This Article Abstract Full Text (PDF) All Versions of this Article: 1541-7786.MCR-05-0165v1 4/3/169    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jung, V. Articles by Wullich, B. Search for Related Content PubMed PubMed Citation Articles by Jung, V. Articles by Wullich, B.

---

Cancer Genes and Genomics

Genomic and Expression Analysis of the 3q25-q26 Amplification Unit Reveals TLOC1/SEC62 as a Probable Target Gene in Prostate Cancer

¹ Clinic of Urology and Pediatric Urology and ² Department of Medical Biochemistry and Molecular Biology, University of the Saarland, Homburg/Saar, Germany and Departments of ³ Urology and ⁴ Pathology, Heinrich-Heine University, Düsseldorf, Germany

Requests for reprints: Bernd Wullich, Clinic of Urology and Pediatric Urology, University of the Saarland, 66421 Homburg/Saar, Germany. Phone: 49-6841-1624700; Fax: 49-6841-1624795. E-mail: urbwul{at}uniklinikum-saarland.de

	Abstract

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined. (Mol Cancer Res 2006;4(3):169–76)

	Introduction

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Gene copy number gain is a common mechanism by which genes achieve overexpression in tumors. For prostate cancer, amplifications of specific genes, such as c-MYC, HER2/neu, and androgen receptor, have been shown to be of prognostic relevance (1). However, most of the chromosomal regions found amplified in prostate cancer by comparative genomic hybridization (CGH) do not harbor known oncogenes. Although the amplification target genes remain mostly unknown, the presence of DNA copy number gains at particular regions may be associated with tumor aggressiveness or clinical outcome as shown for chromosome 7q (2, 3).

Most recently, we reported DNA copy number gains at chromosome 3q25-q26 in 50% of prostate carcinomas using CGH (4). Similar frequencies of 3q gains were described by others, although various sites on 3q are likely to be involved (5, 6). In this report, we describe the fine mapping of the 3q25-q26 amplification unit in prostate cancer and the identification of TLOC1/SEC62 as the gene with the highest frequency of gene copy number gains compared with other positional candidates. Overexpression at the mRNA and protein levels indicates a biological role of TLOC1/SEC62 in prostate cancer development.

	Results

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
DNA Copy Number Gains on 3q25-q26 Are Frequent in Prostate Cancer as Detected by CGH
To assess frequency and extent of genetic alterations on chromosome 3q in prostate cancer, CGH was done on 3 prostate cancer cell lines and 22 primary prostate cancer samples. The CGH findings in the cell lines have been published elsewhere (7). In brief, DNA copy number gains on 3q had been detected in DU145 and DU145MN1 encompassing the region 3q22-qter, whereas PC3 had displayed no copy number changes on 3q. Of the 22 primary tumor samples, 3q gains were documented in 8 (36.4%) cases with the minimal overlapping region mapping to 3q21-q26. Including our previous CGH series with a total of 80 prostate carcinomas (4, 8), the common region of DNA copy number gains can be narrowed down to 3q25-q26. The overall incidence of 3q gains involving 3q25-q26 in prostate cancer attains 31.3%.

Detailed Mapping of the 3q25-q26 Amplicon
We first did fluorescence in situ hybridization on the cell lines DU145, DU145MN1, and PC3 applying 16 BACs within 3q25-q28 (Fig. 1 ). With this approach, the region with the highest signal numbers in the three cell lines could be narrowed down to 1.4 Mb within 3q26.2. This region was then analyzed in more detail using seven overlapping BACs (RP11-627P8, RP11-82C9, RP11-816J6, RP11-362K14, RP11-379K17, RP11-81O18, and RP11-543D10). The region between RP11-82C9 and RP11-816J6 encompassing 500 kb does not contain genes according to the Ensembl database (http://www.ensembl.org) and was therefore not investigated. With this step, the region with highest signal numbers could further be narrowed down to 700 kb between RP11-816J6 and RP11-543D10 at 3q26.2. It is of note that the highest signal numbers for these distinct BACs were detected in DU145 and DU145MN1, whereas PC3 showed only a slight increase above ploidy level.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. 3q copy number determination in the prostate cancer cell lines DU145, DU145MN1, and PC3 using fluorescence in situ hybridization of BAC probes. The area with the highest BAC signal numbers was assigned to 3q26.2 encompassing a length of 700 kb between RP11-816J6 and RP11-543D10.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. DNA copy number quantification of selected genes in 22 prostate cancer samples using real-time PCR. The genes are located within and around the area with the highest BAC signal numbers in the prostate cancer cell lines.

