Colorectal Cancer
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 15, 2004; 10(10): 1431-1435
Published online May 15, 2004. doi: 10.3748/wjg.v10.i10.1431
Loss of heterozygosity on hromosome 1 in sporadic colorectal carcinoma
Chong-Zhi Zhou, Guo-Qiang Qiu, Fang Zhang, Lin He, Zhi-Hai Peng
Chong-Zhi Zhou, Guo-Qiang Qiu, Fang Zhang, Zhi-Hai Peng, Department of General Surgery, Shanghai Jiaotong University, First People’s Hospital, Shanghai 200080, China
Lin He, Shanghai Institute for Biological Science, Chinese Academy of Sciences, Shanghai 200031, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 30080016
Correspondence to: Dr. Zhi-Hai Peng, Department of General Surgery, Shanghai Jiaotong University, First People’s Hospital, 85 Wujin Road, Shanghai 200080, China. pengpzhb@online.sh.cn
Telephone: +86-21-63240090 Ext 3102
Received: June 10, 2003
Revised: August 9, 2003
Accepted: August 16, 2003
Published online: May 15, 2004

Abstract

AIM: Loss of heterozygosity (LOH) on tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer. When it occurs at a tumor suppressor gene locus with abnormal allele, neoplastic transformation happens. In this study, we analyzed the LOH at 21 loci on chromosome 1 in sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis.

METHODS: Twenty-one polymorphic micro-satellite DNA markers were analyzed with PCR both in 83 cases of colorectal cancer and in normal tissues. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. χ2 test was used to compare LOH frequency with clinicopathological data. P < 0.05 was considered as statistically significant.

RESULTS: The average LOH frequency of chromosome 1, short arm and long arm was 19.83%, 18.00% and 21.66%, respectively. The 2 highest LOH loci with a frequency of 36.54% and 32.50% were identified on D1S468 (1p36.33-p36.31) and D1S413 (1q31.3), respectively. On D1S2726 locus, LOH frequency of rectal cancer was 28.57% (6/21), which was higher than that of colon cancer (0.00%, 0/33) (P = 0.002), suggesting that the mechanism of carcinogenisis was different in both groups.

CONCLUSION: Putative tumor suppressor genes on chromosome 1 may relate to sporadic colorectal carcinomas. Tumor-suppressor-genes might locate on 1p36.33-36.31 and/or 1q31.3.




INTRODUCTION

Colorectal cancer is one of the three leading causes of cancer mortality worldwide. The progression of the cancer is due to an accumulation of genetic alteration in controlling growth and proliferation at numerous loci. As a model for both multistep and multipathway carcinogenesis, colorectal neoplastic progression provides paradigms of both oncogenes and tumor suppressor genes[1,2]. The loss of heterozygosity (LOH) on tumor suppressor genes is believed as one of the key steps to carcinogenesis of colorectal cancer[3]. The loss of one allele at a specific locus is caused by a deletion mutation or loss of a chromosome from a chromosome pair[4]. When this occurs at a tumor suppressor gene with an abnormal allele, neoplastic transformation occurs. In colorectal cancers, frequent allelic loss has been identified in chromosome 5q (30%), 8p (40%), 17p (75%-80%), 18q (80%), and 22q (20%-30%)[5,6]. Tumor suppressor genes APC, p53, and DCC were found to be located on chromosome 5q, 17p, and 18q, respectively. LOH analysis became an effective way to find informative loci candidate tumor suppressor genes afterwards[7,8]. In this study we analyzed the LOH at 21 loci on chromosome 1 in sporadic colorectal cancers to identify additional loci involved in colorectal tumorigenesis.

