Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Pathology (December 2006) 38(6), pp. 561–567 EXPERIMENTAL PATHOLOGY Knockdown of Rab25 expression by RNAi inhibits growth of human epithelial ovarian cancer cells in vitro and in vivo FAN YANG, XIN XIAO-YAN, CHEN BI-LIANG AND MA XIANGDONG Department of Obstetrics and Gynecology, Xijing Hospital Fourth Military Medical University, Xi’an, China Summary Aims: Ovarian cancer is the leading cause of cancer death among gynaecological malignancies. Elevated expression of Rab25 has been seen in this malignancy. To better understand its role in maintaining the malignant phenotype, we used RNA interference (RNAi) directed against Rab25 in our study. RNAi provides a new, reliable method to investigate gene function and has the potential for gene therapy. The aim of the study was to examine the anti-tumour effects elicited by a decrease in the level of Rab25 by RNAi and its possible mechanism of action. Methods: According to the Rab25 mRNA sequence in Genbank, a pair of 64 nt oligonucleotides were designed and synthesised, each containing the sites of restriction endonuclease at both ends. Oligonucleotides were annealed and ligated with linearised pSUPER by ligase. The recombinants (named pSUPER/Rab25 siRNA) were finally sequenced and identified by enzyme cutting and sequencing. The human ovarian cell line A2780 was grown without transfection, transfection with empty vector and with pSUPER/Rab25 siRNA with electroporation. The inhibitory effect was examined by RTPCR, MTT, FCM and tumour growth of athymic nude mice. Results: Rab25 siRNA expression vector was successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragment revealed the same sequence as synthesised siRNA oligonucleotides. Cells transfected with Rab25 siRNA can specifically knock down the transcription of Rab25, exhibiting cells with slower proliferation, increased apoptosis, and decreased tumour growth. Conclusions: Rab25 siRNA expression vector has been successfully constructed, and it could inhibit the tumour growth both in vitro and in vivo. Our data suggest that the Rab25 signalling pathway plays a role in the regulation of cell proliferation and apoptosis in ovarian cancer cells, which indicates that the Rab25 gene plays a definite role in the development and aggressiveness of human ovarian cancer and should be further elucidated as a possible therapeutic target of ovarian cancer. Key words: RNA interference, siRNA, Rab25, ovarian cancer, epithelial cell. Received 7 June, revised 30 July, accepted 1 August 2006 INTRODUCTION Ovarian cancer is a leading cause of death among gynaecological malignancies. In spite of advances in surgery and chemotherapy, its mortality rate has remained relatively unchanged during the past several decades.1 Therefore, an understanding of the molecular mechanisms involved in ovarian cancer formation and progression should be helpful in developing more effective treatments for ovarian cancer. Emerging evidence implicates alterations in the Rab small GTPases and their associated regulatory proteins and effectors in multiple human diseases including cancer.2 Rab proteins are Ras-like small GTPases, which are involved in regulating various aspects of the membrane trafficking process.3 More than 60 Rabs are present in distinct membrane compartments in mammalian cells.4 Studies have recently shown that Rab25, located at chromosome 1q22, is amplified at the DNA level and over-expressed at the RNA level in ovarian and breast cancer. These changes correlated with a worse outcome in both diseases. In addition, enforced expression of Rab25 in both breast and ovarian cancer cells decreased apoptosis and increased proliferation and aggressiveness in vivo, potentially explaining the worse prognosis.5 A better understanding of genetic alterations as well as the physiological and pathophysiological roles of Rab GTPases may open new opportunities for therapeutic intervention and better outcomes. Technologies for gene knockout, antisense oligonucleotides and ribozyme have been frequently used to explore new functions of genes, but their low frequencies have limited their applications.6 Fortunately, the