developmental culling. This model is consistent with the prediction, based on epidemiological studies, that at least 20% of pheochromocytomas and neuroblastomas involve a hereditary component (Knudson and Strong 1972; Knudson and Meadows 1976) and could account for previously described patients (Fairchild et al. 1979; Tatekawa et al. 2006) who, like our patient with the S1481N variant, developed both of these otherwise rare tumors as children or young adults.
KIF1B and KIF1Bβ are motor proteins implicated in anterograde transport of mitochondria and synaptic vesicle precursors, respectively (Nangaku et al. 1994; Zhao et al. 2001). The S34L mutation maps to the KIF1Bβ motor domain, although the motor domain is dispensible for KIF1Bβ-induced apoptosis (Figs. 4E, 5B). Conceivably, the S34L mutation affects the folding of KIF1Bβ or eliminates an important phosphorylation site. Clearly, additional studies are now needed to address how, mechanistically, KIF1Bβ regulates apoptosis and to determine how often it is deregulated, epigenetically or genetically, in cancer.
|
Materials and methods |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
EglN3–/– mice were a gift of Regeneron Pharmaceuticals. Sympathetic neurons from P4 rats or mice were isolated from the superior cervical ganglia (SCG) and were cultured in 20 ng/mL NGF (Harlan) as described previously (Palmada et al. 2002). Cerebellar granule neurons were isolated from 4-d-old mouse pups. Cerebella were removed and dissociated by trypsinization and plated on poly-L-ornithine-coated plates. Cells were cultured in neurobasal media (Gibco) supplemented with B27, 0.6% dextrose, 2 mM glutamine, 25 mM KCl, and penicillin/streptomycin.
Immunoblot analysis
Cell extracts were prepared in EBC buffer (50 mM Tris at pH 8.0, 120 mM NaCl, 0.5% NP-40) containing protease inhibitors, unless otherwise noted. Primary neurons were lysed in NP-40 lysis buffer (10% glycerol, 50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride [PMSF], 2 µg/mL leupeptin and aprotinin). For PARP assays, cells were lysed in 62.5 mM Tris HCl (pH 6.8), 6 M urea, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.000125% bromophenol blue, and protease inhibitors. Equal amounts of protein, as measured by the Bradford assay, were immunoblotted as described previously (Lee et al. 2005). Rabbit polyclonal anti-EglN3 sera were generously provided by Dr. Robert Freeman (specific to mouse, rat, and human) (Straub et al. 2003), and rabbit polyclonal antibody against HIF has been described recently (Berra et al. 2003). Rabbit anti-KIF1B antibody specific for the β isoform (cross-reactive with mouse, rat, human, and zebrafish) (sc-28540) or the form (sc-18739) were purchased from Santa Cruz Biotechnology. Antibody raised against cleaved Caspase 3 was purchased from Cell Signaling (Asp715), and antibody raised against PARP was from Biomol International (P9055a).
Neuronal apoptosis assays
Sympathetic neurons from P4 rats or mice were cultured in 20 ng/mL NGF (Harlan) for 2 d, then rinsed twice in Ultraculture medium lacking NGF and once with Ultraculture medium containing anti-NGF (0.1 µg/mL; Chemicon International), and then maintained in NGF-free media for 48 h, at which point cells were fixed in 4% paraformaldehyde (PFA) and stained with DAPI (Vector Laboratories). Approximately 70–100 nuclei were scored for apoptotic changes for each condition, as described before (Kenchappa et al. 2006). In some experiments, cells were pretreated with 1 mM DMOG for 6 h prior to and during NGF withdrawal.
Sympathetic neurons from P4 rat were isolated and transfected with a plasmid encoding GFP alone or cotransfected with pSuper plasmids using the Amaxa Nucleofactor device as described previously (Kenchappa et al. 2006). Neurons were maintained in 20 ng/mL NGF for 4 d and then subjected to NGF withdrawal as above. After fixation, GFP-positive neurons were evaluated for apoptosis as above.