MATERIALS AND METHODS
Materials

From 1998 to 1999, 83 consecutively collected tumors were treated surgically at Surgical Department of First People’s Hospital in Shanghai Jiaotong University. There were 40 males and 43 females with a median age of 66 years (range 31-84). The diagnosis was verified pathologically. The number of Dukes stages A, B, C, D was 8, 21, 40 and 14, respectively. The number of proximal colon cancer, distal colon cancer, and rectal cancer was 33, 21 and 29, respectively. Well-differentiated adenocarcinomas, moderately differentiated adenocarcinomas and poorly differentiated adenocarcinomas were 23, 39 and 6, respectively, and mucinous adenocarcinomas were 15. HNPCC patients were ruled out by Amsterdam criteria[9,10]. Informed consent to use surgical specimens in this study was obtained from patients.

Methods

DNA extraction Thirty min after surgery, fresh cancerous and adjacent normal tissues were cut into approximately 2 mm3 and immediately frozen in liquid nitrogen. DNA was extracted using standard method with proteinase K digestion and phenol/chloroform purification.

Microsatellite markers and PCR Twenty-one fluorescence-labeled primers for polymorphic microsatellite markers (Perkin-Elmer, USA), at a density of approximately one marker every 10 cm (Figure 1), were used to amplify DNAs from normal and tumor tissues for LOH analysis. PCR for DNAs from normal and tumor tissue was done to analyze the polymorphic microsatellite markers. PCR conditions were as follows: 5 μL total volume with approximately 1.4 ng of DNA as a template with 10 × standard buffer, 0.3 μL Mg2+, 0.8 μL deoxynueleotide triphosphetes, 0.3 unit of Hot-start taq polymerase and 0.06 μL of each oligonucleotide primer, with the forward primer fluorescence labeled with HEX, FAM or NED. Cycling conditions consisted of 3 stages: an initial denaturation at 96 °C for 12 min in stage I; 14 cycles each at 94 °C for 20 s, at 63-56 °C for 1 min (0.5 °C decreased per cycle), at 72 °C for 1 min in stage II: 35 cycles each at 94 °C for 20 s, at 56 °C for 1 min, at 72 °C for 1 min in stage III[11-13].

Figure 1
Figure 1 Twenty-one microsatellite markers on chromosome 1.

LOH analysis PCR product (0.5 μL) was mixed with 0.1 μL of Genescan 500 size standard (PE Applied Biosystems, USA) and 0.9 μL of formamide loading buffer. After denaturation at 96 °C for 5 min, products were eletrophoresed on 50 g/L polyacrylamide gels on an ABI 377 DNA sequencer (PE Applied Biosystems, USA) for 3 h. Genotype 2.1 software displayed individual gel lanes as electopherograms with a given size, height, and area for each detected fluorescent peak. Stringent criteria were used to score the samples. Alleles were defined as the two highest peaks within the expected size range. A ratio of T1:T2/N1:N2 less than 0.67 or greater than 1.50 was scored as a loss of heterozygosity (Figure 2). Most amplifications of normal DNA producing two PCR products indicated preserve of heterozygosity. A single fragment amplified from normal DNA (homozygote) and fragments not clearly amplified from PCR reactions were scored as not informative. The LOH frequency of a locus was equal to the ratio of the number between allelic loss and informative cases. The average LOH frequency of chromosome 1 was the mean of the LOH frequency in all loci[14-17].

Figure 2
Figure 2 Typical peak and normal peak of LOH3. A: Typical peak of LOH: Allele ratio = (T1/T2)/(N1/N2) = (190/62)/(341/270) = 2.43 > 1.5, B: The normal peak (no LOH): Allele ratio = (T1/T2)/(N1/N2) = (1172/764)/(350/264) = 1.15, T: Tumor, N: Normal.

Statisticsχ2 test was used to compare LOH with clinicopathological data. P < 0.05 was considered statistically significant.

RESULTS
LOH of 21 microsatellite markers on chromosome 1

The average LOH frequency at chromosome 1, short arm and long arm was 19.83%, 18.00% and 21.66%, respectively. The two highest LOH loci with a frequency of 36.54% and 32.50% were identified on D1S468 (1p36.33-36.31) and D1S413 (1q31.3). Other loci also exhibited higher LOH frequencies, including D1S255 (1p34.1-32.3), D1S2868 (1p22.1), D1S218 (1q24.1-24.3), D1S249 (1q32.2), D1S2800 (1q42.12-42.2) and D1S2836 (1q43-44) (Table 1).

