Colorectal Cancer Open Access
Copyright ©2006 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Feb 21, 2006; 12(7): 1033-1037
Published online Feb 21, 2006. doi: 10.3748/wjg.v12.i7.1033
Loss of heterozygosity of Kras2 gene on 12p12-13 in Chinese colon carcinoma patients
Jun Wan, Hong Li, Yuan Li, Mei-Ling Zhu, Po Zhao
Jun Wan, Hong Li, Yuan Li, Department of Gastroenterology, General Hospital of the Chinese PLA, Beijing 100083, China
Hong Li, In patient Department, General Hospital of the Chinese PLA, Beijing 100083, China
Po Zhao, Department of Pathology, General Hospital of the Chinese PLA, Beijing 100083, China
Supported by the National Natural Science Foundation of China, No.30200326
Correspondence to: Hong Li, In patient Department, South Building of General Hospital of the Chinese PLA, Beijing 100083, China. lihonham@hotmail.com
Telephone: 86-10-66937622
Received: July 10, 2005
Revised: July 20, 2005
Accepted: December 1, 2005
Published online: February 21, 2006

Abstract

AIM: To study the loss of heterozygosity (LOH) on 12p12-13 in Chinese colon carcinoma patients.

METHODS: DNA was extracted from 10 specimens of cancer tissue, 10 specimens of adjacent tissue and 10 specimens of normal tissue, respectively. LOH of Kras2 gene was analyzed by polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis using 11 microsatellite markers on 12p-12-13.

RESULTS: LOH of Kras gene was detected at least on one marker of 12p-12-13 in 30% (3/10) of adjacent tissue specimens. The highest frequency of LOH was identified on D12S1034 in 28.57% (2/7) of adjacent tissue specimens. LOH was detected at least on one marker of 12p12-13 in 60% (6/10) of carcinoma tissue specimens, the most frequent LOH was found on D12S1034 and D12S1591 in 42.86% (3/7) of carcinoma tissue specimens. LOH was detected in 30% (3/10) of carcinoma tissue specimens, 30% (3/10) of adjacent tissue specimens, and no signal in 1% (1/0) carcinoma tissue specimen. The occurrence of LOH did not correlate with sex, age, tumor size and lymph node metastasis.

CONCLUSION: Genomic instability may occur on 12p-12-13 of Kras2 gene in the development and progression of colon carcinoma. The high LOH of Kras2 gene may directly influence the transcription and translation of wild type Kras2 gene.

Key Words: Colon carcinoma, Loss of heterozygosity, Kras2



INTRODUCTION

Clinical and experimental studies have shown that point mutation of Kras2 gene plays an important role in the development and progression of tumors [1-4]. However, it has been reported that wild type Kras2 gene has inhibitory effects on tumor growth and proliferation [5]. Inactivation of cancer suppressor gene is a frequently encountered early event in the development of tumors, leading to loss of heterozygosity (LOH) [6-10]. This study was to investigate the possible genetic variation of wild type Kras2 gene in the carcinogenesis of colon carcinoma by analyzing LOH in tumor and its adjacent tissues.

MATERIALS AND METHODS
Specimens

Ten specimens of primary carcinoma tissue, 10 specimens of its adjacent tissue and 10 specimens of normal tissue at the distal incision margin were taken respectively from patients with pathologically confirmed colon carcinoma before and after surgery at the Department of Surgery, General Hospital of the Chinese PLA from January to December 2003. The patients did not receive radiotherapy and chemotherapy before surgery.

DNA extraction from genome

Specimens of carcinoma and its adjacent tissues as well as normal tissue at the distal incision margin were suspended respectively in 50μL DNA lysate containing 50 mmol/L Tris-HCI, 1mmol/L EDTA, 0.1% Tween 20, 200 mg/L protease K, pH8.0, and digested overnight at 50 oC. Protease K was denatured and inactivated at 95 oC. Then the supernatant was centrifuged and stored at -20oC for use.

Primer design

Microsatellite-labeled primers on 12p12-13 were retrieved from the UniSTS database. The sequences of D12S89, D12S358, D12S310, D12S1606, D12S1596, D12S1592, D12S1617, D12S1057, D12S1591 are listed in Table 1.