emergence of gene ablation technologies based upon the RNA interference (RNAi) phenomenon has provided new opportunities for experimental biology.7,8 Particularly, a generation of vectors directing the synthesis of short hairpin RNAs (shRNAs) that are processed to form small interfering RNAs (siRNAs), enables persistent suppression of endogenous gene expression.9,10 RNA interference (RNAi) can result in sequence-specific gene silencing. This phenomenon is now being exploited as a powerful tool for reverse genetics, and shows great promise for therapeutic applications.11 In the present study, we used RNAi technology to knock down the expression of the Rab25 gene in human ovarian epithelial cancer cells and to examine their effect in human ovarian cancer cells in vitro and on tumour growth in vivo, which may establish candidate status of Rab25mediated signalling for studies in targeted ovarian cancer therapy. ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia DOI: 10.1080/00313020601024037 562 YANG et al. Pathology (2006), 38(6), December MATERIALS AND METHODS siRNA design Three pairs of siRNA were designed according to the human Rab25 sequence in Genbank (BC 009831; Qiagen, Germany) and selected optimal sites of interfering. As shown in Table 1, each pair contained a unique 19-nt double-stranded human Rab25 sequence that was presented as an inverted complementary repeat and separated by a loop of 9 nt spacer. For example: S1 : 50 GAT CCCCðs -N19ÞTTCAAGA GA(as-N19)TTTTTGGAAA 30 ; S2 : 30 GGG ðas-N19Þ A AGTTCTCTðs-N19ÞAAAAACCTTTTCGA 50 human Rab25, sense 5’-GGGGAATGGAACTGAGGAAGA-3’ and antisense 5’-ATTGGTCCGGATGCTGTTCT-3’; for glyceraldehydes-3phosphate dehydrogenase (GAPDH), sense 5’-CAGGGCTGCTTTTAACTCTGG-3’; anti-sense 5’-GTGGACTCCACGACGTACTCA-3’. The newly synthesised cDNA was amplied by PCR. The reaction mixture contained 2 mL cDNA template, 1.5 mM MgCl2, 2.5 U Tag polymerase, and 0.5 mM GAPDH was used as an internal control. Amplification cycles were: 94uC for 3 min, then 33 cycles at 94uC for 1 min, 58uC for 1 min, 72uC for 1.5 min, followed by 72uC for 15 min. Aliquots of PCR product were electrophoresed on 1.5% agarose gels and PCR fragments were visualised by ethidium bromide (EB) staining. All experiments were repeated on at least three occasions. Cell proliferation, MTT assay and flow cytometric analysis Plasmid construction, transformation and extraction Oligonucleotides sequence of Rab25 siRNA was chemically synthesised by TakaRa (Japan). Oligonucleotides were annealed in a buffer containing 10 mM Tris (pH 8.0), 10 mM NaCl and 1 mM EDTA at 95uC for 5 min followed by 70uC for 10 min. Then ligated into pSUPER vector (Ambion, USA) that had been cut with Bgl II and Hind III (TakaRa). The plasmids were then transformed into DH5a competent cells according to the protocol provided by the manufacturer (TakaRa). After selective culture, recombined plasmid was extracted from DH5a. The recombined plasmid was identified via restriction enzyme EcoRI and Hind III (TakaRa) using guidelines provided by the manufacturer. The positive clones were confirmed by sequencing (TakaRa). Reactions were carried out in 20 mL volume containing 2 mL of DNA. Following this, amplification was performed. Medipreps DNA purification system (Promega, USA) was used to extract plasmid DNA according to the protocol. Cell culture and transient transfection We selected epithelial ovarian cancer cells A2780.5 The ovarian cancer cell line A2780 was obtained from the laboratory of the Department of Obstetrics and Gynecology, Xijing Hospital Fourth Military Medical University, Xi’an, China. This was cultured in RPMI 1640 (Invitrogen, USA) supplemented with 10% heat inactivated fetal bovine serum, 100 U/ mL penicillin, and 100 mg/mL streptomycin at 37uC in a humidified atmosphere of 5% CO2 and 95% air. For transfections, cells were seeded in a 6-well plate at a concentration of 16106 cells/well and allowed overnight growth to reach 80–90% confluency.The stable transfections of pSUPER/Rab25 siRNA were performed in A2780 cells using the Lipofectamine 2000 (Gibco, USA) following the guidelines provided by the manufacturer. A pure population of transfected cells can be obtained using a low concentration of G418 (400 mg/mL). