ANTICANCER RESEARCH 26: 299-306 (2006)

CYP2C9 Polymorphisms in Human Tumors

HEIKE KNÜPFER1*, DIRK STANITZ2 and RAINER PREISS1

1University of Leipzig, Department of Clinical Pharmacology, Leipzig; 2Department of Internal Medicine, Hospital of the Paul-Gerhardt-Stiftung, Lutherstadt Wittenberg, Germany

Abstract. The oxazaphosphorines cyclophosphamide (CP) derivative (phosphoramide mustard or ifosphoramide and ifosfamide (IF) are alkylating agents that require mustard) and acrolein. The mustard alkylates DNA and is bioactivation via cytochrome (CYP) P450 isoenzymes considered to be the therapeutically significant cytotoxic including CYP2C9 enzymes. The present study investigated metabolite. CYP2C9 in regard to its allelic variants in 23 tumor samples Multiple human P450 enzymes are capable of activating (10 breast tumors, 1 breast tumor cell line, 5 brain tumors, 7 oxazaphosphorines, among them CYP3A (2-5) and CYP2C glioma cell lines) with restriction fragment length enzymes (6). Four CYP2C have been identified – polymorphism polymerase chain reaction (RFLP-PCR). The CYP2C8, CYP2C9, CYP2C18 and CYP2C19 (7). Of those, mutant alleles of CYP2C9 were residue 144 (Arg (*1)/Cys CYP2C9 is expressed at the highest concentration in human (*2)), residue 358 (Tyr/Cys), residue 359 (Ile/Leu (*3)) and liver (8). CYP2C9 catalyzes the oxidation of diverse xenobiotic residue 417 (Gly/Asp). The frequencies of the CYP2C9*1, chemicals, such as tolbutamide, warfarin, flurbiprofen, CYP2C9*2 and CYP2C9*3 alleles in the cancer samples phenytoin, hexobarbital, diclofenac (8, 9) and also the examined were found to be 0.848, 0.152 and 0.043, anticancer agents ifosfamide and cyclophosphamide (6). respectively. No sample revealed a mutation at residue 358 or The metabolic activation of the oxazaphosphorines is 417. Comparing breast with brain tumors, brain tumors thought to take place primarily in the liver. However, as seemed to reveal a higher incidence of heterozygotes (5/12 CYP enzymes are also found in some extrahepatic tissues compared to 2/11) at residue 144 and, with regard to residue (10), extrahepatic activation, e.g. intratumorally, can be of 359, a lower incidence of heterozygotes (0/12 compared to importance for the bioactivation of some anticancer agents. 2/11). In summary, our data indicate that in tumor material, Indeed, CYP enzymes have been found in different kinds of as in healthy material, the same allelic variants of CYP2C9 tumors, e.g. in lung, colon, liver and breast tumors (11-17), occur. Compared to healthy tissue, tumor material seemed to suggesting that activation of anticancer prodrugs may also reveal a higher incidence of the *2 allele, but a significant occur locally in tumor tissue. Recently we examined the difference could not be established. Our results show that brain CYP2C9 expression in brain and breast tumors and found and breast tumor samples appeared to differ in their frequency all analyzed tumors positive (18-20). We have further shown of heterozygotes at residues 144 and 359. This might also have that breast tumor tissue metabolizes ifosfamide (20). an impact on intratumoral oxazaphosphorine metabolism. Two CYP2C9 variant alleles, designated as CYP2C9*2 and CYP2C9*3, are associated with significant reductions Cyclophosphamide and its isomeric analog, ifosfamide, are in intrinsic clearance of a variety of 2C9 substrates alkylating anticancer prodrugs that require bioactivation to compared with the non-mutated CYP2C9*1 allele. These produce pharmacological activity (1). The 4-hydroxy allelic variants occur at high frequency among Caucasians, metabolite, formed by enzymes, but not among African-American and Asian populations equilibrates with the ring-opened aldophosphamide, which (21). Other allelic variants, designated as CYP2C9*4 to undergoes chemical decomposition to yield a mustard CYP2C9*12, have recently been reported, but the functional significance of these variants is unclear. Such allelic variations may be of substantial pharmacological significance, since striking differences in substrate Correspondence to: Dr. rer. nat. Heike Knüpfer, Universität specificity and catalytic activity can result from as few as Leipzig, Institut für Klinische Pharmakologie, Härtelstr. 16-18, one or two amino acid changes in a P450 enzyme (22-25), 04107 Leipzig, Germany. Tel: +49-341-9724655, Fax: +49-341- 9724569, e-mail: [email protected] e.g. the Leu359 polymorphic variant of CYP2C9 has been shown to be responsible for the poor metabolizer Key Words: RFLP, polymorphism, CYP2C9, allele. phenotype with tolbutamide as substrate (26). Therefore,

