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THE STUDY OF CORRELATION BETWEEN GSTA GENE DELETION AND SUSCEPTIBILITY TO LUNG CANCER Sun Guifan et al Laboratory of Occupational Medicine, Department of Preventive Medicine, China Medical University Shenyang, China INTRODUCTION Studies have confirmed that smoking, air pollution and exposure to occupational carcinogens are the major causes of lung cancer. Research also suggests that polycyclic aromatic hydrocarbons (PAH) may be important carcinogens. Besides these exogenous factors, some investigations have reported that host factors were also important in carcinogenesis. In 1981, Warholm et al first isolated glutathione S-transferase classµ (GSTµ, EC, 2• 5• 1• 18) from human liver, and demonstrated it to be the enzyme with the highest specificity and activity in the bio-transformation of certain type of carcinogens, especially PAH,(1) to inactive matabolites, thus raising the possibility that GSTµ may play a crucial role in the prevention and inhibition of carcinogenesis. The measurement of GSTµ in populations showed that only about 50% of people had the active GSTµ(Z). In 1992, using a molecular biological technique, Brockmoller et al demonstrated that the inactivity of the enzyme was correlated with GSTµ gene deletion in populations(3). In order to study the relationship between GSTµ gene deletion and lung cancer susceptibility, we recruited 175 lung cancer patients with different pathological diagnoses and 104 healthy controls to detect the GSTµ gene using the polymerase chain reaction (PCR) method. The results are reported below.

1. Subjects. 175 lung cancer patients with definite pathological diagnoses in the,Liaoning Cancer Hospital were recruited to be cases and 104 healthy residents living in the same area and being of the same nationality were selected to be controls. Individual investigations included age, sex, occupation, smoking, family history and so on. 2„ Preparation of Genomic DNA and Primer. Three to 5ml of blood were taken by venous puncture and transferred into polystyrene vials containing an appropriate amount of EDTA. 50µ 1 blood were taken and mixed with 0.5 ml TE buffer, centrifuged at 13000g for 10 seconds. The supernatant was discarded and the pellet was washed once more with the TE buffer. The precipitate was finally suspended with 100)u 1 potassium buffer (containing 50 mmol/l KCI, 10--20mmo1/1 Tris-HC1, 2.5mmo1/1 MgC12, 1% Laureth 12 at PH8.3, 0.5% Tween 20 and 100µ 1/ml proteinase K). The suspended solution was warmed at 56°C for 45 min, then at 95°C for 10 min to inactivate proteinase K. The solution was centrifugated at 13000g for 5 min, 10µ 1 supernatant was used for the polymerase chain reaction (PCR). The specific primers for GSTµ were gene selected from the known (GSTm1cDNA sequence according to the homologous rat genomic DNA sequence(4) and were prepared with a DNA synthesizer. A segment of about 250 base pairs covering exon 4 and exon 5 of the GSTµ gene was amplified by PCR in this study (see fig 1).

[Insert Fig. 1 here] Fig. 1. Sketch map of human GSTµ gene and the segment of GSTµ nucleotide in this study (tt). 3. PCR. PCR was performed in 100µ 1 reaction buffer containing 200µ M dNTP, lg M primers, 10µ 1 denatured DNA as described, and 2 units of thermostable Taq polymerase using a heat block instrument (Techne). Thirty cycles of amplification involving a 1 min (denaturation at 94°C), a 1.5 min annealing at 56°C and a 1 min extension, at 72°C were performed. 4. Electrophoresis. The amplification products were subjected to 2% agarose S (sea Kem) gel electrophoresis and identified under UV light. If the GSTµ gene was present, a clear band of GSTE.c gene amplified products was shown in the place about 250 bp, and no band was found if GSTµ gene deletion had occurred. (see fig. 2). [Insert Fig. 2 here] Fig. 2. The PCR amplification products of GSTg nucleotide sequence between exons 4 and 5 using DNA samples from blood. No. 1, 2, 4, 7 showed GSTµ gene presence, and No. 3, 5, 6, 8, 9 showed the gene absence. S: 100 bp DNA fragment ladder.