Analysis of undifferentiated PC12 cells treated with NGF, followed by NGF withdrawal, was as described (Lee et al. 2005). For transfection experiments, undifferentiated PC12 cells were plated onto collagen-coated six-well plates 1 d before transfection with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Transfection mixes contained 500 ng of a plasmid encoding GFP-histone (a gift of Geoffrey Wahl) and 1–2 µg of the plasmid of interest. Seventy-two hours later, 400 GFP-positive cells were scored for the presence of apoptotic nuclei for each set of conditions.
Expression plasmids and siRNA
Adenovirus encoding EglN3 (Ad-EglN3) was a gift from Robert Freeman. The HA-EglN3 and HA-EglN3-H196A expression plasmids were described before (Lee et al. 2005). The NKI pRS hairpin library was described previously (Berns et al. 2004). A KIF1Bβ cDNA was PCR-amplified from a SK-Mel28 cDNA pool and ligated into pcDNA3 via Kpn1 and Xba1 sites to make pcDNA3-Flag-KIF1Bβ. Site-directed mutagenesis to generate pcDNA3-Flag-KIF1Bβ-E646V, S34L, T827I, P1217S, S1481N, E1628K, Y1087C, and V1554M was performed using a QuikChange Site-Directed Mutagenesis kit (Stratagene) using the primers 5'-GGAGATCTTATACAAAAAGGTGAAGGAA GAAGCAGATCTT-3', 5'-TCATTCAGATGCAAGGCAACC TGACCAGTATTATTAACCC-3', 5'-CAAGACGAAAGCGA AACCATTGTGACTGGCAGCGATCCCT-3', 5'-TGAGATC AGTGAACTGGAGTCTACAGGAGAGTATATCCCA-3', 5'-AGCATCCCCAAATCCCTGAACGACTCGTTATCCCCCAG CC-3', 5'-TCAGATTGTCCCAGCTGTGAAAACACCATATTT GGCCCGA-3', 5'-AGTGGAATCCTCCCAGAGTGTGCAGAT ATCTTCTGTCAGT-3', and 5'-CAACAGAGAATTCAGCCA GATGCACGGCAGCGTCAGTGAC-3', respectively.
Retroviruses encoding human KIF1Bβ shRNAs were made using the pRetroSuper plasmid (Brummelkamp et al. 2002b) and the following sequences: (sh) #1, 5'-GGAGCCTCTTTACAG TAAC-3'; (sh) #3, 5'-GCAATGCCGTGTACCTAAA-3'; (sh) #7:,5'-CGAGAGCAGTGGCTATGAT-3'. pRS-shp53 has been described recently (Brummelkamp et al. 2002b).
Plasmids encoding shRNAs for rat EglN3 and rat KIF1Bβ were made using pSuper plasmid (Brummelkamp et al. 2002a) and the following sequences: EglN3, 5'-CAGGTTATGTTCGTCATGT-3'; KIF1Bβ #1, 5'-AGAGCCACTCTCCAGTAAC-3'; KIF1Bβ #2, 5'-CAAGCTGGTTCGGGAGCTG-3'. siRNAs against human targets were EglN1, 5'-AGCUCCUUCUACUGCUGCA-3'; EglN3 #2, 5'-CAGGUUAUGUUCGCCACGU-3'; and EglN3 #3, 5'-UUCUUCUGGUCAGAUCGUA-3'.
Cell culture and retroviral transduction
Undifferentiated PC12 cells were maintained in DMEM containing 5% fetal bovine serum (FBS) (Hyclone) and 10% horse serum (Sigma) in 10% CO2 at 37°C. Human tumor cell lines were maintained in RPMI (neuroblastoma lines) or DMEM (other lines) containing 10% FBS (Hyclone) in the presence of 10% CO2 at 37°C. The production of retroviruses and adenoviruses using Phoenix and 293A packaging cells, respectively, and subsequent infections was carried out as described (Peeper et al. 2002; Lee et al. 2005).
