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Urological Oncology
Mutations of RAS Gene Family in Specimens of Bladder CancerNavaz Karimianpour,1 Parisa Mousavi-Shafaei,1 Abed-Ali Ziaee,1 Mohammad Taghi Akbari,2 Gholamreza Pourmand,3 Amirreza Abedi,3 Ali Ahmadi,4 Hossein Afshin Alavi5 Introduction: Studies have shown different types of RAS mutations in
human bladder tumors with a wide range of mutation frequencies in different patient populations. This study aimed to assess the frequency of specific-point mutations in the RAS gene family of a group of Iranian patients with bladder Methods: We examined the tumor specimens of 35 consecutive patients
with transitional cell carcinoma. The DNA samples were evaluated for the occurrence of HRAS, KRAS, and NRAS activation using a polymerase chain reaction-restriction fragment length polymorphism technique. Results: None of the patients had mutations in the RAS gene family “hot
spots” including codons 12, 13, and 61. Conclusion: We failed to find RAS mutations in our bladder tumor samples.
These observations may reflect the involvement of different etiological factors in the induction of bladder tumor of which RAS mutation might not molecular level, the RAS activating of 3 functional genes, Harvey RAS (HRAS), Kristen RAS (KRAS), and Mutation of RAS in Bladder Cancer—Karimianpour et al Many studies have detected different types of NRAS: 5’-GACATACTGGATACAGCTGGC-3’ RAS mutations in human bladder tumors.(8-14) The results from these studies show a wide range of mutation frequencies. It is not clear whether these The DNA samples were amplified in a total differences are related to the different life styles volume of 50 μL of 10 × PCR buffer (5 μL), of the studied populations, exposure to different dNTP mix (10 mM, 1 μL), each primer (0.5 μL suspected environmental carcinogens, or to the with final concentration of 40 pmol each) and sensitivity of ecogenetic relationships. According Taq polymerase (Fermentas, Burlington, Canada). to these observations, the present study was aimed Amplification of fragments of the studied genes to investigate the frequency of specific point was carried out with a thermal cycler PCR (Geniu mutations of the RAS gene family in a group of system, Boehringer Mannheim, Germany) using Iranian patients suffering from bladder cancer.
the following thermal profile: 95°C for 5 minutes, 32 cycles; HRAS: 95°C for 40 seconds, 6°C for 40 seconds, and 72°C for 45 seconds; KRAS: 94°C for 40 seconds, 60°C for 40 seconds, and 72°C for 50 seconds; NRAS: 94°C for 30 seconds, 60°C for Surgical specimens from 35 patients with 40 seconds, 72°C for 30 seconds, followed by a histologically confirmed transitional cell final extension at 72°C for 2 minutes.
carcinoma were collected and stored at -74°C. The patients were consecutively selected from among admitted patients to Sina Hospital. Age, Restriction endonucleases MspI, BstNI, HphI, sex, and smoking history of the patients were and MscI (Fermentas, Burlington, Canada) obtained from their hospital records.
digested codons 12 (HRAS), 12 and 13 (KRAS), and 61 (NRAS), respectively. Any mutation at these codons disrupts the restriction site for the Genomic DNA was extracted from the tumoral related restriction enzyme. Digestion was carried tissues using proteinase K and phenol extraction out in a total volume of 30 uL that contained 12 methods, and then, it was stored at 4°C.(15) uL of PCR amplicon and 10 IU of restriction endonuclease. Buffers and incubation conditions (overnight at 37°C) were applied as recommended by the manufactures. The digested fragments were electrophoresed on a 6% polyacrylamide gel (1:59 Matched and mismatched oligonucleotide primers bis-acrylamide for HRAS and KRAS genes, and were designed or selected from previous studies 1:19 for NRAS gene) in 0.5 × TBE at 200 V for 1 for amplifying sequences around codon 12 of hour and gels were stained in ethidium bromide. HRAS, codons 12 and 13 of KRAS, and codon 61 In this work, different sizes of DNA fragments of NRAS in order to generate subfragments only of HRAS, NRAS, and KRAS genes (GenBank from wild-type polymerase chain reaction (PCR)- amplified RAS genes (mutant-type destroys the and NM_004985, respectively) were obtained by created restriction site).