Table 1 LOH frequency of 21 microsatellite markers on chromosome 1.
LocusLocationLOH caseNormal caseLOH rate (%)Informative rate (%)
D1S4681p36.33-36.31193336.5462.65
D1S4501p36.3174114.5857.83
D1S26971p36.22-36.1352516.6736.14
D1S2551p34.1-32.3114320.3765.06
D1S27971p32.384614.8165.06
D1S28901p32.3-32.164811.1165.06
D1S28411p31.2114619.3068.67
D1S2071p31.2-22.35637.3581.93
D1S28681p22.1103920.4159.04
D1S2061p22.1-21.273317.5048.19
D1S27261p13.3-13.164811.1165.06
D1S4981q12-21.2114818.6471.08
D1S4841q22105515.3878.31
D1S2181q24.1-24.3164227.5969.88
D1S2381q31.13387.3249.40
D1S4131q31.3132732.5048.19
D1S2491q32.2103621.7455.42
D1S2131q41-42.1284116.3359.04
D1S28001q42.12-42.2124521.0568.67
D1S27851q43104817.2469.88
D1S28361q43-44123625.0057.83
Relationship of clinicopathological features and LOH on chromosome 1

On D1S2726 locus, LOH frequency of rectal cancer was 28.57% (6/21), which was higher than that of colon cancer (0%, 0/33) (P = 0.002). No association between LOH of each marker on chromosome 1 and other clinicopathological data (patient sex, age, tumor size, growth pattern or Dukes stage) was observed. It indicated that LOH on chromosome 1 was a common phenomenon in all kinds of sporadic colorectal cancers (Table 2, Table 3).

Table 2 Relationship between clinicopathological features and LOH of 11 loci on short arm of chromosome 1.
D1S468
D1S450
D1S2697
D1S255
D1S2797
D1S2890
D1S2841
D1S207
D1S2868
D1S206
D1S2726
NLNLNLNLNLNLNLNLNLNLNL
GenderMale188204132197205214206312153173195
Female1511213123244263272265323247164291
Age (yr)> 6026133071943283673763411465327275336
≤ 6076110611131011101201707362150
LocationProximal colon129122132145192223214242136111200
Distal Colon7510142122831119417113284130
Rectum145194811741931521632221321421561
Gross patternMassive159162101157213175204232173171192
Ulcerative13516393224193201184273153103203
Encroaching559261606211083130746391
Size≥ 5 (cm)188215131198244272234311195135243
< 5 (cm)1511202124243224214237324205202243
LN metastasisLN (+)2410265174294286315269385228205284
LN (-)9915281147182171202250172132202
DifferentiationWell8714151103201150124162132143180
Moderately1610195124215147195225313165132186
Poorly3041205040502060310250
Mucinous624060738091102100726070
Dukes stageA3340104132407070502150
B6611271106150131132180122111152
C178203112213205235187265167183204
D72626281818082120612280
Table 3 Relationship between clinicopathological features and LOH of 10 loci on long arm of chromosome 1.
D1S498
D1S484
D1S218
D1S238
D1S413
D1S249
D1S213
D1S2800
D1S2785
D1S2836
NLNLNLNLNLNLNLNLNLNL
GenderMale256257226201147166173199253195
Female2353032010182136204245263237177
Age (yr)> 6037104363014283181028733633103910269
≤ 6011112412210093838212290103
LocationProximal colon146204165151106173152156186165
Distal colon143144144101734212314114265
Rectum202212127131104155143165162142
Gross patternMassive2242331811122145134174173206165
Ulcerative17524315419195135194186203125
Encroaching9284917043101501038182
Size≥ 5 (cm)217277226183116165214218237178
< 5 (cm)2742832010200167205204244253194
LN MetastasisLN (+)3163642611232166226285307317255
LN (-)175196165151117144133155173117
DifferentiationWell1241531138173102132135162105
Moderately264274227190126174185215187186
Poorly20603230212340414021
Mucinous837364826371617110160
Dukes stageA40523251322131404123
B1351441331008512310211513294
C225264189181124155215216236172
D911008251427170918183
DISCUSSION