Morpholino injection in zebrafish
Zebrafish were maintained and bred as described (Westerfield 1993), and were staged according to Kimmel et al. (1995). Microinjections were performed on one-cell-stage embryos according to standard procedures (Westerfield 1993). Based on the published GenBank sequence for zegln3 (gi:47086946), a translation-blocking morpholino was designed by GeneTools. Inc.: zegln3, GTGCTGAAGAAACGGCATTTTGTCC. Zebrafish protein lysates were prepared from 100 3-d-old embryos by homogenizing in lysis buffer (1% NP-40, 0.1% SDS, 100 mM NaCl, 50 mM Tris at pH 7.5, 10 mM EDTA, 0.1% PMSF, supplemented with Roche complete protein inhibitor) using a micropestle, then centrifuged at 15,000 rpm in a microcentrifuge for 10 min at 4°C. The supernatent was transferred to a new tube and stored at –80°C.
Immunofluorescence staining
Primary sympathetic neurons were subjected to NGF withdrawal for 24 h as described above. The cells were then fixed in 4% PFA, permeabilized with 0.1% sodium citrate and 0.1% Triton X-100, blocked with 10% goat serum in PBS, and incubated with the KIF1B antibody (1:100 dilution) or EglN3 antibody (1:100) in PBS containing 0.1% Triton X-100. After incubation with anti-rabbit Alexa 488 (Molecular Probes) and staining with DAPI, images were acquired using a confocal laser imaging system (LSM 510; Carl Zeiss MicroImaging, Inc.) at 400x.
Tumor sample sequencing
Ninety-eight primary neuroblastoma tumor samples were identified from the Children’s Oncology Group (COG) Neuroblastoma Nucleic Acids Bank. Samples were collected after obtaining parental informed consent, and institutional review board (IRB) guidelines were followed for the procurement of each sample. They were obtained at original diagnosis from patients who had received no previous treatment and immediately snap-frozen, and had a tumor cell content of >90% based on differential count, clonal hyperdiploid percentage in some tumors, and direct examination of H&E-stained tumor slides. All 98 patients met the COG criteria for having high-risk disease (Maris 2005). Patients were staged according to the International Neuroblastoma Staging System and histology was analyzed using the Shimada Pathology Classification (Shimada et al. 1984; Brodeur et al. 1993). Loss-of-heterozygosity (LOH) status was determined using conventional microsatellite markers and high-resolution SNP array analysis as described previously (George et al. 2007). DNA was extracted using conventional methods (Qiagen kit) and was sequenced by Agencourt, Inc., by automated sequencing. All 46 coding KIF1Bβ exons were PCR-amplified and sequenced in a duplicate, bidirectional manner. Sequence traces were analyzed to identify potential somatic mutations using the Mutation Surveyor software package (SoftGenetics).
An additional 13 neuroblastoma tumors from the COG, as described above, and 14 medulloblastoma samples obtained at Children’s Hospital in Boston under IRB approval were sequenced for KIF1Bβ at the Broad Institute. Briefly, DNA was extracted from the tumor and matched normal blood sample (Qiagen DNeasy kit), quantified using picogreen (Molecular Probes), and isothermally amplified using the Repli-g whole-genome amplification kit (Amersham). Five nanograms of DNA for each exon of KIF1Bβ were individually PCR-amplified (primer sequences available upon request) with the HotStar Enzyme (Qiagen) and the following cycling parameters: one cycle of 15 min at 95°C; followed by 35 cycles of 20 sec at 95°C, 30 sec at 60°C, and 1 min at 72°C; followed by a final extension of 3 min at 72°C. PCR products were sequenced in a duplicate, bidirectional manner. Sequence traces were analyzed to identify potential somatic mutations using an automated analysis pipeline comprised of the commercial software package Mutation Surveyor (SoftGenetics), PolyPhred 3.5 (Nickerson et al. 1997), and PolyDHAN (D. Richter, pers. comm.). The KIF1Bβ S34SL variant was confirmed by Sequenom mass spectrometric genotyping.
