Interleukin-4 gene intron-3 polymorphism is associated with transitional cell carcinoma of the urinary bladder

Abstract

OBJECTIVE

To evaluate whether polymorphism of the interleukin-4 gene exon 3, and of the interleukin-1β gene exon 5 and promoter region, are associated with transitional cell carcinoma (TCC) of the urinary bladder, as cytokines are hypothesized to be important in cancer formation.

PATIENTS, SUBJECTS AND METHODS

The study included 138 patients with TCC of urinary bladder and 105 healthy controls living in the same area. Each genetic polymorphism was typed using polymerase chain reaction-based restriction analysis. Genotype distribution and allelic frequencies between patients and controls were compared.

RESULTS

There were significant differences in genotype and allelic distribution of intron 3 RP1/RP2 polymorphism (P < 0.001), but no significant difference in genotype distribution or allelic frequencies of the interleukin-1β gene polymorphism between patients with bladder cancer and controls.

CONCLUSION

The interleukin-4 gene intron 3 polymorphism is associated with bladder cancer and is a potential genetic marker in screening for the possible causes of bladder cancer.

Abbreviations

VNTR

variable numbers of tandem repeats.

INTRODUCTION

In Taiwan, urinary bladder cancer is one the most common urological malignancies and most such tumours are TCC; it is a complex and multifactorial disease. Risk factors associated with bladder cancer include cigarette smoking, chemical exposure, and an unknown exposure risk factor of endemic black-foot disease in southern Taiwan [1]. Genetic factors may have a role in the formation of bladder cancer. Some genetic polymorphisms are associated with bladder cancer, e.g. of p53 and p21[2,3].

Cytokines are signalling molecules contributing to the inflammatory response, and protect the body from pathogens and other environmental factors. Cytokines comprise several proteins that are key components in the pathogenesis of many diseases, including cancer. Interleukin-lβ, located at chromosome 2q12, is a potent pro-inflammatory agent which is central in immunoregulation, fever, inflammation and cancer formation [4]. Polymorphisms of the interleukin-1β gene promoter region and exon 5 have been screened for their role in the occurrence and severity of rheumatoid arthritis and osteoporosis [4,5].

Interleukin-4 is a key cytokine that induces the activation and differentiation of B cells, and the development of the Th2 subset of lymphocytes. Th2 cytokines such as interleukin-4, -6 and -10 primarily support antibody production, and many studies have confirmed that patients with cancer have high levels of such cytokines in their serum [6–8]. Interleukin-4 also inhibits macrophage activation and might be involved in cancer formation. The interleukin-4 gene has been mapped to the q arm (q23–31) of chromosome 5 [9], and is in a cluster of cytokine genes (interleukin-3, -5, -9, -13 and -15, granulocyte colony-stimulating factor, and interferon regulatory factor) [10]. A polymorphism of the interleukin-4 gene is located in the third intron, and is composed of a 70-bp sequence of variable numbers of tandem repeats (VNTR) [11].

In the present study we assessed the interleukin-1β and interleukin-4 genes because they have been reported to be associated with several different cancers [12,13]. To elucidate whether these polymorphisms are important in the susceptibility to bladder cancer, we investigated and compared the distribution of these polymorphisms between a control group and patients with bladder cancer, by analysing the results of PCR.

PATIENTS, SUBJECTS AND METHODS

The study comprised 138 patients from central Taiwan (109 men and 42 women, mean age 65.9 years, sd 12.2, range 42–80) with bladder cancer, treated in the authors’ institution, from April 1998 to December 2001. All of the pathological cell types in the cancer group were TCC. Patients were subdivided into those with invasive and noninvasive cancer, according to their pathological grading and clinical course. The patients with noninvasive and invasive cancer were classified as having pathologically superficial (Ta and T1) and invasive (T2a and T2b) tumours, using the American Joint Committee on Cancer staging system; there were 76 patients with noninvasive and 62 with invasive disease. From pathological grading, 36 patients were grade I, 68 grade II and 34 grade III.

