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I 1 I I 1 I I 1 I I 1 I I THE STUDY OF CORRELATION BETWEEN C.STµ GENE DELETION AND SUSCEPTIBILITY TO LUNG CANCER Sun Gui-fan*, Pi Jing-bo*, Zheng Quan-mei** and Zheng Mei-zhen*** * Laboratory of Occupational Medicine, Department of Preventive Medicine, China Medical University, Shenyang, China ** Cancer Prevention Center of China Medical University, Shenyang, China *** Liaoning Cancer Hospital, Shenyang, China Introduction Studies have confirmed that smoking, air pollution and exposure to occupational carcinogens are the major risk factors for lung cancer. Research also suggests that polycyclic aromatic hydrocarbons (PAH) may be important carcinogens. In addition to these exogenous factors, some investigations have reported that host factors were also important in carcinogenesis. In 1981, Warholm eta l. (1) first isolated glutathione S-transferase classµ (GSTµ, EC, 2• 5• I• 18) from human liver, and demonstrated it to be the enzyme with the highest specificity and activity in the bio-transformation of certain types of carcinogens, especially PAH, to inactive metabolites, thus raising the possibility that GSTµ may play a crucial role in the prevention and suppression of carcinogenesis. The measurement of GSTµ in populations showed that only about 50% of people had the active GSTµ(2). In 1992, using a molecular biological technique, Brockmoller et al.(3) demonstrated that the inactivity of the enzyme was correlated with GSTµ gene deletion in certain populations. 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 GSTp gene using the polymerase chain reaction (PCR) method. The results are reported below. Materials and Methods 1. Subjects. One hundred and seventy-five lung cancer patients in the Liaoning Cancer Hospital with confirmed pathological diagnoses were recruited as cases and 104 healthy residents living in the same area and with the same nationality were selected to be controls. Individual investigations included age, sex, occupation, smoking, family history, etc. 2. PreXaration of Genomic DNA and Primer. Three to 5 ml of blood were taken by venous puncture and transferred into polystyrene vials containing an appropriate amount of EDTA. Fifty µl of blood were taken and mixed with 0.5 ml TE buffer and centrifuged at 13,000g 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 µl buffer (containing 50 tnmol/L KCI, 10-2ommol/L Tris-HCI, 2.5 mmol/L MgClz, 1% Laureth 12 at pH 8.3, 0.5% Tween 20 and 100 µl/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 13,000g for 5 min,

10 µ1 of supernatant was used for the polymerase chain reaction (PCR). The specific primers for GSTµ gene selected from the known GSTmI cDNA sequence according to the homologous rat genomic DNA sequence(4) were prepared with a DNA synthesizer. A segment of about 250 base pairs covering exon 4 and exon 5 of the GSTg gene was amplified by PCR in this study (Fig 1). , E1 EZ E3 E4 ES E6 E7 E8 3, rr Fig. 1 Sketch map of human GSTµ gene and the segment of GSTµ nucleotide in this study ( t}). 3. PCR. PCR was performed in 100 µl reaction buffer containing 200 µM dNTP, 1 µM primers, 10 µl denatured DNA 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 separated on 2% agarose S (sea Kem) gels and identified under W light. The presence of GSTµ gene was identified by a clear band of GSTµ gene amplified products migrating to a position of 250 bp. No band was found if GSTp gene deletion had occurred. (Fig. 2). S t a a 4 e s 7 8 9 Fig. 2 The PCR amplification products of GSTfc 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. -2- I I I I I I I I I I I I I I I

I I I I I I I I I I I I I I I I Results 1. Table 1 shows 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 was significantly higher than the 51.9°k in controls. (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 X'=10.37 P<0.005 OR=2.3 95%Cl 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. In the small cell cancer group, 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 Pmsence Absence Patholo8ic ----- ----- X, ~ • P . OR . .~. 95% Ct Cell Types . . Cases (%) CaSes (%)' .::.... Squamous cell carcinoma 22 (29.7) 52 (70.3) 5.78 <0.05 2,19 1.16d.15 Adenocan:inoma 19 (31.1) 42 (68.9) 4.31 <0.05 2.05 1.04d.04 Small cell carcinoma 9(22.5) 31 (77.5) 7.57 <0.05 3.19 1.40-Z29 3. Table 3 shows the stratified analysis of the GSTµ gene deletion rate, according to smoking status. The data show that the deletion rate of the GSTµ gene in lung cancer patients was significantly higher than in the controls, no significant difference was observed between the smoking and nonsmoking groups in either lung cancer cases 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 subjects 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 the controls. The stratified analysis in each group showed that in controls, the GSTµ gene deletion rate had no correlation with age, but in lung cancer patients, the deletion -3- I