Table 1 Primer sequences on 12p12-13 microsatellite markers.
MarkersPrimer sequencesLength(bp)
D12S89D12S89-F:5’-ATTTGAGAGCAGCGTGTTTT-3’254-288
D12S89-R:5’-CCATTATGGGGAGTAGGGGT-3’
D12s358D12s358-F:5’-GCCTTTGGGAAACTTTGG-3’238-270
D12s358-R:5’-TAAGCCCTTTTATTTTTCCTAAC-3’
D12s310D12s310-F:5’-GAAAACTAATTGCCCCTTAC-3’243-253
D12s310-R:5’-TTTAGATTCTCCAAATGCC-3’
D12S1606D12S1606-F:5’-ATGAGAGGCCAAATTAAAG-3’271-279
D12S1606-R:5’-CTACTGTTGTGTCAGGGTCA-3’
D12s1596D12s1596-F:5’-TCATGTGGCTGGTAGAGAAG-3’204-222
D12s1596-R:5’-CGTGAAGCAGATTTATCGTG-3’
D12s1617D12s1617-F:5’-AGCCTGAGGGGCCACAT-3’215-261
D12s1617-R:5’-TGGGCAACTTGGATAAGAAACA-3’
D12s1592D12s1592-F:5’-GCTGAGTGTGGTGGCAC-3’238-280
D12s1592-R:5’-GAGACTATTCCAACAGTGTATTTTC-3’
D12S1057D12S1057-F:5’-AGAGACAGAAGCAGTAGGATGG-3’227
D12S1057-R:5’-GGTTCTGACGTTTTAAGACTCG-3’
D12S1591D12S1591-F:5’-GCGCTTTGTACTATGTTACATTT-3’234-276
D12S1591-R:5’-GAAGACGTTGGGTGAATC-3’
D12S823D12S823-F:5’-GGGCAATAGAGTGAGATTCTG-3’119
D12S823-R:5’-CCTACCTCCTTCCCCATCC-3’
D12S1034D12S1034-F:5’-TTTTCAAAAAGTGTGACTGTGC-3’280-302
D12S1034-R:5’-CAGCCTAGTAAAAAATTTAAATTGG-3’
Polymerase chain reaction (PCR)

One μL 10xPCR buffer, 0.5 mmol/L magnesium ion, 0.2 mmol/L 4xDNTP, 0.4 μmol/L upstream primer, 0.4 μmol/L downstream primer, 1U Taq DNA polymerase/reaction and 0.5 μLDNA template were added into 10 μL PCR system at 94 oC for 5 min. Fourteen PCR cycles of amplification were performed at 94 oC for 30 s, at 60 oC for 30 s, at 72 oC for 30 s, followed by 16 cycles at 94 oC for 30 s, at 55 oC for 30 s, at 72 oC for 30 s, and a final extension at 72 oC for 5 min.

Denaturing polyacrylamide gel electrophoresis

PCR products (0.3μL) were added to the loading buffer containing GENESCAN-500 molecular weight as internal control and mixed with formamide, then denatured at 72oC for 2-3 min and electrophoresed by the AB1377 fluorescence sequencer. The standard sequencing PAGE 64-well denaturing polyacrylamide gel was used for electrophoresis. The electrophoresis was performed at the temperature higher than 42 oC for 2.5-3 h. The electrophoresis channels were analyzed using GENESCAN version 3.1 Software. The types and intensity of fluorescence were collected for each channel, the size of amplified PCR products was determined using the molecular weight as internal control. Data of the type, intensity and molecular weight of the amplified microsatellite DNA fluorescence were obtained using GENOTYPE version 2.0 Software. The fluorescence intensity could indicate the amplified DNA within the linearity (relative fluorescence intensity was 200-300).

LOH analysis

Based on the principles of fluorescence labeling of primers, a fluorescence labeled primer group-matched sequence was linked to the 5 ' end of a specific primer, so that the PCR products were labeled with the fluorescence group in the process of PCR. The fluorescence labeled PCR products were electrophoresed using the AB 1377 fluorescence sequencer. Data collected by the electrophoresis were analyzed using the GENESCAN and GENOTYPE Softwares to obtain the peak and size of the map. Gene typing was performed. The peak was found in 2 allelic gene segments and compared to the normal value of the adjacent channels. The allelic ratio was calculated according to the following formula: allelic ratio=peak ratio of carcinoma tissue/peak ratio of normal tissue. LOH was considered when the allelic ratio was higher than 1.5 or lower than 0.67. Microsatellite instability (MSI) was considered when no abnormal peak point was found in DNA of carcinoma tissue compared to normal tissue.