(16) The primer sequences used were as follows: HRAS: 5’-GACGGAATATAAGCTGGTGG-3’ KRAS: 5’-ACTGAATATAAACTTGTGGTAG The mean age of the patients was 65.8 ± 11.8 years (range, 34 to 85 years), and 74.3% of them were older than 60 years. Twenty-nine patients were men (82.8%) and 18 were smokers (51.4%). Analysis of the pathological grades showed that Mutation of RAS in Bladder Cancer—Karimianpour et al 23 specimens (65.7%) were low grade (2 low malignant potential, 21 low grade, and 12 high Polymerase Chain Reaction AmplificationThe lengths of the RAS amplified fragments according to the designed primers were 420 bp, 65 bp, 144 bp for HRAS, NRAS, and KRAS genes, Codon 12To determine any point mutation at codon 12 of HRAS, the restriction enzyme MspI was used. Only the wild-type amplicon containing the Figure 2. NRAS gene product analysis in bladder cancer
endonuclease recognition site could be cut off by polymerase chain reaction-restriction fragment length polymorphism. A, undigested DNA from a healthy person and give rise to 390-bp and 30-bp fragments. No (65-bp); Ad, MscI-cut polymerase chain reaction product of the point mutation on codon 12 of HRAS was found healthy person (44-bp and 21-bp); Lane 8 and 9, undigested DNA of patients 8 and 9, respectively; and Lane 8d and 9d, digested products of patients 8 and 9, respectively.
nucleotide change just before codon 61. In case of any mutation, the restriction enzyme MscI would be unable to cut the PCR fragment to 21-bp and The restriction enzyme MscI was used for digestion of the codon 61 of NRAS. The proper cutting site (TGG↓CCA) was created with the help of the forward primer, which led to a single Codons 12 and 13The restriction enzyme BstNI was used for codon 12 of KRAS gene digestion. A primer was designed, so that the cutting site was created just before codon 12. Only the wild-type KRAS PCR product would be cut by BstN1, yielding 2 fragments around 115-bp and 29-bp oligonucleotides. For codon 13, GGTGA7/8↓ is the recognition site for HphI and is cut off by the enzyme. This site does not exist naturally, but it would appear in any type of mutation. Digestion reaction was carried out for each sample and no mutation was detected for KRAS (Figure 3). DISCUSSIONStudies on a variety of tumors have demonstrated Figure 1. HRAS gene product analysis in bladder cancer
some “hot spots” in RAS gene family that are by polymerase chain reaction-restriction fragment length susceptible to point mutations. The frequent polymorphism. A, undigested DNA from a healthy person mutations are changes of glycine to valine at (420-bp); Ad, MspI-cut polymerase chain reaction product of the healthy person (390-bp and 30-bp); Lanes 15 and 18, codon 12, glycine to cysteine at codon 13, and undigested DNA of patients 15 and 18, respectively; and glutamine to arginine/lysine/leucine at codon Lane 15d and 18d, digested products of patients 15 and 18, 61.(17) The incidence of RAS mutation varies and is Mutation of RAS in Bladder Cancer—Karimianpour et al other hand, various levels of RAS mutation at codon 12 have recently been reported in bladder cancer. While Zhu and associates and Buyru and coworkers showed 46.7% and 39% point mutation of HRAS at codon 12, respectively,(3,12) Cattan and associates detected only 1% of such alterations.(13) Furthermore, Przybojewska and colleagues found the HRAS mutation in 84% of patients with bladder cancer using a PCR-restriction fragment length polymorphism assay.(16) In contrast to the above discussed investigations showing RAS activation, our study detected no mutation in the RAS gene family in any grades of bladder cancer in the 35 studied patients. It should be mentioned that the RAS protein dysfunction may occur not only as a result of mutations in the RAS gene, but also due to changes in the protein level. Quantitative alterations in the expression Figure 3. KRAS gene product analysis in bladder cancer
due to gene amplification or overexpression could by polymerase chain reaction-restriction fragment length lead to continuous proliferative signals needed for polymorphism. A, undigested DNA from a healthy person (144-bp); cell propagation. Previous studies demonstrated Ad, BstNI-cut polymerase chain reaction product of the healthy person (115-bp and 29-bp); Lane 27, undigested DNA from patient 27; and Lane 27d, the digested product from patient 27.