Fifty-two pheochromocytoma or paraganglioma samples were used to sequence the KIF1Bβ gene under an IRB-approved protocol. Fragments were obtained from the core of the tumor and contained >70% tumor cells. Samples with a clear adjacent cortical component were macrodissected. Specimens were snap-frozen at the time of surgical resection and stored at –70°C or in liquid nitrogen until processed. Diagnosis of pheochromocytoma and/or paraganglioma was confirmed by histology in every case. Eleven of these tumors came from individuals with hereditary disease (two MEN2A, one MEN2B, one VHL, two familial paraganglioma syndromes type 4-PGL4/SDHB, and five familial cases without an identifiable primary mutation in pheochromocytoma susceptibility genes). The remaining 41 tumors were sporadic or had an unknown familial history. Four tumors were recurrent or malignant (the latter were defined by the detection of metastasis at nonchromaffin sites), while the others were considered benign or had short follow-up.
Two approaches were used for sequencing these samples. Genomic DNA was isolated from 36 tumors using standard methods (Qiagen). Ten nanograms were used to amplify 50 amplicons spanning the 46 coding exons and exon–intron boundaries of the KIF1Bβ gene (primer sequences available upon request). For 16 tumors, only cDNA (prepared using Applied Biosystems Reverse Transcription kit) was available. Twenty primer pairs spanning the entire coding region of the longest KIF1Bβ transcript were used for PCR and sequencing of these samples.
PCR was performed using HotMaster Enzyme (Eppendorf). PCR conditions were as follows: one cycle of 5 min at 95°C; followed by 35 cycles of 30 sec at 95°C, 30 sec at 59°C, and 45 sec at 72°C; followed by a final extension of 5 min at 72°C. PCR products were purified and sequenced in both directions by Agencourt Bioscience using dye terminator technology. Sequence traces were analyzed using the commercial software Mutation Surveyor (SoftGenetics) and were manually verified. Variants were confirmed by the sequence of an independent sample.
The copy number of KIF1Bβ was determined by real-time PCR. Pooled results from three reference housekeeping genes with distinct genomic locations (β2 microglobulin, Albumin, and TRIM43) were used to calculate the copy number using the 
Ct method as described previously. Primer sequences are available upon request.
Frequency of KIF1Bβ S34L, E646V, T827I, P1217S, S1481N, E1628K, Y1087C, andV1554M in 270 controls of diverse ethnic backgrounds was determined by Sequenom mass spectrometric genotyping of the HapMap collection of normal DNA (Thorisson et al. 2005).
|
Acknowledgments |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
|
Footnotes |
|---|
E-MAIL william_kaelin{at}dfci.harvard.edu; FAX (617) 632-4760 
Supplemental material is available at http://www.genesdev.org.
Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.1648608.
|
References |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
Bagchi, A., Papazoglu, C., Wu, Y., Capurso, D., Brodt, M., Francis, D., Bredel, M., Vogel, H., and Mills, A.A. 2007. CHD5 is a tumor suppressor at human 1p36. Cell 128: 459–475.[CrossRef][Medline]
Benn, D.E., Dwight, T., Richardson, A.L., Delbridge, L., Bambach, C.P., Stowasser, M., Gordon, R.D., Marsh, D.J., and Robinson, B.G. 2000. Sporadic and familial pheochromocytomas are associated with loss of at least two discrete intervals on chromosome 1p. Cancer Res. 60: 7048–7051.