Statistical analysis

The correlation between LOH and clinical and pathological parameters was evaluated by chi-square test. All statistical analyses were carried out by SPSS 10.0. P  <  0.05 was considered statistically significant.

RESULTS
Frequency of LOH in carcinoma adjacent tissue

No LOH was detected in 70 (7/10) of adjacent tissue specimens (1, 2, 4-6, 8, 9) on all markers. LOH was detected in 30% (3/10) of adjacent tissue specimens (3,7,10) at least on one marker, 28.5% (2/7) of adjacent tissue specimens on D12S1034, 25% (1/4) on D12S1617, 14.29% (1/7) on D12S1596, 12.5% (1/8) on D12S89, and 0% on other markers (Table 2 and Table 3, Figure 1).

Figure 1
Figure 1 LOH on D12S1591 (A) and gene type (B) in carcinoma and normal tissues. The arrowhead shows the allele loss. 10-1: tumor, 10-2: adjacent tumor, 10-3: normal.
Table 2 Frequency of LOH on the 11 markers of 12p12-13 in colon carcinoma and its adjacent tissues.
Adjacent tumor tissueTumor tissue
Markers
LOH (n)
Signal (n)
Frequency ofLOH (%)
Heterozygo-sity (%)
LOH (n)
Signal (n)
Frequency ofLOH (%)
Heterozy-gosity (%)
D12S82305050152050
D12S10342728.57703742.8870
D12S1596142540142540
D12S15911714.29703742.8870
D12S35808080282580
D12S310030301333.3330
D12S15920404004040
D12S891812.5801812.580
D12S16171714.29701714.2970
D12S1057070702728.5770
D12S1606060601616.6760
Table 3 Distribution of LOH on 12p12-13 in microsatellite-labeled primers.
12345678910
BiomarkerATATATATATATATATATAT
D12S823
D12S1034
D12S1596
D12S1591
D12S358
D12S310
D12S1592
D12S89
D12S1617
D12S1057
D12S1606
Frequency of LOH in carcinoma tissue

No LOH was detected in 40% (4/10) 0f primary colon carcinoma tissue specimens (1, 4-6) on all markers. LOH was detected in 60% (6/10) of colon carcinoma tissue specimens (2, 3, 7-10) at least on one marker, 42. 86% (3/7) on D12S1034 and D12S1591, 33.33% (1/3) on D12S310, and 0% (0/4) on D12S1592 (Tables 2 and 3).

Correlation between LOH on 12p12-13 and clinico-pathological parameters

Chi-square test was used to evaluate the correlation between LOH and clinico-pathological parameters. The results showed that LOH did not correlate with age, sex, tumor size and lymph node metastasis (Table 4).

Table 4 Correlation between LOH on chromosome 12p12-13 and clinical pathologic factors.
Clinical characterLOH
TotalRatio (%)P
+-
Tissue class
Adjacent Tumor3710300.17753
Tumor641060
Age (yr)
≤50437700.77816
>5021330
Sex
Male527700.25979
Female12330
Tumor size (cm)
<5314400.42919
≥533660
Lymph node matastasis
Yes426600.598161
No22440
DISCUSSION

RASp21consisting of Hras1, Nras and Kras2, is a GTP-coupled protein and can transfer signals from cell surface into cells. Its normal expression is necessary to maintain the normal physiological activities of cells. Activated Ras proto-oncogenes, especially Kras2, play an important role in the carcinogenesis of human and rodent tumors. Mutations of Kras2 gene have been found in tumor tissues of human organs, including bladder[11], breast[12], rectum[13], kidney[14], liver[15], lung[16], ovary[17], pancreas[18], stomach[19] and hematopoietic system[20]. In general, about 30% cancers display ras gene mutations, while the highest mutation rate is found in colonic and pancreatic cancer [21-25]. In samples of mutated ras gene, most mutations occur in Kras2 gene. Mutation and activation of ras gene usually occur at codens 12 and 13 or 61, leading to the transformation of proto-oncogene to oncogene[26]. This kind of activation can up-regulate the expression of ras/ErK signal channel in the absence of external stimuli and further increase the abnormality of associated signal channels, leading to malignant transformation of cells. Activated ras gene is usually considered as the dominant oncogene because of the existing expression of wild type ras and malignant transformation of activated ras[27]. However, is still controversial the effect of the dominant gene-ras is still controversial since wild type ras has been found in human and mouse pulmonary adenocarcinomas[28,29].