The control group comprised 105 healthy volunteers (65 men and 40 women, mean age 53.5 years, sd 10.3, range 40–87) from the same area; none had a history of cancer. Routine urinary tests were used to exclude any control subjects who may have had microscopic haematuria. Informed consent was obtained from all patiensts and subjects participating in the study. Genomic DNA was prepared from peripheral blood using the Genomaker DNA Extractor kit (Blooms, Taiwan).

The total PCR volume was 25 µL and was composed of 2.5–10 pmol of each primer, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.0 mmol/L MgCl₂, 0.2 mmol/L of each deoxyribonucleotide triphosphate, and 1 unit of AmpliTaq DNA polymerase (Perkin Elmer, Forster City, CA, USA). Primers for the interleukin-4 gene intron 3 polymorphism were: upstream 5′-AGGCTGAAAGGGGGAAAGC-3′, and downstream 5′-CTGTTCACCTCAACTGCTCC-3′[11,14]. The cycling conditions were 95°C for 30 min, 55°C for 30 min and 72°C for 30 min. The interleukin-4 gene intron 3 polymorphism PCR products, including the 70-bp VNTR region, were directly analysed by 3% agarose gel electrophoresis, and each allele recognized according to its size. The RP1 and RP2 alleles were 183 bp and 253 bp, respectively.

The primers for the interleukin-1β gene promoter region − 511C/T polymorphism were: upstream 5′-TGGCATTGAT CTGGTTCATC-3′, and downstream 5′-GTTTAGGAATCTGGACCAGA-3′[4]. The cycling conditions were as given above. The restriction enzyme for the analysis was Ava I (New England Biolabs, Beverly, USA); 304 bp of PCR product was digested into 190 bp + 114 bp if the restriction site was present (‘C’ allele). This polymorphism was detected by restriction analysis according to the report by Cantagrel et al.[4].

Primers for the interleukin-1β gene exon 5 polymorphism were: upstream 5′-GTTGTCATCAGACTTTGACC-3′, and downstream 5′-TTCAGTTCATATGGACCAGA-3′. The cycling conditions were as given above. The region containing the polymorphic site within exon 5 of the interleukin-1β gene was amplified and then digested by Taq I (New England Biolabs). Class ‘E1’ was 135 bp + 114 bp and ‘E2’ was 249 bp, as shown on electrophoresis. For quality control, part of the samples were sequenced to confirm the results. Before sequencing, PCR fragments were purified from the agarose gel using QIAEX II kit (Qiagen, Germany). Direct sequencing used a d-rhodamine DyeDeoxy Terminator Sequencing kit (PE Applied Biosystems, Foster City, CA) with an ABI Prism 377 DNA Sequencer (PE Applied Biosystems).

The allelic frequency and genotype distributions of these polymorphisms in both groups were analysed using the chi-square test. When the assumption of the chi-square test was violated (i.e. when one cell had an expected count of <1, or >20% of the cells had an expected count of <5), Fisher's exact test was used; in both P < 0.05 was to indicate statistical significance. Odds ratios (OR) were calculated from allelic frequencies and carriage rates, with 95% CI.

RESULTS

The genotype distributions and allelic frequencies of the intron 3 regions in the interleukin-4 gene obtained from patients and controls are shown in Table 1; there were significant differences in genotype distribution of this polymorphism between the groups (P < 0.001), with significantly more patients with the RP1 homozygote (86%) than controls (64%). The OR (95% CI) for the RP1 homozygote and heterozygote were 8.88 (1.02–77.16) (P = 0.018) and 2.73 (0.289–25.73) (P = 0.359), respectively. The patients were further categorized into three groups according to pathological grading, but there were no significant differences in this polymorphism among the groups (Table 2). There was a significant difference in the genotype distribution of interleukin-4 intron 3 RP1/RP2 polymorphism between the noninvasive and invasive group (P = 0.002, Fisher's exact test); the distribution of RP1 in the invasive was significantly higher than that in the noninvasive group.