Berns, K., Hijmans, E.M., Mullenders, J., Brummelkamp, T.R., Velds, A., Heimerikx, M., Kerkhoven, R.M., Madiredjo, M., Nijkamp, W., Weigelt, B., et al. 2004. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428: 431–437.[CrossRef][Medline]
Berra, E., Benizri, E., Ginouves, A., Volmat, V., Roux, D., and Pouyssegur, J. 2003. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1 in normoxia. EMBO J. 22: 4082–4090.[CrossRef][Medline]
Brodeur, G.M. 1994. Molecular pathology of human neuroblastomas. Semin. Diagn. Pathol. 11: 118–125.[Medline]
Brodeur, G.M., Sekhon, G., and Goldstein, M.N. 1977. Chromosomal aberrations in human neuroblastomas. Cancer 40: 2256–2263.[CrossRef][Medline]
Brodeur, G.M., Pritchard, J., Berthold, F., Carlsen, N.L., Castel, V., Castelberry, R.P., De Bernardi, B., Evans, A.E., Favrot, M., Hedborg, F., et al. 1993. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J. Clin. Oncol. 11: 1466–1477.
Brummelkamp, T.R., Bernards, R., and Agami, R. 2002a. A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550–553.
Brummelkamp, T.R., Bernards, R., and Agami, R. 2002b. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2: 243–247.[CrossRef][Medline]
Caren, H., Ejeskar, K., Fransson, S., Hesson, L., Latif, F., Sjoberg, R.M., Krona, C., and Martinsson, T. 2005. A cluster of genes located in 1p36 are down-regulated in neuroblastomas with poor prognosis, but not due to CpG island methylation. Mol. Cancer 4: 10. doi: 10.1186/1476-4598-4-10.[CrossRef][Medline]
Cheng, N.C., Van Roy, N., Chan, A., Beitsma, M., Westerveld, A., Speleman, F., and Versteeg, R. 1995. Deletion mapping in neuroblastoma cell lines suggest two distinct tumor suppressor genes in the 1p36 region, only one of which is associated with N-myc amplification. Oncogene 10: 291–297.[Medline]
Fairchild, R., Kyner, J., Hermreck, A., and Schimke, R. 1979. Neuroblastoma, pheochromocytoma, and renal cell carcinoma. Occurrence in a single patient. JAMA 242: 2210–2211.[Abstract]
George, R.E., Attiyeh, E.F., Li, S., Moreau, L.A., Neuberg, D., Li, C., Fox, E.A., Meyerson, M., Diller, L., Fortina, P., et al. 2007. Genome-wide analysis of neuroblastomas using high-density single nucleotide polymorphism arrays. PLoS ONE 2: e255. doi: 10.1371/journal.pone.0000255.[CrossRef]
Haag, M.M., Soukup, S.W., and Neely, J.E. 1981. Chromosome analysis of a human neuroblastoma. Cancer Res. 41: 2995–2999.
Ichimiya, S., Nimura, Y., Kageyama, H., Takada, N., Sunahara, M., Shishikura, T., Nakamura, Y., Sakiyama, S., Seki, N., Ohira, M., et al. 1999. p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent. Oncogene 18: 1061–1066.[CrossRef][Medline]
Johnson, M.R., Look, A.T., DeClue, J.E., Valentine, M.B., and Lowy, D.R. 1993. Inactivation of the NF1 gene in human melanoma and neuroblastoma cell lines without impaired regulation of GTP.Ras. Proc. Natl. Acad. Sci. 90: 5539–5543.
Kenchappa, R.S., Zampieri, N., Chao, M.V., Barker, P.A., Teng, H.K., Hempstead, B.L., and Carter, B.D. 2006. Ligand-dependent cleavage of the P75 neurotrophin receptor is necessary for NRIF nuclear translocation and apoptosis in sympathetic neurons. Neuron 50: 219–232.[CrossRef][Medline]
Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., and Schilling, T.F. 1995. Stages of embryonic development of the zebrafish. Dev. Dyn. 203: 253–310.[Medline]
Knudson, A.G. and Meadows, A.T. 1976. Developmental genetics of neuroblastoma. J. Natl. Cancer Inst. 57: 675–682.[Medline]
Knudson, A.G. and Strong, L.C. 1972. Mutation and cancer: Neuroblastoma and pheochromocytoma. Am. J. Hum. Genet. 24: 514–532.[Medline]
Krona, C., Ejeskar, K., Abel, F., Kogner, P., Bjelke, J., Bjork, E., Sjoberg, R.M., and Martinsson, T. 2003. Screening for gene mutations in a 500 kb neuroblastoma tumor suppressor candidate region in chromosome 1p; mutation and stage-specific expression in UBE4B/UFD2. Oncogene 22: 2343–2351.[CrossRef][Medline]
Lee, M., Draoui, M., Zia, F., Gazdar, A., Oie, H., Bepler, G., Bellot, F., Tarr, C., Kris, R., and Moody, T. 1992. Epidermal growth factor receptor monoclonal antibodies inhibit the growth of lung cancer cell line. J. Natl. Cancer Inst. Monogr. 13: 117–123.