RESULTS 1. Table 1 showed the comparison of GSTµ gene deletion in lung ; cancer patients and controls. The results indicated that the GSTµ gene deletion rate in lung cancer patients was 71.4% which is significantly higher than that in controls, 51.9%. (p<0.005). Table 1. Frequency of the presence and absence of the GSTµ gene in lung cancer patients and controls. group presence (%) absence (%) total lung cancer 50 (28.6) 125 (71.4) 175 controls 50 (48.1) 54 (51.9) 104 xz=10.37 P<0.005 OR=2.3 95%CI 1.39-3.82 2. The stratified analysis of GSTµ gene deletion, according to the pathological types of lung cancer, indicated that in all ,squamous, adenocarcinoma and small cell carcinoma groups, the GSTµ gene deletion rate was markedly higher than that in controls, especially in the small cell cancer group, where the deletion rate reached 77.5% (see table 2).

Table 2. Frequency of the presence and absence of the GSTµ gene in different pathological types of lung cancer presence absence pathology xz P OR 95%CI cases(%) cases (%) squamous 22 (29.7) 52 (70.3) 5.78 <0.05 2.19 1.16-4.15 adenocarcinoma 19 (31.1) 42 (68.9) 4.31 <0.05 2.05 1.04-4.04 small cell carcinoma 9 (22.5) 31 (77.5) 7.57 <0.05 3.19 1.40-7.29 3. Table 3 was the stratified analysis of the GSTµ gene deletion rate, according to smoking status. The data showed that while the deletion rate of the GSTµ gene in lung cancer patients was significantly higher than the controls, respectively. No significant difference was observed between the smoking or non-smoking groups in either lung cancer subjects or controls. 4. Both the patients and controls were divided into two age groups to analyze the rate of GSTµ gene deletion. The older age group included subjects above 50 years of age and the younger group included subj ects below 50 years of age. The results showed that the frequency of GSTµ gene

deletion in lung cancer patients in both the older group and the younger group was significantly higher than that in controls. The stratified analysis in each group showed that in controls, the GSTg gene deletion rate had no correlation with age, but in lung cancer patients, the deletion rate in the younger group reached 85.3% which is signifiantly higher than that of the older group, 68.1% (see table 4). D]:SGUSSION With the invention and application of the technique of molecular genetics, especially the PCR method, Seidegard and Brockmoller confirmed i that GSTI.c genotypes were completely identical with phenotypes (i.e., the GSTµ activity could be detected in the liver and other tissues of the one with the GSTA gene and could not be detected in the body of the one without the GSTµ gene)(3-5). As the genotype determination showed a high specialty with which other methods could not be compared, the appliance of the PCR technique to detect the GSTU gene was one of the most reliable methods to determine whether the body could synthesize GSTµ. In this study, 175 lung cancer patients and 104 healthy controls had been detected by this method, and the results showed that GSTµ gene deletion in lung cancer patients was as high as 71.4% which was significantly higher than the rate in controls, and the OR value reached 2.3.

From these data, we can infer that GSTµ gene deletion may be an important marker of the susceptibility of the host to lung cancer. In 1990, Seidegard first reported that GSTµ gene deletion correlated with an increased risk of lung cancer,(6) and later, the same data were confirmed by other studies.(7"S) However, most of the studies reported that the GSTp gene deletion in small cell carcinoma and squamous cell cancer ranked at the top but that GSTµ gene deletion had no significant correlation with adenocarcinoma, especially in smokers. In our study, the data indicated that GSTµ gene deletion in the lung cancer patients with all the three pathologic types were markedly higher than that in controls, although the highest rate (77.5%) was in small cell carcinoma. However, when we stratified groups by smoking, there was no apparent relationship between smoking and GSTµ gene deletion. These results suggest that there may be some other factors besides smoking which may also be potentially associated with lung cancer development. Since the specificity and activity of GSTµ in catalyzing reactions involving PAH was the highest, and many environmental carcinogens (including the products of smoking) belong to the PAH compound, which were represented by BAP, we can infer that the deletion of the GSTµ gene may be one of the important host factors for susceptibility to lung cancer. Lafuente et al. reported that susceptibility to cancer due to GSTp gene deletion may manifest