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis or other experiments were performed. We plated cells at a density of 16105 cells/35-mm dish. Cells were then cultured in the presence of 10% FBS for 8 days. We harvested and counted total cells. Cells were grown in a 96-well plate with a concentration of 26105 and transfected with plasmids (pSUPER/Rab25 siRNA) using Lipofectamine 2000 (a total of 50 mL). The tetrazolium salt MTT solution (5 mg/mL; Sigma, USA) was added into each well and incubated at 37uC for 4 h. The media were carefully aspirated. The blue formazan product converted from MTT was dissolved by the addition of 150 mL/well dimethyl sulfoxide (DMSO; Sigma) and optical density (OD) readings were obtained using an autoreader (Tecan, Austria) at 490 nm. Each experiment was repeated three times. Flow cytometry (Beckman-Coulter, USA) analysis was used to determine apoptosis and cell cycle of the cells. In brief, A2780 cells were transfected with Rab25 siRNA or without Rab25 siRNA; cells (56105,16106) were harvested, washed twice with PBS, and resuspended in 100 mL PBS and stained with propidium iodine (PI; Sigma) for apoptosis and cell cycle analysis. Tumour growth Equal numbers (106,26106) of A2780 cells, without transfection, transfection empty vector, and transfection with pSUPER/Rab25 siRNA cells were harvested by trypsinisation, washed twice with 16PBS, and resuspended in 0.1 mL saline. The suspensions were then injected subcutaneously into 4- to 6-week-old athymic nude mice. A total of 15 mice was used for each cell line infected with the above three cells. The mice were kept in pathogen-free environments and checked every 2 days for 1.5 months. The date at which a grossly visible tumour first appeared and the size of the tumour were recorded. The mice were killed when tumours reached 2.5 cm in diameter. Statistical analysis RNA extraction and RT-PCR analysis Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The reverse transcription reaction was performed using the Superscript First-Strand Synthesis System (Invitrogen Life Technologies, USA) according to the protocol. After incubation at 42uC for 80 min, the reverse transcription reaction was terminated by heating at 70uC for 15 min. DNA primer sequences were designed as follows: for TABLE 1 Oligonucleotides sequence of Rab25 specific siRNA Name pSUPER/ Rab25siRNA 1 pSUPER/ Rab25siRNA 2 pSUPER/ Rab25siRNA 3 Sequence of siRNA Target nucleotides sites TCCCTCTGGCTGCAGAAGT 75–93 bp GGTGGTGCTGATCGGCGAA 265–283 bp TGCCATCACTCTGG GCAGT 787–805 bp Data are expressed as mean¡SEM. The significance of differences was determined by ANOVA and post-hoc multiple comparison test using SPSS 11.5 software (SPSS, USA). p,0.05 was considered to be statistically significant. RESULTS Plasmid identification We designed and synthesised three Rab25 siRNA sequences, and ligated the sequences into pSUPER vector. The recombined plasmid (named pSUPER/Rab25 siRNA) was finally sequenced and identified by enzyme cutting and sequencing. The result of sequencing confirmed that siRNA had been ligated to the vector. The transfected vector extracted was digested with EcoRI and Hind III, then checked with 1.5 % agarose gel electrophoresis. The length of the fragments of 291 kb (recombined plasmid) and 227 kb (empty plasmid) showed the success of construction as expected (Fig. 1). RNA INTERFERENCE INHIBITS EXPRESSION OF RAB25 563 TABLE 2 Decrease in cell proliferation by Rab25 siRNA A2780 103.5¡7.5 Empty vector Rab25 siRNA 106.9¡7.4 38.5¡3.5* *p,0.01. Cell counts were performed on day 8 in stable cells lines. Numbers represent cell number 610 000 cells/mL. Each sample is presented¡SEM of three replicates from one of three representative experiments. Results were confirmed with multiple subclones of each cell line. Clearly, cells transfected with Rab25 siRNA showed significantly decreased cell proliferation and cell counts were significantly less than in the controls. Fig. 1 Identification of interfering vector by enzyme cutting. 