During tumorigenesis, loss of wild-type alleles (inherited from the non-mutation-carrying parents) is frequently observed. Loss of heterozygosity (LOH) on tumor suppressor genes played a key role in colorectal cancer transformation, and LOH analysis of sporadic colorectal cancers could help discover unknown tumor suppressor genes[7,8]. In this study, LOH scanning was analyzed by Genotyper software in 83 sporadic colorectal cancer samples with 21 highly polymorphic markers, the ratio of the fluorescence intensity of alleles was studied to identify additional loci involved in colorectal tumorigenesis.

In this study, the average LOH frequency at chromosome 1 (19.83%), short arm (18.00%) and long arm (21.66%) was consistent with the previous study[5]. The two highest LOH loci with a frequency of 36.54% and 32.50% was identified on D1S468 (1p36.33-36.31) and D1S413 (1q31.3). There were few reports about the relationship between the long arm of chromosome 1 and colorectal cancer. But some previous studies showed that the 1q31-32 region frequently presented allelic loss in breast cancers and medulloblastomas. Pietsch et al[18] found that 36% of medulloblastomas showed loss of heterozygosity (LOH) on chromosome 1q. The study of Benitez J showed more than 60% of breast tumors exhibited allelic loss in the 1q31-32 region[19]. These results suggested that putative tumor suppressor genes might locate on the 1q31-32 region. Our study also found that D1S413 (1q31.3) exhibited a higher LOH frequency and that the LOH frequency of long arm of chromosome 1 was higher than that of short arm[5]. If D1S413 could be excluded, the LOH frequency of long arm was nearly equal to that of short arm. Thus, we hypothesized that the higher LOH frequency of D1S413 might be the reason why the LOH frequency of long arm of chromosome 1 was higher than that of short arm, suggesting the presence of a tumor suppressor gene in this region. This gene might be involved in the neoplastic process of colorectal cancer, breast cancer and medulloblastoma.

Previous studies showed that the 1p36 region frequently presented allelic loss in various cancers, such as colon cancer[20], neuroblastoma[21], hepatocellular carcinomas[22], lung cancer[23], and breast cancer[24]. But only NB gene was confirmed to be the tumor suppressor gene of neuroblastomas. In 1993, Tanaka et al[25] believed that a normal chromosome 1p36 might contain a tumor suppressor gene of colon carcinogenesis. By database referring, we found TP73 gene (1p36) might be the known candidate tumor-suppressor genes related to colon cancer in this region. TP73, a novel family member of p53, was predicted to encode a protein with significant amino acid sequence similarity to p53[29]. TP73 could inhibit cell growth in a p53-like manner by inducing apoptosis[27]. Kaghad et al[26] regarded TP73 as a tumor suppressor gene. But Sunahara found that allelic loss of p73 occurred only in 17% of colorectal carcinomas, and suggested that p73 might not play a role as a tumor suppressor in colorectal carcinoma at least not in a classic Knudson manner[28]. In our study, the highest LOH frequency was exhibited in 1p36.33-36.31, and colorectal cancer related tumor suppressor gene (s) might locate in the region. TP73gene is a member of p53 family, its effect on colorectal carcinogenisis is not certain and requires further study. Due to many genes located in the region of 1p36.33-36.31, further LOH scanning with high-density microsatellite markers in the region is necessary in order to find new candidate genes.

No association between LOH markers on chromosome 1 and the clinicopathological data was found, indicating that LOH was a common phenomenon in all sporadic colorectal cancers. However, we found that on D1S2726 locus, LOH frequency of rectal cancer was high, no LOH was found in colon cancer. In 2001, Kapiteijn et al[29] proposed that rectal cancer had more significant expression of p53 and more nuclear beta-catenin than colon cancer, and considered that the mechanism of carcinogenisis in distal colon was different from that in proximal colon. Our results could show that the mechanism of carcinogenisis in distal colon and rectum was not completely the same as in proximal colon.