0250-7005/2006 $2.00+.40 299 ANTICANCER RESEARCH 26: 299-306 (2006) different allelic variants of CYP2C members should have Table I. Restriction digestion conditions. an impact on the activation of oxazaphosphorines with /amino Enzyme Concentration Incubation Incubation poor (PM) and extensive (EM) metabolizers, even in acid position temperature time tumor material. In general, allelic variations were analyzed using healthy 2C9/144 Ava II 3.0 U 37ÆC 5 hours tissue samples. Little is known about the possible 2C9/358 Nsi I 5.0 U 37ÆC 3 hours differences in allelic frequencies between cancer patients 2C9/359 Ile Nsi I 5.0 U 37ÆC 3 hours 2C9/359 Leu Kpn I 15.0 U 37ÆC 5 hours and healthy people, which might also have an impact on 2C9/417 Hph I 5.0 U 25ÆC 5 hours anticancer pro-drug activation, although knowledge about intratumoral activation of anticancer drugs is increasing. Concerning tumors of the breast and brain, nothing is known about polymorphic alleles in these tumor entities. For this purpose, we examined 11 breast (10 breast tumors, PCR. The final volume of the PCR reaction was 50 Ìl. All PCR 1 breast tumor cell line) and 12 brain tumors (5 brain reactions were performed with 200 ng of genomic DNA, 5 Ìl tumors, 7 brain tumor cell lines) with regard to allelic 10xPCR buffer (200 mM Tris-HCl (pH 8.4); 500 mM KCl, 2 Ìl variants of CYP2C9, by RFLP-PCR methods. 50 mM MgCl2, 5 Ìl 2 mM dNTP, either 10 Ìl (359Leu and 359Ile) or 15 Ìl (144, 358 and 417) of 2 ÌM of each primer Materials and Methods respectively, and 1 unit Taq-Polymerase (Gibco, Germany). For each gene investigated, either 35 (144, 359Ile, 359Leu) or 40 (358, 417) PCR cycles were performed, respectively. All of the Materials. The chemicals and reagents were obtained from Sigma primers were previously published by Wang et al. (29) and (Deisenhofen, Germany). The cell culture reagents were obtained synthesized by Gibco. The PCR reaction mixtures were treated from Gibco/BRL (Eggenstein, Germany), while the restriction with 2 Ìl of loading buffer (30% (w/v) glycerine; 0.1 mM EDTA, enzymes (AvaII, NsiI, KpnI) were from Boehringer Mannheim pH 7.5; 0.1% (w/v) xylencyanol-FF), electrophorized at 100 V (Germany) and HphI from ICN (Eschwege, Germany). and stained with ethidium bromide (0.5 Ìg/ml). For size definition the 50-bp-ladder from Gibco was used. Tissue samples. A total of 10 breast tumor samples and 5 brain tumor samples was collected for this study. After resection, the Restriction-enzyme digestion. The PCR products were analyzed by samples were immediately frozen in liquid nitrogen. Additionally, selecting restriction enzymes that recognized the single nucleotide cell culture cell lines were also used (MCF-7, breast tumor cells; difference at the corresponding sites and were unique to the A171, 86HG39, T98G, 85HG66, U87MG, GOS1, GOS2, brain regions amplified. The restriction digestion conditions are tumor cells). DNA from human liver material and from human summarized in Table π. The digested products were analyzed on leukocytes served as positive controls. agarose gels (1 – 3.5%). The theoretical background for the PCR-RFLP tests of the Cell culture. The adherent growing permanent human cell lines different loci is described by Wang et al. (29). In brief, substitution → used in this study (A172, T98G, U87MG) were obtained from the of a single base (T416 C416) in exon 3 of CYP2C9 results in an → American Type Culture Collection, Rockville, MD, USA. amino acid change of the CYP2C9 (Cys144 Arg144) 86HG39 and 85HG66 were a gift from T. Stavrou (Neurosurgery, creating the recognition sequence of Ava II and restriction of the University of Hamburg, Germany). The characterizations of PCR product (420 bp) into 2 fragments of 363 and 57 bp. The 86HG39 and 85HG66 have been published (27, 28). The GOS1 PCR-RFLP test for CYP2C9/358 (Tyr/Cys) in exon 7 requires the and GOS2 cells were a gift from H. Halfter (University of amino acid Ile at position 359, because the recognition sequence Münster, Germany). The MCF-7 breast tumor cells were kindly of Nsi I includes both codons. The exchange from A1059 to G1059 provided by Antje Berthold (Children`s Hospital, Leipzig, in the sequence ATACATT creates a recognition sequence of Nsi Germany). All the cell lines were grown in monolayer culture in I and restriction of the PCR product (93 bp) into 2 fragments of complete medium consisting of RPMI 1640 supplemented with 46 and 47 bp. 10% fetal calf serum, 100 U/ml penicillin, 100 Ìg/ml streptomycin A mismatched PCR primer (29) was used to detect codon and 2 mM L-glutamine. The cell lines were maintained at 37ÆC Ile359. The A1061 in the sequence together with a mismatched and 5% CO2. The cells were harvested on reaching 90% codon (G1059) creates the recognition site of Nsi I. After PCR confluency and immediately frozen in liquid nitrogen until DNA amplification (131 bp) and digestion, samples with A1061 gave 110 extraction. To exclude the possibility of mycoplasma and 21 bp bands. Another mismatched primer (29) was used to contamination, some cell lines were spot-checked. detect codon Leu359. A C1061 in the sequence together with a mismatched codon (G1057) creates the recognition site of Kpn I. DNA isolation. Tissue and cell culture samples were After PCR amplification (131 bp) and digestion, samples with homogenized with 10-20 strokes using a glass tube and a glass C1061 gave 110 and 21 bp bands. pestle. Total genomic DNA was isolated with TRIzol Reagent The exchange of a single base (G1236 → A1236) in the sequence (Gibco/BRL), as recommended by the manufacturers using the GGTGGC (CYP2C9/417 wild-type, Gly/Gly) creates the recognition DNA isolation protocol. Afterwards, the DNA was dissolved in sequence of Hph I. After PCR amplification (160 bp) and digestion, 8 mM NaOH. samples with A1236 (mutant, Asp/Asp) gave 90 and 70 bp bands.