1, DNA marker; 2, pSUPER/Rab25 siRNA3; 3, empty vector. The knock-down efficiencies of pSUPER/Rab25 siRNA in A2780 cells were first evaluated using RT-PCR. After transfection, relative Rab25 levels were normalised against mRNA levels of an internal control gene, GAPDH, performed in the same run. As shown in Fig. 2, cells transfected with pSUPER/Rab25 siRNA showed a significantly reduced transcription of Rab25 mRNA when compared with empty vector and cells without transfectants, respectively. In 1–4 months, the suppression of pSUPER/Rab25 siRNA 3 in the cultured transfection cells was more powerful than the others. Further examination was carried out with MTT and flow cytometry to observe the effect of decreasing Rab25 levels. Slower proliferation and increased apoptosis in Rab25 silenced cells Under 10% fetal bovine serum (FBS) conditions, cells with transfected pSUPER/Rab25 siRNA 3 decreased cell numbers stably (Table 2). We examined the viability of Rab25 specific siRNA transfectants. The results showed that RNAi directed against Rab25 significantly decreased the growth rate of A2780 cells. As shown in Fig. 3, pSUPER/Rab25 siRNA transfected A2780 cells began to show a decreased viability at 48 hours after transfection and the decline slowed down and became mild at 72 hours. The two control cell lines grew rigorously with the cell number increased at 48 hours and the increase proceeding until 72 hours (Fig. 3). The knock-down of Rab25 in A2780 cells could significantly inhibit the growth of tumour cells in vitro. Apoptosis analysis indicated that the number of apoptotic cells increased in A2780 cells transfected with pSUPER/ Rab25 siRNA compared with the controls (Fig. 4). pSUPER/Rab25 siRNA transfected A2780 cells were most prone to apoptosis with an apoptotic rate of 12.3%, higher than the rate of 0.9% in the A2780 control cells. Thus, treatment with siRNA against Rab25 could activate apoptotic pathways. Different cell cycle phases (G1, S or G2/M phase) are characterised by different DNA contents. Table 3 shows differences in cell cycle phases in A2780 cells transfected with or without Rab25 siRNA. It was clear that pSUPER/ Rab25 siRNA transfection induced an increase in the number of cells in G1 phase and a decrease in the number of cells in S phase compared with the control (Table 3; Fig 5). Decreases in transformation efficiency in vitro and tumour growth in vivo Injections of A2780 cells in nude mice demonstrated relevant tumour biology. In this study, tumour growth was inhibited by treatment with Rab25 siRNA. Nude mice were randomly selected for treatment with Rab25 siRNA which would affect tumour growth in vivo. A2780 cells transfected with or without the empty vector produced Fig. 2 RT-PCR analysis. (A) Rab25 and (B) GAPDH. RT-PCR analysis showed a lower mRNA level in A2780 cells transfected with Rab25 siRNA. M, marker; 1, A2780; 2, empty vector; 3, Rab25 siRNA3. 564 YANG et al. Fig. 3 Growth curve of A2780 cells after Rab25 siRNA transfection. It can be seen that, when compared with both the empty vector and A2780, cells transfected with Rab25 siRNA show a remarkable reduction of cell proliferation, in keeping with the observations of decreased expression of Rab25 in these transfectants. tumours 25 mm in diameter within 4 weeks of their subcutaneous injection into nude mice, while A2780 cells transfected with Rab25 siRNA showed only minimal tumour growth at 4 weeks (Fig. 6). Furthermore, Rab25 siRNA signicantly inhibited production of A2780 in the tumours. Therefore, transfected Rab25 siRNA is a signicantly novel way to inhibit tumour growth in vivo. DISCUSSION As far as we know, cancer is caused by abnormalities in DNA sequence, copy number, rearrangements, or Pathology (2006), 38(6), December expression. The accumulation of multiple changes in critical genes within a single cell is required to escape from normal controls on cell growth and proliferation, allowing development into a clinically evident tumour.11 Cancer cells often show abnormal signal transduction pathways,12 leading to proliferation in response to external signals.13 Oncogene over-expression14 is a common phenomenon in the development and progression of many human cancers. Therefore, oncogenes provide a potential target15 for cancer gene therapy. We know that