In conclusion, colorectal cancer associated candidate genes are likely to locate on D1S468 and D1S413. Further LOH scanning with high-density microsatellite markers in the region may provide much more genetic information and discover novel tumor suppressor genes.

Footnotes

Edited by Ren SY and Wang XL Proofread by Xu FM

References
1.  Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759-767.  [PubMed]  [DOI]
2.  Hardy RG, Meltzer SJ, Jankowski JA. ABC of colorectal cancer. Molecular basis for risk factors. BMJ. 2000;321:886-889.  [PubMed]  [DOI]
3.  Kataoka M, Okabayashi T, Johira H, Nakatani S, Nakashima A, Takeda A, Nishizaki M, Orita K, Tanaka N. Aberration of p53 and DCC in gastric and colorectal cancer. Oncol Rep. 2000;7:99-103.  [PubMed]  [DOI]
4.  Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature. 1998;396:643-649.  [PubMed]  [DOI]
5.  Vogelstein B, Fearon ER, Kern SE, Hamilton SR, Preisinger AC, Nakamura Y, White R. Allelotype of colorectal carcinomas. Science. 1989;244:207-211.  [PubMed]  [DOI]
6.  Weber TK, Conroy J, Keitz B, Rodriguez-Bigas M, Petrelli NJ, Stoler DL, Anderson GR, Shows TB, Nowak NJ. Genome-wide allelotyping indicates increased loss of heterozygosity on 9p and 14q in early age of onset colorectal cancer. Cytogenet Cell Genet. 1999;86:142-147.  [PubMed]  [DOI]
7.  Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM, vanTuinen P, Ledbetter DH, Barker DF, Nakamura Y. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science. 1989;244:217-221.  [PubMed]  [DOI]
8.  Kinzler KW, Nilbert MC, Vogelstein B, Bryan TM, Levy DB, Smith KJ, Preisinger AC, Hamilton SR, Hedge P, Markham A. Identification of a gene located at chromosome 5q21 that is mutated in colorectal cancers. Science. 1991;251:1366-1370.  [PubMed]  [DOI]
9.  Vasen HF, Mecklin JP, Khan PM, Lynch HT. The International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC). Dis Colon Rectum. 1991;34:424-425.  [PubMed]  [DOI]
10.  Vasen HF, Watson P, Mecklin JP, Lynch HT. New clinical crite-ria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology. 1999;116:1453-1456.  [PubMed]  [DOI]
11.  Zhou CZ, Peng ZH, Zhang F, Qiu GQ, He L. Loss of heterozygosity on long arm of chromosome 22 in sporadic colorectal carcinoma. World J Gastroenterol. 2002;8:668-673.  [PubMed]  [DOI]
12.  Peng Z, Zhou C, Zhang F, Ling Y, Tang H, Bai S, Liu W, Qiu G, He L. Loss of heterozygosity of chromosome 20 in sporadic colorectal cancer. Chin Med J (Engl). 2002;115:1529-1532.  [PubMed]  [DOI]
13.  Zhang F, Zhou C, Ling Y, Qiu G, Bai S, Liu W, He L, Peng Z. [Allelic analysis on chromosome 5 in sporadic colorectal cancer patients]. Zhonghua Zhongliu Zazhi. 2002;24:458-460.  [PubMed]  [DOI]
14.  Xu SF, Peng ZH, Li DP, Qiu GQ, Zhang F. Refinement of heterozygosity loss on chromosome 5p15 in sporadic colorectal cancer. World J Gastroenterol. 2003;9:1713-1718.  [PubMed]  [DOI]
15.  Peng Z, Zhang F, Zhou C, Qiu G, Bai S, Liu W, He L. [Loss of heterozygosity analysis to define putative region involved in tumor differentiation and metastases in sporadic colorectal cancer patients]. Zhonghua Waike Zazhi. 2002;40:776-779.  [PubMed]  [DOI]