As the most interesting candidate, TLOC1/SEC62 gene expression was further tested in 6 benign prostatic hyperplasia samples, which revealed a >3-fold up-regulation in only 2 samples (3.9- and 4.6-fold). The remainder varied between 0.69- and 2.85-fold.

Expression Analyses of the TLOC1/SEC62 Gene by Western Blot Analyses
To confirm the PCR data at the protein level and to address the question of functionality of the quantified mRNA, Western blot analysis was done. Similar amounts of protein that were derived from various cell lines were subjected to SDS-PAGE and Western blot analysis, employing an antipeptide antibody directed against the COOH terminus of TLOC1/Sec62 protein. In addition, the blots were analyzed with antibodies that were directed against Sec61p, an unrelated, ubiquitously expressed protein of the endoplasmic reticulum, and ß-tubulin, a cytoskeleton-associated protein. The primary antibodies were visualized by luminescence imaging (Fig. 3 ). The ratio of TLOC1/Sec62 protein to ß-tubulin and Sec61p, respectively, was determined for the benign prostate hyperplasia cell line BPH1 (DSMZ no. ACC 143, German Resource Centre for Biological Material, Braunschweig, Germany) as well as for the prostate cancer cell lines PC3, DU145, and DU145MN1 in six independent analyses. Then, each ratio for the BPH1 cells was independently set to 1 and the ratios for the cancer cell lines that were analyzed on the same blot were normalized to the BPH1 ratios. On average, there was an 1.5-fold increase in the ratios between TLOC/Sec62 protein and the control proteins in the case of the PC3 cells, whereas in the other two cancer cell lines the increase was 3.5-fold. Thus, increased synthesis of TLOC1/Sec62 protein was observed in all tested tumor cell lines albeit to varying degree.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. SDS-PAGE and Western blot analysis of prostate cancer (DU145MN1, DU145, and PC3) and benign prostate hyperplasia (BPH1) cell lines. Cytoskeleton-associated (ß-tubulin) and endoplasmic reticulum–resident (Sec61p) proteins were quantified with respect to the Sec62p content. A single experiment is depicted as original together with its luminescence values (divided by 1,000). In addition, averaged and normalized values are shown as diagram (n = 6). The ratios of the detected proteins for BPH1 cells were set to 1.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Box plot showing the relation between TLOC1/SEC62 gene copy number in the tumor samples and clinical/biochemical progression after radical prostatectomy in 22 prostate cancer patients (15 nonprogressors and 7 progressors; P = 0.024). Dotted lines, cutoff values for significant TLOC1/SEC2 gene copy number deviation from normal.

	Discussion

Top Notes Abstract Introduction Results Discussion Materials and Methods References

Using fluorescence in situ hybridization, we could narrow down the minimally amplified region in the prostate cancer cell lines to a locus encompassing 700 kb at 3q26.2. To test for putative target genes of amplification within and around this locus, quantitative real-time PCR was done revealing copy number gains for the genes IL12A, MDS1, hTERC, SAMD7, TLOC1/SEC62, SKIL, SLC2A2, or PIK3CA in at least one of the prostate cancer cell lines analyzed. Determination of the copy number status of these genes in 22 prostate cancer samples revealed the gene TLOC1/SEC62 to be most frequently increased in 50% of the samples. Less frequently increased were the genes SAMD7 and SLC2A2 in 32% and 27%, respectively, and the genes hTERC and PIK3CA in 9% each. There were no SKIL gene copy number gains detected. Fitting these findings, the mRNA overexpression frequency was highest for TLOC1/SEC62 (100% of the prostate cancer samples) followed by SLC2A2 (62%) PIK3CA (54%), and hTERC (39%). SAMD7 was not found to be expressed at all. As TLOC1/SEC62 mRNA overexpression was not restricted to tumors with TLOC1/SEC62 gene copy number gain, other regulatory mechanisms have to be assumed emphasizing a general importance of this gene in prostate cancer development or growth. This assumption is in accord to other findings showing a generalized overexpression of genes, such as E2F3, in bladder cancer but with a gene copy number increase in only a subset of tumors (15).