Ras proteins are oncogenes and Rab proteins are Ras-like small GTPases, which have crucial roles in vesicle trafficking, signal transduction and receptor recycling, which in turn regulate normal cellular activity.3 Approximately 60 human Rab genes are encoded by the human genome. However, the complexity of the Rab protein family might be even greater as there is evidence that alternative splicing of Rab mRNAs results in the production of functionally distinct isoforms.16 Recent studies have shown multiple links between Rab GTPase dysfunction and associated regulatory proteins in human diseases, including cancer.17–21 Most Rab proteins are constitutively expressed in all mammalian cells, although several Rab proteins have been shown to be differentially expressed in epithelia and neurons, perhaps fulfilling specialised transport functions of these polarised cells. Therefore, some Rab protein functions may be ubiquitous. Rab5 has been localised to the early endosomal fraction in a number of cell systems.22 Other Rab proteins may have different functions in Fig. 4 Rab25 gene silencing increases early-stage apoptosis in A2780 cells. A2780 cells were transfected with RabR25 siRNA or empty vector. Cells were then collected by trypsinisation.The apoptotic cells were determined by flow cytometry. (A) A2780; (B) empty vector; (C) Rab25 siRNA; (D) diagram summarising the differences among the above groups. A2780 cells transfected with Rab25 siRNA show much greater rates of early apoptosis compared with the control (p,0.01). RNA INTERFERENCE INHIBITS EXPRESSION OF RAB25 TABLE 3 Cell cycle phase distribution of A2780 cells after transfection of Rab25 siRNA Group A2780 Empty vector Rab25 siRNA G1 % S% G2/M % 50.2 48.4 88.6 36.3 37.7 8.1 13.5 14.0 3.3 The cells with Rab25 gene silencing revealed an increase in the percentage of G1 phase and a decrease in S phase. different cellular systems. Thus, while Rab2 is associated with the intermediate endoplasmic reticular-Golgi compartment in Madin–Darby canine kidney (MDCK) cells,23 it is associated with the H,K-ATPase-containing tubulovesicles in gastric parietal cells.24 In addition to the ubiquitous small GTP-binding proteins, some demonstrate tissuespecific distribution, including Rab3A,25,26 Rab3D,27 and Rabl5.28 Rabl7 is expressed only in the kidney, intestines, and liver,29 and its distribution suggests a possible role in transcytosis. Fig. 5 565 Rab25, which shares 68% sequence identity with Rab11a, is expressed exclusively in epithelial cells, suggesting it regulates aspects of vesicular transport that are unique to epithelia.30,31 The sequence of the GTP-binding site of Rab25 is unique among Rab proteins, in that the P-3 domain consensus sequence WDTAGQE contains a leucine residue in place of the conserved glutamine. An equivalent substitution in Ras creates a dominant-active GTPase.32 Casanova et al. have found that Rab25 over-expression alters transcytosis and apical recycling,33 and Cheng et al.5 have reported that Rab25 controls tumour progression, aggressiveness, and potentially chemosensitivity, and is amplified at the DNA level and over-expressed at the RNA level in ovarian cancer. Rab25 over-expression markedly increases cancer cell proliferation, preventing apoptosis. Schaner et al.34 reported similar results and also found that ovarian cancer patients with elevated Rab25 either failed to enter a disease-free state following surgery and chemotherapy, or exhibited short durations of disease-free survival. Therefore, specific down-regulation of Rab25 might be a potential therapeutic strategy against human ovarian cancer. Cell cycle of A2780 cells analysed by flow cytometry. (A) A2780; (B) empty vector; (C) Rab25 siRNA. 