Table 1.  Comparison of the genotype distributions and allelic frequencies for interleukin-4 gene intron 3 region, and interleukin-1β gene promoter and exon 5, in patients with bladder cancer or in healthy control subjects

Genotype	Controls	Cancer	P
Interleukin-4 gene intron 3
N	105	138
Genotype			<0.001*
RP1/RP1	67 (64)	119 (86)
RP1/RP2	33 (31)	18 (13)
RP2/RP2	5 (5)	1 (1)
Allelic frequencies			<0.001*
RP1	167 (80)	256 (93)
RP2	43 (21)	20 (7)
Interleukin-1 promoter
N	105	130
Genotype − 511			0.338*
C/C	28 (27)	25 (20)
C/T	50 (48)	74 (60)
T/T	27 (26)	24 (20)
Allelic frequencies			0.988*
C	106 (51)	124 (50)
T	104 (49)	122 (49)
Interleukin-1 exon 5
Genotype			0.812¶
E1/E1	102 (97)	127 (98)
E1/E2	3 (3)	2 (2)
E2/E2	0	1 (1)
Allelic frequencies			1.0¶
E1	207 (98.6)	256 (98.5)
E2	3 (1.4)	4 (1.5)

*Chi-square test; ¶Fisher's exact test.

Table 2.  Distribution of interleukin-4 gene polymorphism genotypes in patients with bladder cancer according to pathological grading and clinical staging

Group	RP1/RP1	RP1/RP2	RP2/RP2	Total	P*
* Fisher's exact test.
Grade, n (%)					0.69
I	27 (75)	9 (25)	0	36
II	62 (91)	5 (7)	1 (1.4)	68
III	30 (88)	4 (12)	0	34
Total	119	18	1	138
Invasiveness					0.002*
Non-invasive	60 (79)	16 (21)	0 (0)	76
Invasive	59 (95)	2 (3)	1 (1.6)	62
Total	119	18	1	138

The genotype frequencies of the promoter and exon 5 regions of the interleukin-1β gene in the two groups are also shown in Table 1. There were neither significant differences in the genotype distributions nor allelic frequencies of the interleukin-1β gene promoter and exon 5 polymorphism between the patients and controls.

DISCUSSION

The interleukin-4 gene intron 3 polymorphism showed significantly different distributions between normal controls and patients with cancer, but there was no association between the interleukin-1 gene and bladder cancer, indicating that this gene and its inflammatory response is less likely to be associated with bladder cancer. The association between bladder cancer and various genetic markers has helped to increase knowledge of the genetics of the immune response to and pathogenesis of bladder cancer.

In the present analysis, we chose to screen polymorphisms in the interleukin-4 gene; the RP1/RP2 polymorphism in intron 3 may enhance cancer formation either through an IgE pathway, or its transcription activity. The function of the RP1/RP2 intron 3 polymorphism is unknown; possibly distinct numbers of VNTR copies might affect the transcriptional activity of the interleukin-4 gene. Why the RP1/RP2 intron 3 polymorphism of this gene is associated with bladder cancer and tumour invasiveness should be further clarified; future studies could lead to immunotherapy for bladder cancer. However, the relationship between bladder cancer and interleukin-4 gene polymorphism has been studied less; to our knowledge, the present is the first study of the association of interleukin-4 gene intron3 RP1/RP2 polymorphism with bladder cancer. The relationship between cytokines and the severity of bladder cancer is worth further investigation.

Seddighzadeh et al.[15] studied 164 bladder tumours and found a large variation in mRNA levels of interleukin-1α; the association of interleukin-1α expression and cancer-specific survival was not statistically significant. The evidence for a mechanism of tumour growth inhibition by interleukin-1α is weak. Kuo et al.[16] also found no statistical significance of interleukin-1α and -1β activities in blood cultures from patients with bladder cancer when compared with control subjects; the present data are compatible with their findings.

In the present study, that interleukin-1 was not significantly associated with bladder cancer formation may have been because there was an incomplete match between the serum level and its expression in tissue, or because interleukin-1 influenced tumour progression but not formation. This problem provides a target for further studies.

In conclusion, the interleukin-4 gene intron 3 polymorphism is associated with bladder cancer but the mechanism remains unclear. However, the interleukin-4 but not interleukin-1 gene might be a potential genetic marker in screening for the possible causes of bladder cancer.

ACKNOWLEDGEMENTS

This study was supported by a grant from the China Medical University Hospital (DMR-90–043).

CONFLICT OF INTEREST

None declared.