Considering potential clinical implications, the TLOC1/SEC62 gene copy number turned out to be significantly increased in tumors from patients who did not develop progression following radical prostatectomy compared with those who did. Splitting the patients into two subgroups defined by the presence or absence of TLOC1/SEC62 gene copy number gains, log-rank analysis revealed a lower risk of and a longer time to progression for patients with a TLOC1/SEC62 gene copy number gain. However, because of the small number of patients and the short follow-up time, precaution should be used in the interpretation of these results until larger studies and longer follow-up intervals have confirmed these data.

The high frequency of gene copy number gain and mRNA overexpression makes the gene TLOC1/SEC62 the best candidate within the 3q25-q26 amplification unit in prostate cancer. This assumption was strongly supported by Western blot analyses, which showed an up to 3.5-fold increase of TLOC1/Sec62 protein production in the DU145 and DU145MN1 cell lines, both revealing TLOC1/SEC62 gene amplification, whereas the TLOC1/Sec62 protein expression in PC3, for which no TLOC1/SEC62 gene copy number gain could be documented but that showed increased TLOC1/SEC62 mRNA content, was increased by a factor of 1.5 compared with benign prostate hyperplasia cell line BPH1.

TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery. First described in the yeast Saccharomyces cerevisiae, the protein transport across the endoplasmic reticulum membrane is mediated by a membrane protein complex that consists of the trimeric Sec61p complex and the Sec62p-Sec63p subcomplex (16, 17). Although the association of TLOC1/Sec62 protein with the Sec61 complex in mammalian cells indicates involvement of this protein in transport processes similar to those done by the yeast Sec complex (18, 19), the precise function of the TLOC1/Sec62 protein in mammalian cells is still unknown. Our finding of TLOC1/Sec62 overexpression in prostate cancer cells is the first description of TLOC1/SEC62 up-regulation in tumor cells at all. Additional work will be required to precisely define its pathogenetic role, if any, in prostate cancer development. It is noteworthy that the orthologue of TLOC1/SEC62 in Drosophila, termed DTRP1, is maximally expressed at certain stages of embryonic and pupal development and that production of one of the two DTRP1 transcripts is by far the highest in the paragonial glands, which are somewhat equivalent organs to prostate glands (20). Furthermore, we note that TLOC1/Sec62 protein is classified as a potential nonselective cation channel by the protein families database of alignments and that another nonselective cation channel, TRPV6, is overexpressed in human prostate cancer cells (21).

In addition to prostate cancer, gain of sequences at 3q has also been identified in other malignancies, including tumors of the cervix (22), ovary (23), lung (10), head and neck (24), and esophagus (9). Although the region 3q26 seems to be consistently involved, the molecular counterpart seems less consistent in the different tumor types. Whereas SKIL is a likely target in esophageal carcinoma (9), PIK3CA and hTERC seem to play a biological role in cancer of the ovary and cervix, respectively (11, 22). We could document gene copy number gain and overexpression of the gene TLOC1/SEC62 in prostate cancer but could exclude SKIL and PIK3CA as amplification targets in this tumor type. It seems most likely that the 3q26 region harbors various genes that contribute differentially to carcinogenesis in different organs. The biological role of TLOC1/SEC62 in prostate carcinogenesis and the value of TLOC1/SEC62 gene copy number gain as a diagnostic or prognostic marker in prostate cancer remain to be clarified in future studies.

	Materials and Methods

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Cell Lines and Tissue Samples of Prostate Carcinoma
For fine mapping of the 3q25-q26 amplification unit, prostate cancer cell lines DU145 (ATCC no. HTB-81) and PC3 (ATCC no. CRL-1435) as well as the derived subline DU145MN1 (7) were used. Quantitative gene copy number and expression measurements of positional candidates were done on primary prostate adenocarcinoma samples, which were obtained after radical prostatectomy from thus far untreated prostate cancer patients. The specimens represent a subset of a previously described series (25) that was selected by the criteria of a complete standardized patient follow-up. Following prostatectomy, the specimens were dissected by a pathologist, snap frozen, and stored at –80°C. Only samples containing >50% tumor cells were included in the study. In the present subset of 22 prostate cancer samples, 6 carcinomas were staged as pT₂ and 16 as pT₃ according to the tumor-node-metastasis classification system, 5th revision (26). Concerning Gleason sum, 2 were graded as <7, 11 as 7, and 9 as >7. Lymph node metastases were present in 6 cases. All patients were clinically staged as M₀. Following surgery, clinical status and serum prostate-specific antigens were monitored every 3 months during the first year, every 6 months in the second year, followed by yearly monitoring. The end point for calculating the survival time was defined by time from radical prostatectomy to either biochemical (prostate-specific antigen > 0.2 ng/mL or rising prostate-specific antigen in three consecutive measurements) or clinical progression. The study was approved by the local ethics committee.