566 YANG et al. Pathology (2006), 38(6), December Fig. 6 Tumour growth in A2780 cells transfected with Rab25 siRNA. (A) Rab25 siRNA significantly inhibits A2780 cell growth in nude mice (p,0.05). (B) Rab25 siRNA significantly inhibits A2780 cell proliferation in nude mice. We investigated the effects of Rab25 by in vivo subcutaneous injection into nude mice. Three weeks after transfection there was a tiny tumour in the mouse on the right which received transfected Rab25 siRNA. Techniques based upon RNAi principle35 have become powerful and indispensable tools in the molecular toolkit, being honoured as ‘the breakthrough of 2002’.35,36 By means of the RNAi method, cellular growth assays in vitro were used to determine the functional consequences of RNAi-mediated decreases of Rab25 in established ovarian carcinoma cells. Our results demonstrate that RNAi can effectively down-regulate Rab25 over-expression with great specificity and that the plasmids endogenously expressing siRNA can successfully deplete Rab25 expression in A2780 cells after transfection. Furthermore, the tumour inhibition effects persist after transfection as shown by experiments in vitro and in vivo. Therefore, it is not surprising that we have shown that reduction of the Rab25 level by Rab25 siRNA can significantly inhibit the growth rate of cancer cells. Many anticancer agents induce apoptosis. Our data also suggest that knock-down of Rab25 by Rab25 siRNA in A2780 cells can increase the sensitivity of these cells to apoptosis. This was probably one of the reasons for the anti-tumour effect. Moreover, in cell cycle, increases in G1 and decreases in S further confirm a role for Rab25 in regulating cellular apoptosis. A recent study has provided direct evidence that multiple signalling molecules including AKT, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase, associate on endocytic vesicles and mediate their function by facilitating translocation of the vesicles to the nucleus, implicating Rab GTPases in conducting cell survival signals.37 However, the pathways that Rab25 controls and/or that are involved in the observed apoptosis need further study. All of the nude mice injected with Rab25 transfected cells developed subcutaneous tumours. However, mice injected with Rab25 transfected A2780 cells developed obviously smaller tumours than the controls, and the controls developed larger tumours in a shorter time. This further demonstrates that Rab25 plays a master role in tumour progression and aggressiveness. These studies have marked a new target in the genetic manipulation of ovarian cancer development by allowing Rab25 to be down-regulated by RNAi. In conclusion, Rab25 siRNA substantially reduces the expression of Rab25 mRNA, slows cell proliferation, increases apoptosis, suppresses transformation, and inhibits tumour growth, demonstrating that Rab25 siRNA silencing is a powerful approach for dissecting oncogenic signals in human ovarian cancer. Our data implicate Rab25 in the aggressiveness of ovarian carcinomas suggesting that Rab25 can be used to predict patient outcome and provide a novel therapeutic target. These studies also implicate Rab25 and, by extension, the Rab GTPase family in tumour aggressiveness. This adds the genes encoding the Rab family to other members of the Ras oncogene superfamily as tumorigenic mediators. Address for correspondence: Dr Y. Fan, Department of Obstetrics and Gynecology, The Fourth Military Medical University, 17, The West Road, Xi’an, Xi’an Shaanxi Province, 710032, China. E-mail: [email protected] References 1. Ozols RF. Progress in ovarian cancer: an overview and perspective. EJC Supplements 2003; 1: 43–55. 2. Cheng KW, Lahad JP, Gray JW, Mills GB. Emerging role of RAB GTPases in cancer and human disease. Cancer Res 2005; 65: 2516–9. 3. Wang X, Kumar R, Navarre J, Casanova JE, Goldenring JR. Regulation of vesicle trafficking in Madin-Darby canine kidney cells by Rab11a and Rab25. J Biol Chem 2000; 275: 29138–46. 4. Hales CM, Griner R, Hobdy-Henderson KC, et al. Identification and characterization of a family of Rab11-interacting proteins. J Biol Chem 2001; 276: 39067–75. 5. Cheng KW, Lahad JP, Kuo WL, et al. The Rab25 small GTPase determines aggressiveness of ovarian and breast cancers. Nat Med 2004; 10: 1251–6. 6. Gudkov AV, Zelnick CR, Kazarov AR, et al. Isolation of genetic suppressor elements, inducing resistance to topoisomerase II—interactive cytotoxic drugs, from human topoisomerase II cDNA. Proc Natl Acad Sci USA 1993; 90: 3231–5. 7. Novina CD, Sharp PA. The RNAi revolution. Nature 2004; 430: 161–4. 8. Matzke M, Matzke A, Kooter MJ. RNA: guiding gene silencing. Science 2001; 293: 1980–3. 