16.  Peng Z, Ling Y, Bai S. [Loss of heterozygosity on chromosome 3 in sporadic colorectal carcinoma]. Zhonghua Yixue Zazhi. 2001;81:336-339.  [PubMed]  [DOI]
17.  Zhang F, Zhou C, Ling Y, Bai S, Liu W, Qiu G, He L, Peng Z. High frequency of LOHon chromosome 18 in Chinese sporadic colorectal cancer patients. Zhonghua Shiyan Waike Zazhi. 2002;19:320-321.  [PubMed]  [DOI]
18.  Pietsch T, Koch A, Wiestler OD. Molecular genetic studies in medulloblastomas: evidence for tumor suppressor genes at the chromosomal regions 1q31-32 and 17p13. Klin Padiatr. 1997;209:150-155.  [PubMed]  [DOI]
19.  Benítez J, Osorio A, Barroso A, Arranz E, Díaz-Guillén MA, Robledo M, Rodríguez de Córdoba S, Heine-Suñer D. A region of allelic imbalance in 1q31-32 in primary breast cancer coincides with a recombination hot spot. Cancer Res. 1997;57:4217-4220.  [PubMed]  [DOI]
20.  Praml C, Finke LH, Herfarth C, Schlag P, Schwab M, Amler L. Deletion mapping defines different regions in 1p34.2-pter that may harbor genetic information related to human colorectal cancer. Oncogene. 1995;11:1357-1362.  [PubMed]  [DOI]
21.  White PS, Maris JM, Beltinger C, Sulman E, Marshall HN, Fujimori M, Kaufman BA, Biegel JA, Allen C, Hilliard C. A region of consistent deletion in neuroblastoma maps within human chromosome 1p36.2-36.3. Proc Natl Acad Sci USA. 1995;92:5520-5524.  [PubMed]  [DOI]
22.  Yeh SH, Chen PJ, Chen HL, Lai MY, Wang CC, Chen DS. Frequent genetic alterations at the distal region of chromosome 1p in human hepatocellular carcinomas. Cancer Res. 1994;54:4188-4192.  [PubMed]  [DOI]
23.  Nomoto S, Haruki N, Tatematsu Y, Konishi H, Mitsudomi T, Takahashi T, Takahashi T. Frequent allelic imbalance suggests involvement of a tumor suppressor gene at 1p36 in the patho-genesis of human lung cancers. Genes Chromosomes Cancer. 2000;28:342-346.  [PubMed]  [DOI]
24.  Bièche I, Khodja A, Lidereau R. Deletion mapping of chromosomal region 1p32-pter in primary breast cancer. Genes Chromosomes Cancer. 1999;24:255-263.  [PubMed]  [DOI]
25.  Tanaka K, Yanoshita R, Konishi M, Oshimura M, Maeda Y, Mori T, Miyaki M. Suppression of tumourigenicity in human colon carcinoma cells by introduction of normal chromosome 1p36 region. Oncogene. 1993;8:2253-2258.  [PubMed]  [DOI]
26.  Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon P, Lelias JM, Dumont X. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell. 1997;90:809-819.  [PubMed]  [DOI]
27.  Jost CA, Marin MC, Kaelin WG. p73 is a simian [correction of human] p53-related protein that can induce apoptosis. Nature. 1997;389:191-194.  [PubMed]  [DOI]
28.  Sunahara M, Ichimiya S, Nimura Y, Takada N, Sakiyama S, Sato Y, Todo S, Adachi W, Amano J, Nakagawara A. Mutational analysis of the p73 gene localized at chromosome 1p36.3 in colorectal carcinomas. Int J Oncol. 1998;13:319-323.  [PubMed]  [DOI]
29.  Kapiteijn E, Liefers GJ, Los LC, Kranenbarg EK, Hermans J, Tollenaar RA, Moriya Y, van de Velde CJ, van Krieken JH. Mechanisms of oncogenesis in colon versus rectal cancer. J Pathol. 2001;195:171-178.  [PubMed]  [DOI]