Comparative Genomic Hybridization
CGH was done as described (7). Applying nick translation, 1 µg test DNA was labeled with biotin-16-dUTP and control DNA with digoxigenin-11-dUTP. Biotinylated DNA was visualized with streptavidin-FITC, and digoxigenin-haptenized DNA was visualized with anti-digoxigenin-rhodamine. Chromosomes were embedded in Antifade with 0.15 µg 4',6-diamidino-2-phenylindole. CGH analysis was done using the software package ISIS (MetaSystems, Altlusheim, Germany). Chromosomal imbalances were scored as losses or gains when the profile differed from the mean by at least 3 SDs. A minimum of 12 to 15 metaphase spreads for each sample was analyzed.

Fluorescence In situ Hybridization
BAC clones from the human RPCI-11 and RPCI-13 BAC libraries were hybridized to interphase nuclei and metaphase spreads of the cell lines (Fig. 1). Physical locations of BAC clones were based on the information available at the University of California at Santa Cruz Human Genome Browser Gateway (http://genome.ucsc.edu; RP11-286N6, RP11-91L9, RP11-209H21, RP11-67F24, RP11-90M7, RP11-190F16, RP11-148D23, RP11-292L5, RP11-245C23, RP11-259I19, RP11-102G2, RP11-12L14, RP11-125E8, RP11-63M3, and RP11-79K10) and the Ensembl database (http://www.ensembl.org; RP11-627P8, RP11-82C9, RP11-816J6, RP11-362K14, RP11-379K17, RP13-81O8, and RP11-543D10). The clones were obtained from BACPAC Resource Center Children's Hospital (Oakland Research Institute, Oakland, CA). Bacterial cultures and DNA isolation were done according to the BAC-PAC Miniprep protocol (http://www.biologia.uniba.it/rmc). Alu-PCR products of the BACs were used as probes and were biotinylated using nick translation. A digoxigenin-labeled D3Z1 probe specific for the centromeric region of chromosome 3 (Qbiogene, Illkirch, France) served as internal control. Dual-color fluorescence in situ hybridization and detection of fluorescence signals were done as described previously (7) with the following modifications: the hybridization mixture contained 50% formamide, 10% dextran sulfate, 2x SSC, 10 µg/µL human Cot-1 DNA, 500 ng BAC probe DNA, and 0.5 µL D3Z1 probe DNA. This mixture was denatured at 75°C for 5 minutes and preannealed in a 37°C water bath for 15 minutes before it was applied to the slides.

Quantitative Real-time PCR
Quantitative real-time PCR for gene copy number measurement was done as described recently (27) using the LightCycler system (Roche, Mannheim, Germany) and the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany). Relative gene copy number was calculated from the real-time PCR efficiencies (E), which were determined for each individual run, and the crossing point (CP) deviations of the target and reference genes in a test sample versus a control following the equation: E_target^{CPtarget (control – sample)} / E_reference^{CPreference (control – sample)} (28). Because the gene copy number is 2 in normal diploid DNA, the relative copy number was multiplied by 2 to achieve the copy number in the test sample for the target gene. PCR was done at least in duplicates with 20 µL reaction volumes containing 10 µL of 2x QuantiTect SYBR Green PCR Master Mix, 0.5 µmol/L of each primer, 2.5 to 5 mmol/L MgCl₂, and 25 ng genomic DNA. Primer sequences and cycling variables are listed in Table 1 . GAPDH, which is located at 12p13, served as reference gene. The region 12p13 has been shown not to be involved in prostate cancer. The existence of specific primer binding sites in pseudogenes of GAPDH was excluded by use of the BLASTN algorithm.