Lee, S., Nakamura, E., Yang, H., Wei, W., Linggi, M.S., Sajan, M.P., Farese, R.V., Freeman, R.S., Carter, B.D., Kaelin, W.G., et al. 2005. Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: Developmental culling and cancer. Cancer Cell 8: 155–167.[CrossRef][Medline]
Lipscomb, E., Sarmiere, P., Crowder, R., and Freeman, R. 1999. Expression of the SM-20 gene promotes death in nerve growth factor-dependent sympathetic neurons. J. Neurochem. 73: 429–432.[CrossRef][Medline]
Maris, J.M. 2005. The biologic basis for neuroblastoma heterogeneity and risk stratification. Curr. Opin. Pediatr. 17: 7–13.[CrossRef][Medline]
Nagai, M., Ichimiya, S., Ozaki, T., Seki, N., Mihara, M., Furuta, S., Ohira, M., Tomioka, N., Nomura, N., Sakiyama, S., et al. 2000. Identification of the full-length KIAA0591 gene encoding a novel kinesin-related protein which is mapped to the neuroblastoma suppressor gene locus at 1p36.2. Int. J. Oncol. 16: 907–916.[Medline]
Nakamura, E. and Kaelin, W.G. 2006. Recent insights into the molecular pathogenesis of pheochromocytoma and paraganglioma. Endocr. Pathol. 17: 97–106.[CrossRef][Medline]
Nangaku, M., Sato-Yoshitake, R., Okada, Y., Noda, Y., Takemura, R., Yamazaki, H., and Hirokawa, N. 1994. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79: 1209–1220.[CrossRef][Medline]
Nickerson, D.A., Tobe, V.O., and Taylor, S.L. 1997. PolyPhred: Automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res. 25: 2745–2751.
Ohira, M., Kageyama, H., Mihara, M., Furuta, S., Machida, T., Shishikura, T., Takayasu, H., Islam, A., Nakamura, Y., Takahashi, M., et al. 2000. Identification and characterization of a 500-kb homozygously deleted region at 1p36.2–p36.3 in a neuroblastoma cell line. Oncogene 19: 4302–4307.[CrossRef][Medline]
Opocher, G., Schiavi, F., Vettori, A., Pampinella, F., Vitiello, L., Calderan, A., Vianello, B., Murgia, A., Martella, M., Taccaliti, A., et al. 2003. Fine analysis of the short arm of chromosome 1 in sporadic and familial pheochromocytoma. Clin. Endocrinol. (Oxf.) 59: 707–715.[CrossRef][Medline]
Palmada, M., Kanwal, S., Rutkoski, N.J., Gustafson-Brown, C., Johnson, R.S., Wisdom, R., and Carter, B.D. 2002. c-jun is essential for sympathetic neuronal death induced by NGF withdrawal but not by p75 activation. J. Cell Biol. 158: 453–461.