9. Tuschl T. Expanding small RNA interference. Nat Biotechnol 2002; 20: 446–8. 10. Agami R. RNAi and related mechanisms and their potential use for therapy. Curr Opin Chem Biol 2002; 6: 829–34. 11. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2002; 100: 57–70. 12. Adjei AA, Hidalgo M. Intracellular signal transduction pathway proteins as targets for cancer therapy. J Clin Oncol 2005; 6: 27. 13. Ukegawa JI, Takeuchi Y, Kusayanagi S, Mitamura K. Growth promoting effect of muscarinic acetylcholine receptors in colon cancer cells. J Cancer Res Clin Oncol 2003; 129: 272–8. 14. Onel K, Cordon-Cardo C. MDM2 and prognosis. Mol Cancer Res 2004; 2: 1–8. RNA INTERFERENCE INHIBITS EXPRESSION OF RAB25 15. Buolamwini JK, Addo J, Kamath S, Patil S, Mason D, Ores M. Small molecule antagonists of the MDM2 oncoprotein as anticancer agents. Curr Cancer Drug Targets 2005; 5: 57–68. 16. Stein MP, Dong J, Wandinger-Ness A. Rab proteins and endocytic trafficking: potential targets for therapeutic intervention. Adv Drug Deliv Rev 2003; 14: 1421–37. 17. Menasche G, Pastural E, Feldmann J, et al. Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nat Genet 2000; 25: 173–6. 18. Calvo A, Xiao N, Kang J, et al. Alterations in gene expression profiles during prostate cancer progression: functional correlations to tumorigenicity and downregulation of selenoprotein-P in mouse and human tumors. Cancer Res 2002; 62: 5325–35. 19. Croizet-Berger K, Daumerie C, Couvreur M, Courtoy PJ, van den Hove MF. The endocytic catalysts, Rab5a and Rab7, are tandem regulators of thyroid hormone production. Proc Natl Acad Sci USA 2002; 99: 8277–82. 20. Mor O, Nativ O, Stein A, et al. Molecular analysis of transitional cell carcinoma using cDNA microarray. Oncogene 2003; 22: 7702–10. 21. Wang W, Wyckoff JB, Frohlich VC, et al. Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling. Cancer Res 2002; 62: 6278–88. 22. Gorvel JP, Chavrier P, Zerial M, Gruenberg J. Rab5 controls early endosome fusion in vitro. Cell 1991; 4: 915–25. 23. Chavrier P, Parton RG, Hauri HP, Simons K, Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell 1990; 62: 317–29. 24. Tang LH, Stoch SA, Modlin 1M, Goldenring JR. Identification of rab2 as a tubulovesicle-membrane-associated protein in rabbit gastric parietal cells. Biochem J 1992; 285: 715–9. 25. Darchen F, Zahraoui A, Hammel F, Monteils MP, Kvitian A, Scherman D. Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules. Proc Natl Acad Sci USA 1990; 87: 5692–6. 567 26. Mollard GFV, Mignery GA, Baumert M, et al. Rab3 is a small GTPbinding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci USA 1990; 87: 1988–92. 27. Baldini G, Hohl T, Lin HY, Lodiah H. Cloning of a Rab3 isotype predominantly expressed in adipocytes. Proc Natl Acad Sci USA 1992; 89: 5049–52. 28. Elferink LA, Anzai K, Scheller RH. Rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain. J Biol Chem 1992; 267: 5768–75. 29. Lutke A, Jansson S, Parton RG, et al. Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization. J Cell Biol 1993; 121: 553–64. 30. Goldenring JR, Shen KR, Vaughan HD, Modlin IM. Identification of a small GTP-binding protein, Rab25, expressed in the gastrointestinal mucosa, kidney and lung. J Biol Chem 1993; 268: 18419–22. 31. Calhoun BC, Lapierre LA, Chew CS, Goldenring JR. Rab11a redistributes to apical secretory canaliculus during stimulation of gastric parietal cells. Am J Physiol Cell Physiol 1998; 275: C163–70. 32. Haubruck H, McCormick F. Ras p21: effects and regulation. Biochem Biophys Acta 1991; 1072: 215–29. 33. Casanova JE, Wang X, Kumar R, et al. Association of Rab25 and Rab11a with the apical recycling system of polarized Madin–Darby canine kidney cells. Mol Biol Cell 1999; 10: 47–61. 34. Schaner ME, Ross DT, Ciaravino G, et al. Gene expression patterns in ovarian carcinomas. Mol Biol Cell 2003; 14: 4376–86. 35. Fire A, Xu S, Montgomery MK. Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806–11. 36. Dorsett Y, Tuschl T. siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov 2004; 3: 318–29. 37. Delcroix JD, Valletta JS, Wu C, et al. NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals. Neuron 2003; 39: 69–84.