Quantitative Real-time Reverse Transcription-PCR
Quantitative real-time PCR for mRNA expression was done using the LightCycler system and the QuantiTect SYBR Green PCR kit. Total RNA was isolated using the RNeasy Mini kit (Qiagen). RNA quality was controlled by spectrophotometrical quantification and agarose gel electrophoresis. Reverse transcription of 1 µg total RNA was done with SuperScript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) using oligo(dT)_12-18 primer (Invitrogen) and RNase OUT (Invitrogen) after pretreatment with RNase-free DNase I (Stratagene, Amsterdam, the Netherlands). PCR was done at least in duplicates with 20 µL reaction volumes containing 10 µL of 2x QuantiTect SYBR Green PCR Master Mix, 0.5 µmol/L of each primer, and 2 µL cDNA (1:20 dilution). Primer sequences and cycling variables are listed in Table 2 .

Western Blot Analysis
The rabbit antibodies were directed against the COOH-terminal peptide of TLOC1/Sec62 protein (CGETPKSSHEKS), COOH-terminal peptide of Sec61p (CKEQSEVGSMGALLF), and ß-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA). The antibodies reacted only with the expected proteins in immunoblots. The samples were analyzed by electrophoresis in 12.5% polyacrylamide gels and subsequent electroblotting to polyvinylidene fluoride membranes followed by incubation with specific antibodies and peroxidase conjugate of goat anti-rabbit IgG. The antibodies were visualized by incubation of the blots with enhanced chemiluminescence (Amersham Biosciences, Freiburg, Germany) and subsequent analysis with a Lumi-Imager F1 (Roche).

Statistical Analysis
For all variables complying with normal Gaussian distribution, Student's t test was applied for comparison of independent sample sets. Variables not complying with normal Gaussian distribution were analyzed by Mann-Whitney U test. All Ps were based on two-sided tests and the threshold to accept statistical significance was set at the level 0.05. Log-rank test was applied as statistical hypothesis test procedure for the comparison of time to progression after surgical therapy in the independent groups of tumors with a TLOC1/SEC62 gene copy number gain and those without. Analyses were done with the Statistical Software Package SPSS version 10.0 (SPSS, Chicago, IL).

	Notes

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Grant support: Deutsche Krebshilfe 70-2936-Wu I (B. Wullich) and 70-3193-Schu I (W.A. Schulz) and Deutsche Forschungsgemeinschaft SFB 530 (R. Zimmermann).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: V. Jung and R. Kindich contributed equally to this work.

Received 9/ 2/05; revised 1/24/06; accepted 1/27/06.