In vivo and in vitro experiments[28] have shown that tumors are found more frequently in normal mice with 2 wild type Kras2 copies than in those with LOH of one wild type Kras2 copy after they are treated with 2 carcinogens. The occurrence of tumor is 50-fold higher in mice with LOH of one wild type Kras copy than in normal mice with 2 wild type Kras copies. The tumor in the former group of mice is poorly-differentiated adenocarcinoma, while the tumor in the later group of mice is adenoma. Zhang et al[28] reported that wild type Kras2 gene can inhibit cell growth , formation of clones, and induce tumors in naked mice. In addition, LOH has been found in pulmonary adenocarcinoma induced by various chemical carcinogens. Point mutation of Kras2 gene is detected in 67-100% mice with LOH of wild type Kras 2 gene. These important findings will certainly query the established carcinogenesis of dominant Kras2 gene.

It was reported that the development and progression of colon carcinoma are a process involving multiple genes and factors, and characterized by its stages: normal mucosa→atypical hyperplasia including intestinal metaplasia→adenoma→adenocarcinoma[30]. Kras2 gene as a dominant oncogene due to its point mutation plays an important role in the progression of canceration, which is one of the reasons why the inhibitory effect of wild type kras2 gene on cancer is concealed. Since cancer suppressor gene can be inactivated by deleting mutation, we studied LOH of Kras 2 gene on 12p12-13 in primary colon carcinoma. LOH was detected in 30% (3/10) of adjacent tissue specimens at least on one marker, 28.5% (2/7) on D12S1034, suggesting that 12p12-13 is genomically insin precancerous cells, and that LOH on 12p12-13 may influence the expression of wild type Kras2 gene in early colon carcinoma. LOH was detected in 60% colon carcinoma tissue specimens at least on one marker, which was higher than that in adjacent tissue, and 42.86% (3/7) on D12S1034, suggesting that the genomic instability of 12p12-13 exacerbates with the progression of carcinoma as demonstrated not only by the increasing number of allelic gene loss points but also by the increasing frequency of allelic gene loss at the same point. Such changes lead to a decrease of Kras2 gene copies used for transformation. When another point mutation occurs in the process of carcinogenesis and produces dominant oncogene effects, the cancer suppressing function of wild type Kras2 gene can be completely covered up.

In conclusion, Kras2 gene can exert inhibitory effects on the proliferation of colon carcinoma cells. LOH on 12p12-13 does not correlate with the clinical and pathological parameters obtained from colon carcinoma.