I I rate in the younger group reached 85.3% which is significantly higher than that of the older group, 68.1 % (Table 4). Table 3. Comparison of GSTµ gene deletion between lung cancer patients and controls stratified by smoking GSTP(+) _..(S) GSTr(•) (x) ' GSPY(fl (Y) GSTP(.) (9) Smaki~ 36 (Z/.6) 89 (R.4) 14 (46,Y) 16 (53.3) 4.u3 <0AJ 2.29 LOY5.13 Nmsm~3iny __.. 16 (326) 36 (69.2) 36 (B.6) .,d (51.4) 4D2 0.05 2.13 1.02i.46 K2-O.IB P>U.05 22-0.03 P>0.05 Cmnpuism & GSTM lmm Eeletim Ddxeen fmotiy md mnsmakicg Table 4. Comparison of GSTµ gene deletion between lung cancer patients and controls stratified by age nsc Uiwq tmig Cuw 95% CI QtTr (-) ('L) GStP (-) (E) GSfx (1) . , .1A) G51'e (-)c..'_ (%) <Sp 5 (14.3) 29 (9S3) 28 (49.1) 19 (509) 9.4t <0AOOS 5.60 LlT16Jl 250 _.. . 45 (31.9) 96 (619) 22 (<6.W 25 (51.2) 3.0 005 1.91 1.013.A6 y2-3.91 P<0.05 x2-0.06 P>U.LLS CmnPuum d GSfM ~ 6e4tian hetwan elkr mC Ywngc Discussion With advances and new applications developed in molecular genetic techniques, especially the PCR method, Seidegard and Brockmo"ller confirmed that GSTµ genotypes were completely identical with phenotypes (i.e., the GSTµ activity was detected in the liver and other tissues of individuals carrying the GSTµ gene but not in individuals lacking the GSTµ gene)(3-5). Since genotypic characterization has the precision unmatched by other methods, application of the PCR technique for detecting the GSTµ gene becomes a most reliable method to determine whether the capacity for the broaynthesis of GSTµ is present. In this study, GSTµ gene in 175 lung cancer patients and 104 healthy controls was evaluated by this method. The results show that GSTµ gene deletion in lung cancer patients was as high as 71.4 % and was significantly higher than the rate in controls (OR 2.3, 95 % CI 1.39-3.82). Thus it can be inferred that GSTµ gene deletion is an important marker of the susceptibility of the host to lung cancer development. In 1990, Seidegard first reported that GSTµ gene deletion correlated with an increased risk of lung cancer(6), a finding which was subsequently confirmed by other studies (7,8). All published studies reported that the GSTµ gene deletion was most pronounced in small cell carcinoma and squamous cell -4- I I I I I I I I I I NI C 00 i -4 ta ~ °° I y I I I I I

I I I I I I I , I I I I I I carcinoma. No study has reported GSTµ gene deletion being significantly correlated with adenocarcinoma, especially in smokers. Our study shows that GSTµ gene deletion in the lung cancer patients were markedly higher than that in controls. All three pathologic types of lung cancer had elevated GSTµ gene deletions although the highest rate (77.5%) was seen in the small cell carcinoma group. However, when stratified groups by smoking, no apparent relationship between smoking and GSTµ gene deletion was found. These results suggest that there may be factors other than smoking which may have a potential association with lung cancer development. Since GSTµ has the highest specificity and activity for the bio-transformation of PAH and many environmental carcinogens (including the products of smoking and B(a)P belonging to the PAH family of compounds), to inactive metabolites, we can infer that the deletion of the GSTµ gene may be one of the most important host factors for susceptibility to lung cancer. Lafuente et al.(9) reported that susceptibility to cancer due to GSTµ gene deletion may manifest itself in an earlier age of cancer development and a more malignant form of carcinoma. In this study, when the groups were stratified by age, there was no relationship between GSTµ gene deletion and age found in the controls. However, in the lung cancer groups, the GSTµ gene deletion rate of the younger age group was 85.3 % which was markedly higher than that of the older group. Although the causes of lung cancer are becoming clear, the process of carcinogenesis is a complex interaction of multiple factors. These factors can include exposure to carcinogens, the degree of exposure, and the host conditions, all of which may converge in some yet-to-be-defined mechanism in causing the onset of cancer. The discovery of the GSTµ gene and its involvement in bio-inactivation of carcinogens provides an important lead for exploring 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 significantly elevated, compared to controls. A prospective cohort study involving GSTµ gene deletion assessment, in combination with data on exposure to environmental carcinogens may further clarify the role of GSTµ gene in lung carcinogenesis. -5- I

I References 1. Warholm, M, etal., 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, etal. 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, eta l. 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 CYPIAI and glutathione S-transferase µ gene associated with susceptibility to lung cancer in relation to cigarette dose in a Japanese population. Cancer Res. 1993; 53:2994-2999 8. Nazar-Stewart, V, et al. The glutathione transferase polymorphism as a marker for susceptibility to lung carcinoma. Cancer Res. 1993; 53:2313-2318 9. Lafuente, A, et al. Human glutathione transferase µ(GSTµ) deficiency as a marker for the susceptibility to bladder and larynx cancer among smokers. Cancer Letters. 1993; 68:49-54 -6- I I I I I I I I I I I 1 I I

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