	References

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 

1. Porkka KP, Visakorpi T. Molecular mechanisms of prostate cancer. Eur Urol 2004;45:683–91.[CrossRef][Medline]
2. Alers JC, Rochat J, Krijtenburg PJ, et al. Identification of genetic markers for prostatic cancer progression. Lab Invest 2000;80:931–42.[Medline]
3. Chu LW, Troncoso P, Johnston DA, Liang JC. Genetic markers useful for distinguishing between organ-confined and locally advanced prostate cancer. Genes Chromosomes Cancer 2003;36:303–12.[CrossRef][Medline]
4. Sattler HP, Lensch R, Rohde V, et al. Novel amplification unit at chromosome 3q25-27 in human prostate cancer. Prostate 2000;45:207–15.[CrossRef][Medline]
5. Cher ML, Bova GS, Moore DH, et al. Genetic alterations in untreated metastases and androgen-independent prostate cancer detected by comparative genomic hybridization and allelotyping. Cancer Res 1996;56:3091–102.[Abstract/Free Full Text]
6. Strohmeyer DM, Berger AP, Moore DH, et al. Genetic aberrations in prostate carcinoma detected by comparative genomic hybridization and microasatellite analysis: association with progression and angiogenesis. Prostate 2004;59:43–58.[CrossRef][Medline]
7. Lensch R, Götz C, Andres C, et al. Comprehensive genotypic analysis of human prostate cancer cell lines and sublines derived from metastases after orthotopic implantation in nude mice. Int J Oncol 2002;21:695–706.[Medline]
8. Rahnenführer J, Beerenwinkel N, Wullich B, Schulz WA, Deimling von A, Lengauer T. Estimating cancer survival and clinical outcome based on genetic tumor progression scores. Bioinformatics 2005;21:2438–46.[Abstract/Free Full Text]
9. Imoto I, Pimkhaokham A, Fukuda Y, et al. SKL is a probable target for gene amplification at 3q26 in squamous-cell carcinomas of the esophagus. Biochem Biophys Res Commun 2001;286:559–65.[CrossRef][Medline]
10. Brass N, Racz A, Heckel D, Remberger K, Sybrecht GW, Meese EU. Amplification of the genes BCHE and SLC2A2 in 40% of squamous cell carcinoma of the lung. Cancer Res 1997;57:2290–4.[Abstract/Free Full Text]
11. Campbell IG, Russell SE, Choong DYH, et al. Mutation of the PIK3CA gene in ovarian and breast cancer. Cancer Res 2004;64:7678–81.[Abstract/Free Full Text]
12. Li XL, Eishi Y, Bai YQ, et al. Expression of the SRY-related HMG box protein SOX2 in human gastric cancer. Int J Oncol 2004;24:257–63.[Medline]
13. Wolf M, Mousses S, Hautaniemi S, et al. High-resolution analysis of gene copy number alterations in human prostate cancer using CGH on cDNA microarrays: impact of copy number on gene expression. Neoplasia 2004;6:240–7.[CrossRef][Medline]
14. Paris PL, Andaya A, Fridlyand J, et al. Whole genome scanning identifies genotypes associated with recurrence and metastasis in prostate tumors. Hum Mol Genet 2004;13:1303–13.[Abstract/Free Full Text]
15. Wu Q, Hoffmann MJ, Hartmann FH, Schulz WA. Amplification and overexpression of the ID4 gene at 6p22.3 in bladder cancer. Mol Cancer 2005;4:16.[CrossRef][Medline]
16. Deshaies RJ, Sanders SL, Feldheim DA, Schekman R. Assembly of yeast Sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex. Nature 1991;349:806–8.[CrossRef][Medline]
17. Wittke S, Dünnwald M, Johnsson N. Sec62p, a component of the endoplasmic reticulum protein translocation machinery, contains multiple binding sites for the Sec-complex. Mol Biol Cell 2000;11:3859–71.[Abstract/Free Full Text]
18. Tyedmers J, Lerner M, Bies C, et al. Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes. Proc Natl Acad Sci U S A 2000;97:7214–9.[Abstract/Free Full Text]
19. Wirth A, Jung M, Bies C, et al. The Sec61p complex is a dynamic precursor activated channel. Mol Cell 2003;12:261–8.[CrossRef][Medline]
20. Noel PJ, Cartwright IL. A Sec62p-related component of the secretory protein translocon from Drosophila displays developmentally complex behavior. EMBO J 1994;13:5253–61.[Medline]
21. Fixemer T, Wissenbach U, Flockerzi V, Bonkhoff H. Expression of the Ca²⁺-selective cation channel TRPV6 in human prostate cancer: a novel prognostic marker for tumor progression. Oncogene 2003;22:7858–61.[CrossRef][Medline]
22. Heselmeyer-Haddad K, Sommerfeld K, White NM, et al. Genomic amplification of the human telomerase gene (TERC) in Pap smears predicts the development of cervical cancer. Am J Pathol 2005;166:1229–38.[Abstract/Free Full Text]
23. Arnold N, Hagele L, Walz L, et al. Overrepresentation of 3q and 8q material and loss of 18q material are recurrent findings in advanced human ovarian cancer. Genes Chromosomes Cancer 1996;16:46–54.[CrossRef][Medline]
24. Singh B, Gogineni SK, Sacks PG, Shaha JP, Stoffel A, Rao P. Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement of 3q amplification. Cancer Res 2001;61:4506–13.[Abstract/Free Full Text]
25. Florl AR, Steinhoff C, Müller M, et al. Coordinate hypermethylation at specific genes in prostate carcinoma precedes LINE-1 hypomethylation. Br J Cancer 2004;91:985–94.[CrossRef][Medline]
26. Sobin LH, Wittekind C. TNM classification system of malignant tumours, 5th ed. Berlin: Springer-Verlag; 1997. p. 162–5.
27. Kindich R, Florl AR, Jung V, et al. Application of a modified real-time PCR technique for relative gene copy number quantification to the determination of the relationship between NKX3.1 and MYC gain in prostate cancer. Clin Chem 2005;51:649–52.[Free Full Text]
28. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.[Abstract/Free Full Text]

This article has been cited by other articles:

				D. N. Hebert and M. Molinari In and Out of the ER: Protein Folding, Quality Control, Degradation, and Related Human Diseases Physiol Rev, October 1, 2007; 87(4): 1377 - 1408. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 1541-7786.MCR-05-0165v1 4/3/169    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jung, V. Articles by Wullich, B. Search for Related Content PubMed PubMed Citation Articles by Jung, V. Articles by Wullich, B.