Footnotes

S- Editor Guo SY L- Editor Wang XL E- Editor Cao L

References
1.  Rapallo A, Sciutto A, Geido E, Orecchia R, Infusini E, Pujic N, d'Amore ES, Monaco R, Risio M, Rossini FP. K-ras2 activation and genome instability increase proliferation and size of FAP adenomas. Anal Cell Pathol. 1999;19:39-46.  [PubMed]  [DOI]  [Cited in This Article: ]
2.  Spandidos DA, Sourvinos G, Tsatsanis C, Zafiropoulos A. Normal ras genes: their onco-suppressor and pro-apoptotic functions (review). Int J Oncol. 2002;21:237-241.  [PubMed]  [DOI]  [Cited in This Article: ]
3.  Geido E, Sciutto A, Rubagotti A, Oliani C, Monaco R, Risio M, Giaretti W. Combined DNA flow cytometry and sorting with k-ras2 mutation spectrum analysis and the prognosis of human sporadic colorectal cancer. Cytometry. 2002;50:216-224.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 17]  [Cited by in F6Publishing: 12]  [Article Influence: 0.9]  [Reference Citation Analysis (0)]
4.  Sommerer F, Vieth M, Markwarth A, Röhrich K, Vomschloss S, May A, Ell C, Stolte M, Hengge UR, Wittekind C. Mutations of BRAF and KRAS2 in the development of Barrett's adenocarcinoma. Oncogene. 2004;23:554-558.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 52]  [Cited by in F6Publishing: 40]  [Article Influence: 3.1]  [Reference Citation Analysis (0)]
5.  Li J, Zhang Z, Dai Z, Plass C, Morrison C, Wang Y, Wiest JS, Anderson MW, You M. LOH of chromosome 12p correlates with Kras2 mutation in non-small cell lung cancer. Oncogene. 2003;22:1243-1246.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 42]  [Cited by in F6Publishing: 27]  [Article Influence: 2.3]  [Reference Citation Analysis (0)]
6.  Hirayama R, Sawai S, Takagi Y, Mishima Y, Kimura N, Shimada N, Esaki Y, Kurashima C, Utsuyama M, Hirokawa K. Positive relationship between expression of anti-metastatic factor (nm23 gene product or nucleoside diphosphate kinase) and good prognosis in human breast cancer. J Natl Cancer Inst. 1991;83:1249-1250.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 77]  [Cited by in F6Publishing: 44]  [Article Influence: 2.6]  [Reference Citation Analysis (0)]
7.  Rollins LA, Leone-Kabler S, O'Sullivan MG, Miller MS. Role of tumor suppressor genes in transplacental lung carcinogenesis. Mol Carcinog. 1998;21:177-184.  [PubMed]  [DOI]  [Cited in This Article: ]  [Reference Citation Analysis (0)]
8.  Fodde R. The APC gene in colorectal cancer. Eur J Cancer. 2002;38:867-871.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 229]  [Cited by in F6Publishing: 77]  [Article Influence: 12.1]  [Reference Citation Analysis (0)]
9.  Bruewer M, Schmid KW, Krieglstein CF, Senninger N, Schuermann G. Metallothionein: early marker in the carcinogenesis of ulcerative colitis-associated colorectal carcinoma. World J Surg. 2002;26:726-731.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 15]  [Cited by in F6Publishing: 11]  [Article Influence: 0.8]  [Reference Citation Analysis (0)]
10.  Nagothu KK, Jaszewski R, Moragoda L, Rishi AK, Finkenauer R, Tobi M, Naumoff JA, Dhar R, Ehrinpreis M, Kucuk O. Folic acid mediated attenuation of loss of heterozygosity of DCC tumor suppressor gene in the colonic mucosa of patients with colorectal adenomas. Cancer Detect Prev. 2003;27:297-304.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 16]  [Cited by in F6Publishing: 6]  [Article Influence: 0.9]  [Reference Citation Analysis (0)]
11.  Ayan S, Gokce G, Kilicarslan H, Ozdemir O, Yildiz E, Gultekin EY. K-RAS mutation in transitional cell carcinoma of urinary bladder. Int Urol Nephrol. 2001;33:363-367.  [PubMed]  [DOI]  [Cited in This Article: ]  [Reference Citation Analysis (0)]
12.  Hulit J, Di Vizio D, Pestell RG. Inducible transgenics. New lessons on events governing the induction and commitment in mammary tumorigenesis. Breast Cancer Res. 2001;3:209-212.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 8]  [Cited by in F6Publishing: 4]  [Article Influence: 0.4]  [Reference Citation Analysis (0)]
13.  Frattini M, Balestra D, Suardi S, Oggionni M, Alberici P, Radice P, Costa A, Daidone MG, Leo E, Pilotti S. Different genetic features associated with colon and rectal carcinogenesis. Clin Cancer Res. 2004;10:4015-4021.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 153]  [Cited by in F6Publishing: 59]  [Article Influence: 9.6]  [Reference Citation Analysis (0)]
14.  Kozma L, Kiss I, Nagy A, Szakáll S, Ember I. Investigation of c-myc and K-ras amplification in renal clear cell adenocarcinoma. Cancer Lett. 1997;111:127-131.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 28]  [Cited by in F6Publishing: 6]  [Article Influence: 1.2]  [Reference Citation Analysis (0)]