Peeper, D.S., Shvarts, A., Brummelkamp, T., Douma, S., Koh, E.Y., Daley, G.Q., and Bernards, R. 2002. A functional screen identifies hDRIL1 as an oncogene that rescues RAS-induced senescence. Nat. Cell Biol. 4: 148–153.[CrossRef][Medline]
Schwab, M., Praml, C., and Amler, L.C. 1996. Genomic instability in 1p and human malignancies. Genes Chromosomes Cancer 16: 211–229.[CrossRef][Medline]
Shimada, H., Chatten, J., Newton, W.A., Sachs, N., Hamoudi, A.B., Chiba, T., Marsden, H.B., and Misugi, K. 1984. Histopathologic prognostic factors in neuroblastic tumors: Definition of subtypes of ganglioneuroblastoma and an age-linked classification of neuroblastomas. J. Natl. Cancer Inst. 73: 405–416.[Medline]
Sommer, L. and Rao, M. 2002. Neural stem cells and regulation of cell number. Prog. Neurobiol. 66: 1–18.[CrossRef][Medline]
Stoler, A. and Bouck, N. 1985. Identification of a single chromosome in the normal human genome essential for suppression of hamster cell transformation. Proc. Natl. Acad. Sci. 82: 570–574.
Straub, J.A., Lipscomb, E.A., Yoshida, E.S., and Freeman, R.S. 2003. Induction of SM-20 in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c in the cytosol, and caspase-dependent cell death. J. Neurochem. 85: 318–328.[Medline]
Takeda, O., Homma, C., Maseki, N., Sakurai, M., Kanda, N., Schwab, M., Nakamura, Y., and Kaneko, Y. 1994. There may be two tumor suppressor genes on chromosome arm 1p closely associated with biologically distinct subtypes of neuroblastoma. Genes Chrom. and Cancer 10: 30–39.[CrossRef]
Tatekawa, Y., Muraji, T., Nishijima, E., Yoshida, M., and Tsugawa, C. 2006. Composite pheochromocytoma associated with adrenal neuroblastoma in an infant: A case report. J. Pediatr. Surg. 41: 443–445.[CrossRef][Medline]
The, I., Murthy, A.E., Hannigan, G.E., Jacoby, L.B., Menon, A.G., Gusella, J.F., and Bernards, A. 1993. Neurofibromatosis type 1 gene mutations in neuroblastoma. Nat. Genet. 3: 62–66.[CrossRef][Medline]
Thorisson, G.A., Smith, A.V., Krishnan, L., and Stein, L.D. 2005. The International HapMap Project Web site. Genome Res. 15: 1592–1593.
Wang, Q., Diskin, S., Rappaport, E., Attiyeh, E., Mosse, Y., Shue, D., Seiser, E., Jagannathan, J., Shusterman, S., Bansal, M., et al. 2006. Integrative genomics identifies distinct molecular classes of neuroblastoma and shows that multiple genes are targeted by regional alterations in DNA copy number. Cancer Res. 66: 6050–6062.
Westerfield, M. 1993. The zebrafish book. University of Oregon Press, Eugene, OR.
White, P.S., Thompson, P.M., Gotoh, T., Okawa, E.R., Igarashi, J., Kok, M., Winter, C., Gregory, S.G., Hogarty, M.D., Maris, J.M., et al. 2005. Definition and characterization of a region of 1p36.3 consistently deleted in neuroblastoma. Oncogene 24: 2684–2694.[CrossRef][Medline]
Yang, H.W., Chen, Y.Z., Takita, J., Soeda, E., Piao, H.Y., and Hayashi, Y. 2001. Genomic structure and mutational analysis of the human KIF1B gene which is homozygously deleted in neuroblastoma at chromosome 1p36.2. Oncogene 20: 5075–5083.[CrossRef][Medline]
Zhao, C., Takita, J., Tanaka, Y., Setou, M., Nakagawa, T., Takeda, S., Yang, H.W., Terada, S., Nakata, T., Takei, Y., et al. 2001. Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bβ. Cell 105: 587–597.[CrossRef][Medline]
Zhu, Y. and Parada, L.F. 2002. The molecular and genetic basis of neurological tumours. Nat. Rev. Cancer 2: 616–626.[CrossRef][Medline]
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