itself in an earlier age of cancer development and a more malignant form of qarcinoma.(9) In this study, having stratified the groups by age, we found that in controls, there was no relationship between GSTµ gene deletion and age. However, in lung cancer groups, the GSTµ gene deletion rate of the younger group reached 85.3% which was markedly higher than that of the older group. Although the causes of lung cancer were relatively clear, the process of carcinogenesis was a mutual action of complex multiple factors. Factors, including exposure to carcinogens, the degree of exposure, and the host conditions, all may play important roles in causing the onset of cancer. Nevertheless, the discovery of the GSTµ gene and its physiologic and biochemical characteristics on catalyzing reactions involving carcinogens provided an important method to explore host susceptibility to lung cancer at the molecular level. In this research, we used a case- control study to demonstrate that the GSTµ gene deletion rate in lung cancer patients was abnormally increased. If a prospective cohort study of the onset of lung cancer in a population with GSTIi, gene deletion that included data on the exposure level of environmental carcinogens was conducted, the role of GSTµ gene deletion in causing lung cancer would be further clarified.

Table 3. Comparison of GSTg gene deletion between (ung cancer patients and controls stratified by s+moking smoking Lung Cancer Controls X2 P conditions GSTg (+) (X) GSTµ (-) (X) GSTA (+) (X) GSTg (-) (%) . smoking 34 (27.6) 89 (72.4) 14 (46.7) 16 (53.3) 4.05 <0.05 non-smoking 16 (3.08) 36 (69.2) 36 (48.6) 38 (51.4) 4.02 <0.05 X =0.18 P>0.05 X =0.03 P>0.05 OR 95%CI 2.29 1.02-5.13 2.13 1.02-4.46 * Cortparison of GSTµ gene deletion between smoking and non-smoking Table 4. Comparison of GSTg gene deletion between lung cancer patients and controls stratified by age. Lung Cancer Controls 2 age (year) X P OR 95XCI GSTµ (+) (X) GSTg (-) (X) GSTµ (+) (X) GSTµ (-) (X) < 50 5 (14.7) 29 (85.3) 28 (49.1) 29 (50.9) 9.48 <0.005 5.60 1.87-16.77 > 50 45 (31.9) 96 (61.8) 22 (46.8) 25 (53.2) 3.89 <0.05 1.97 1.01-3.86 X =3.97 P>0.05 X`=0.06 P>0.05 * Comparison of GSTg gene deletion between elder and younger tRzGy OSzO7

References 1. Warholm M, et al., Purification of a new glutathione S- transferase (transferase µ) from human liver having high activity with benzo(a)pyrene-4, 5-oxide. Biochen Biophys Res Commun 1981; 98(2):512-519 2. Brockmoller J, et al. Genotype and phenotype of glutathione S-transferase class µ isoenzyme and in lung cancer and controls. Cancer Res 1993; 53: 1004-1011. 3. Brockmoller J, et al. Correlation between trans-stilbene oxide-glutathione conjugation activity and the deletion mutation in the glutathione-transferase class gene detected by polymerase chain reaction. Biochem Pharmacol 1992; 43: 647- 650 4. Lai H-C J, et al. Gene expression of rat glutathione S- transferase. J. Biol. Chem. 263:11389-11395, 1989 5. Seidegard J, et al. Hereditary difference in the expression of the human glutathione transferase active on trans-stilbene oxide are due to a gene deletion. Pro Natl Acad Sci USA 1988; 85:7298-7297 6. Seidefard J, et al. Isoenzymes of glutathione transferase (class µ) as a marker for the susceptibility to lung cancer: a follow up study. Carcinogenesis 1990; 11:33-36 7. Nadachi K, et al. Polymorphisms of the CYP1A1 and glutathione S-transferase µ gene associated with susceptibility to lung