15.  Bai F, Nakanishi Y, Takayama K, Pei XH, Inoue K, Harada T, Izumi M, Hara N. Codon 64 of K-ras gene mutation pattern in hepatocellular carcinomas induced by bleomycin and 1-nitropyrene in A/J mice. Teratog Carcinog Mutagen. 2003;Suppl 1:161-170.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 12]  [Cited by in F6Publishing: 7]  [Article Influence: 0.7]  [Reference Citation Analysis (0)]
16.  Mascaux C, Iannino N, Martin B, Paesmans M, Berghmans T, Dusart M, Haller A, Lothaire P, Meert AP, Noel S. The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer. 2005;92:131-139.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 414]  [Cited by in F6Publishing: 254]  [Article Influence: 25.9]  [Reference Citation Analysis (0)]
17.  Semczuk A, Postawski K, Przadka D, Rozynska K, Wrobel A, Korobowicz E. K-ras gene point mutations and p21ras immunostaining in human ovarian tumors. Eur J Gynaecol Oncol. 2004;25:484-488.  [PubMed]  [DOI]  [Cited in This Article: ]
18.  Zheng M, Liu LX, Zhu AL, Qi SY, Jiang HC, Xiao ZY. K-ras gene mutation in the diagnosis of ultrasound guided fine-needle biopsy of pancreatic masses. World J Gastroenterol. 2003;9:188-191.  [PubMed]  [DOI]  [Cited in This Article: ]
19.  Yashiro M, Nishioka N, Hirakawa K. K-ras mutation influences macroscopic features of gastric carcinoma. J Surg Res. 2005;124:74-78.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 19]  [Cited by in F6Publishing: 12]  [Article Influence: 1.2]  [Reference Citation Analysis (0)]
20.  Barletta E, Gorini G, Vineis P, Miligi L, Davico L, Mugnai G, Ciolli S, Leoni F, Bertini M, Matullo G. Ras gene mutations in patients with acute myeloid leukaemia and exposure to chemical agents. Carcinogenesis. 2004;25:749-755.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 20]  [Cited by in F6Publishing: 12]  [Article Influence: 1.1]  [Reference Citation Analysis (0)]
21.  Maire F, Micard S, Hammel P, Voitot H, Lévy P, Cugnenc PH, Ruszniewski P, Puig PL. Differential diagnosis between chronic pancreatitis and pancreatic cancer: value of the detection of KRAS2 mutations in circulating DNA. Br J Cancer. 2002;87:551-554.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 92]  [Cited by in F6Publishing: 69]  [Article Influence: 4.8]  [Reference Citation Analysis (0)]
22.  Feng D, Han T, Jiang Y, Yuan Z, Wang X, Jiang Z, Zhang S. [Detection of K-ras gene mutations in DNA extracted from the plasma of patients with pancreatic cancer]. Zhonghua Wai Ke Za Zhi. 2000;38:767-770.  [PubMed]  [DOI]  [Cited in This Article: ]
23.  Talar-Wojnarowska R, Gasiorowska A, Smolarz B, Romanowicz-Makowska H, Strzelczyk J, Janiak A, Kulig A, Malecka-Panas E. Clinical significance of K-ras and c-erbB-2 mutations in pancreatic adenocarcinoma and chronic pancreatitis. Int J Gastrointest Cancer. 2005;35:33-41.  [PubMed]  [DOI]  [Cited in This Article: ]  [Reference Citation Analysis (0)]
24.  Wan J, Zhang ZQ, You WD, Sun HK, Zhang JP, Wang YH, Fu YH. Detection of K-ras gene mutation in fecal samples from elderly large intestinal cancer patients and its diagnostic significance. World J Gastroenterol. 2004;10:743-746.  [PubMed]  [DOI]  [Cited in This Article: ]
25.  Higashidani Y, Tamura S, Morita T, Tadokoro T, Yokoyama Y, Miyazaki J, Yang Y, Takeuchi S, Taguchi H, Onishi S. Analysis of K-ras codon 12 mutation in flat and nodular variants of serrated adenoma in the colon. Dis Colon Rectum. 2003;46:327-332.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 16]  [Cited by in F6Publishing: 14]  [Article Influence: 0.9]  [Reference Citation Analysis (0)]
26.  Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779-827.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 3174]  [Cited by in F6Publishing: 1898]  [Article Influence: 93.4]  [Reference Citation Analysis (0)]
27.  Bos JL. ras oncogenes in human cancer: a review. Cancer Res. 1989;49:4682-4689.  [PubMed]  [DOI]  [Cited in This Article: ]
28.  Zhang Z, Wang Y, Vikis HG, Johnson L, Liu G, Li J, Anderson MW, Sills RC, Hong HL, Devereux TR. Wildtype Kras2 can inhibit lung carcinogenesis in mice. Nat Genet. 2001;29:25-33.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 211]  [Cited by in F6Publishing: 137]  [Article Influence: 10.6]  [Reference Citation Analysis (0)]
29.  De Gregorio L, Manenti G, Incarbone M, Pilotti S, Pastorino U, Pierotti MA, Dragani TA. Prognostic value of loss of heterozygosity and KRAS2 mutations in lung adenocarcinoma. Int J Cancer. 1998;79:269-272.  [PubMed]  [DOI]  [Cited in This Article: ]  [Reference Citation Analysis (0)]
30.  Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759-767.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 7557]  [Cited by in F6Publishing: 2170]  [Article Influence: 243.8]  [Reference Citation Analysis (0)]