carcinoma in situ and high-grade tumors, but not in hyperplasia or low-grade tumors when greatly dependent on the tissue or cell type from immunohistochemical technique was applied.(14) which the cancer cells are derived. Although RAS Vageli and associates reported an increase in RAS mutations occur in 75% to 95% of pancreatic transcripts in about 40% of the bladder cancers, carcinomas and 50% of colon carcinomas, they as well.(25) All these studies indicate that the are rare in several other neoplasms.(18-20) The precise frequency of RAS mutations in human HRAS mutation was first detected in the human bladder cancer is still unclear. The observed bladder cancer cell line T24.(7) Subsequent studies discrepancies in the mutation pattern of RAS demonstrated that HRAS mutations were more gene family among different populations suffering frequently observed in urinary tract tumors from bladder cancer may either reflect different than the KRAS or NRAS genes.(21) This initial etiological mechanisms involved in disease expectation has been materialized, since later progression or alternative RAS dysfunction such analysis of uncultured bladder tumors showed as gene amplification and/or overexpression.(14,25) that only about 10% of the samples contained Notably, it is not surprising that Iranian patients a mutated HRAS.(22-24) However, later reports have a specific mutation pattern for P53 gene showed higher frequencies. While Fitzgerald and as it has been reported for esophageal cancer.(26) associates reported mutations in the HRAS gene Ecogenetic relationships and cultural conditions in 44% of urine sediments from bladder cancer of may somehow explain the absence of RAS gene patients,(9) Czerniak and coworkers observed family mutation in our patients. Although the HRAS mutation specifically in connection to results so far reported still remain controversial, codon 12 in 45% of the bladder cancers.(10) Also, activation of the RAS oncogene by point in a recent study by Jebar and colleagues on 98 mutation or overexpression may be important bladder tumors and 31 bladder cell lines, RAS in the carcinogenesis and progression of human mutation was detected in 13% of both types of samples.(11) In total, there were 10 mutations in HRAS, 4 in KRAS, and 4 in NRAS. On the Smoking is an established risk factor for bladder Mutation of RAS in Bladder Cancer—Karimianpour et al cancer.(27) Consistent with the epidemiological 8. Oxford G, Theodorescu D. The role of Ras superfamily evidence for an association between bladder proteins in bladder cancer progression. J Urol. cancer and smoking, we found that about 51% of 9. Fitzgerald JM, Ramchurren N, Rieger K, et al. our patients were smokers, which shows a direct Identification of H-ras mutations in urine sediments correlation between smoking and the incidence complements cytology in the detection of bladder of bladder cancer. However, the group under tumors. J Natl Cancer Inst. 1995;87:129-33.
our investigation is too small in number to be 10. Czerniak B, Cohen GL, Etkind P, et al. Concurrent mutations of coding and regulatory sequences of considered for epidemiological conclusions. the Ha-ras gene in urinary bladder carcinomas. Hum The other related risk factor of bladder cancer is age. Our data showed nearly three-fourth of 11. Jebar AH, Hurst CD, Tomlinson DC, Johnston C, our patients were over 60 years of age. This is in Taylor CF, Knowles MA. FGFR3 and Ras gene mutations are mutually exclusive genetic events in accordance with the previous data showing more urothelial cell carcinoma. Oncogene. 2005;24:5218- than 65% of bladder cancer patients in the United 12. Zhu D, Xing D, Shen X, Liu J. A method to quantitatively detect H-ras point mutation based on electrochemiluminescence. Biochem Biophys Res We failed to find RAS gene mutation in our 13. Cattan N, Saison-Behmoaras T, Mari B, et al. patients with bladder tumors. This observation Screening of human bladder carcinomas for the presence of Ha-ras codon 12 mutation. Oncol Rep. may reflect the involvement of different etiological factors in the induction of this tumor. 14. Viola MV, Fromowitz F, Oravez S, Deb S, Schlom Due to the reported studies and possibility of the J. ras Oncogene p21 expression is increased involvement of various etiological factors, it is in premalignant lesions and high grade bladder carcinoma. J Exp Med. 1985;161:1213-8.
interesting to study the situation among Iranian patients suffering from bladder cancer with 15. Raply R. The nucleic acid protocols hand book. Totowa, New Jersey: Human Press; 2000.
various pathological low-grade and high-grade 16. Przybojewska B, Jagiello A, Jalmuzna P. H-RAS, tumors regarding the status of these three genes.
