Loss of heterozygosity of Kras2 gene on 12p12-13 in Chinese colon carcinoma patients

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 -20^oC 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.

Markers	Primer sequences	Length(bp)
D12S89	D12S89-F:5’-ATTTGAGAGCAGCGTGTTTT-3’	254-288
	D12S89-R:5’-CCATTATGGGGAGTAGGGGT-3’
D12s358	D12s358-F:5’-GCCTTTGGGAAACTTTGG-3’	238-270
	D12s358-R:5’-TAAGCCCTTTTATTTTTCCTAAC-3’
D12s310	D12s310-F:5’-GAAAACTAATTGCCCCTTAC-3’	243-253
	D12s310-R:5’-TTTAGATTCTCCAAATGCC-3’
D12S1606	D12S1606-F:5’-ATGAGAGGCCAAATTAAAG-3’	271-279
	D12S1606-R:5’-CTACTGTTGTGTCAGGGTCA-3’
D12s1596	D12s1596-F:5’-TCATGTGGCTGGTAGAGAAG-3’	204-222
	D12s1596-R:5’-CGTGAAGCAGATTTATCGTG-3’
D12s1617	D12s1617-F:5’-AGCCTGAGGGGCCACAT-3’	215-261
	D12s1617-R:5’-TGGGCAACTTGGATAAGAAACA-3’
D12s1592	D12s1592-F:5’-GCTGAGTGTGGTGGCAC-3’	238-280
	D12s1592-R:5’-GAGACTATTCCAACAGTGTATTTTC-3’
D12S1057	D12S1057-F:5’-AGAGACAGAAGCAGTAGGATGG-3’	227
	D12S1057-R:5’-GGTTCTGACGTTTTAAGACTCG-3’
D12S1591	D12S1591-F:5’-GCGCTTTGTACTATGTTACATTT-3’	234-276
	D12S1591-R:5’-GAAGACGTTGGGTGAATC-3’
D12S823	D12S823-F:5’-GGGCAATAGAGTGAGATTCTG-3’	119
	D12S823-R:5’-CCTACCTCCTTCCCCATCC-3’
D12S1034	D12S1034-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 72^oC 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 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 tissue					Tumor tissue
Markers	LOH (n)	Signal (n)	Frequency ofLOH (%)	Heterozygo-sity (%)	LOH (n)	Signal (n)	Frequency ofLOH (%)	Heterozy-gosity (%)
D12S823	0	5	0	50	1	5	20	50
D12S1034	2	7	28.57	70	3	7	42.88	70
D12S1596	1	4	25	40	1	4	25	40
D12S1591	1	7	14.29	70	3	7	42.88	70
D12S358	0	8	0	80	2	8	25	80
D12S310	0	3	0	30	1	3	33.33	30
D12S1592	0	4	0	40	0	4	0	40
D12S89	1	8	12.5	80	1	8	12.5	80
D12S1617	1	7	14.29	70	1	7	14.29	70
D12S1057	0	7	0	70	2	7	28.57	70
D12S1606	0	6	0	60	1	6	16.67	60

Table 3 Distribution of LOH on 12p12-13 in microsatellite-labeled primers.

	1		2		3		4		5		6		7		8		9		10
Biomarker	A	T	A	T	A	T	A	T	A	T	A	T	A	T	A	T	A	T	A	T
D12S823	○	○	◎	◎	◎	◎	○	○	○	○	◎	◎	◎	●	○	○	◎	◎	○	○
D12S1034	◎	◎	○	○	●	●	○	○	◎	◎	◎	◎	◎	●	○	○	◎	◎	●	●
D12S1596	◎	◎	◎	◎	○	○	○	○	○	○	○	○	●	●	○	○	○	○	◎	◎
D12S1591	○	○	◎	●	◎	◎	○	○	◎	◎	○	○	●	●	◎	◎	◎	◎	◎	●
D12S358	◎	◎	○	○	◎	◎	○	○	◎	◎	◎	◎	◎	●	◎	◎	◎	●	◎	◎
D12S310	◎	◎	○	○	○	○	○	○	○	○	○	○	○	○	◎	●	○	○	◎	○
D12S1592	○	○	◎	◎	○	○	○	○	◎	◎	○	○	◎	◎	◎	◎	○	○	○	○
D12S89	◎	◎	◎	◎	◎	◎	○	○	◎	◎	◎	◎	●	○	◎	●	○	○	◎	◎
D12S1617	○	○	◎	◎	◎	◎	○	○	◎	◎	◎	◎	●	●	◎	◎	○	○	◎	◎
D12S1057	◎	◎	◎	◎	◎	◎	○	○	○	○	◎	◎	◎	●	◎	◎	◎	●	○	○
D12S1606	◎	◎	○	○	◎	◎	○	○	◎	◎	○	○	◎	◎	◎	◎	○	○	◎	●

◎: Normal; ○: No signal; ●: LOH; A: adjacent tissue T: cancer tissue

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 character	LOH		Total	Ratio (%)	P
	+	-
Tissue class
Adjacent Tumor	3	7	10	30	0.17753
Tumor	6	4	10	60
Age (yr)
≤50	4	3	7	70	0.77816
>50	2	1	3	30
Sex
Male	5	2	7	70	0.25979
Female	1	2	3	30
Tumor size (cm)
<5	3	1	4	40	0.42919
≥5	3	3	6	60
Lymph node matastasis
Yes	4	2	6	60	0.598161
No	2	2	4	40

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.