K-RAS, and N-RAS gene activation in human bladder cancers. Cancer Genet Cytogenet. 2000;121:73-7.
17. Levesque P, Ramchurren N, Saini K, Joyce A, Libertino J, Summerhayes IC. Screening of human bladder tumors and urine sediments for the presence of H-ras mutations. Int J Cancer. 1993;55:785-90.
18. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of 1. Borden LS, Jr., Clark PE, Hall MC. Bladder cancer. the exocrine pancreas contain mutant c-K-ras genes. 2. Jemal A, Murray T, Ward E, et al. Cancer statistics, 19. Smit VT, Boot AJ, Smits AM, Fleuren GJ, Cornelisse 2005. CA Cancer J Clin. 2005;55:10-30.
CJ, Bos JL. KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic 3. Buyru N, Tigli H, Ozcan F, Dalay N. Ras oncogene mutations in urine sediments of patients with bladder cancer. J Biochem Mol Biol. 2003;36:399-402.
20. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N 4. Bos JL. ras oncogenes in human cancer: a review. 21. Rabbani F, Cordon-Cardo C. Mutation of cell cycle 5. Varras MN, Koffa M, Koumantakis E, et al. ras gene regulators and their impact on superficial bladder mutations in human endometrial carcinoma. Oncology. cancer. Urol Clin North Am. 2000;27:83-102, ix.
22. Fujita J, Srivastava SK, Kraus MH, Rhim JS, Tronick 6. Shinohara N, Koyanagi T. Ras signal transduction SR, Aaronson SA. Frequency of molecular alterations in carcinogenesis and progression of bladder affecting ras protooncogenes in human urinary tract cancer: molecular target for treatment? Urol Res. tumors. Proc Natl Acad Sci U S A. 1985;82:3849-53.
23. Knowles MA, Williamson M. Mutation of H-ras is 7. Capon DJ, Chen EY, Levinson AD, Seeburg PH, infrequent in bladder cancer: confirmation by single- Goeddel DV. Complete nucleotide sequences of the strand conformation polymorphism analysis, designed T24 human bladder carcinoma oncogene and its restriction fragment length polymorphisms, and direct normal homologue. Nature. 1983;302:33-7.
sequencing. Cancer Res. 1993;53:133-9.
Mutation of RAS in Bladder Cancer—Karimianpour et al 24. Saito S, Hata M, Fukuyama R, et al. Screening of of the esophagus in Iran. Oncogene. 2001;20:7368- H-ras gene point mutations in 50 cases of bladder carcinoma. Int J Urol. 1997;4:178-85.
27. Dolin PJ. An epidemiological review of tobacco use 25. Vageli D, Kiaris H, Delakas D, Anezinis P, Cranidis and bladder cancer. J Smoking Rel Dis. 1991; 2 : 129- A, Spandidos DA. Transcriptional activation of H-ras, K-ras and N-ras proto-oncogenes in human bladder tumors. Cancer Lett. 1996;107:241-7.
28. Jung I, Messing E. Molecular mechanisms and pathways in bladder cancer development and 26. Sepehr A, Taniere P, Martel-Planche G, et al. Distinct progression. Cancer Control. 2000;7:325-34.
pattern of TP53 mutations in squamous cell carcinoma CLINICAL TRIAL REGISTRATIONThe Iranian Registry of Clinical Trials (IRCT) has been launched (http://www.irct.ir/) thanks to the sponsorship by the Iranian Ministry of Health. We strongly encourage researchers who would like to publish reports of their clinical trial in Urology Journal to register their studies in the IRCT or other registries that are proposed by the World Health Organization and the International Committee of Medical Journal Editors. Registration of clinical trials before starting the research project is now considered a primary requirement by these organizations, and it is also emphasized by the World Medical Association Declaration of Helsinki. This helps to ensure that decisions about healthcare are informed by all of the available evidence, ensure that a trial and its results are publicly disclosed, avoid unnecessary duplication, facilitate recruitment of participants, identify gaps in research, encourage collaboration among researchers, and make it possible to identify potential problems and improve clinical trials.
The links below would help you to find clinical trial registries and useful information on this issue:www.icmje.org/clin_trialup.htm

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