300 Knüpfer et al: CYP2C9 Polymorphisms in Human Tumors

Figure 2. PCR-RFLP strategy and analysis for substitution of the CYP2C9358 Tyr/Cys. The results of the PCR-RFLP analysis for the presence of the wild-type and the mutant allele for 11 breast and 12 brain tumor samples are presented (10 breast tumors, 1 breast tumor cell line, 5 brain tumors, 7 glioma cell lines). The amplification products were Figure 1. PCR-RFLP strategy and analysis for substitution of the CYP2C9 digested with Nsi I. This strategy yielded a 93-bp band for wild-type DNA Arg/Cys allele. The results of the PCR-RFLP analysis for the presence of (Tyr/Tyr), 46 and 47 bp for the mutant DNA (Cys/Cys), and all bands the wild-type and the mutant allele for 11 breast and 12 brain tumor with DNA from heterozygous individuals (Tyr/Cys). samples are presented (10 breast tumors, 1 breast tumor cell line, 5 brain K=undigested control; HL=human liver; BPL= ladder; tumors, 7 glioma cell lines). The amplification products were digested with 1=breast tumor cell line, 2-11=breast tumor tissues; 12-18=malignant Ava II. This strategy yielded a 420-bp band for mutant DNA (Cys/Cys), glioma cell lines, 19-23=brain tumor tissues; Leu=human leukocytes. 363 bp and 57 bp for the wild-type DNA (Arg/Arg), and all bands with DNA from heterozygous individuals (Cys/Arg). K=undigested control; HL=human liver; BPL= base pair ladder; 1=breast tumor cell line, 2-11=breast tumor tissues; 12-18=malignant glioma cell lines, 19-23=brain tumor tissues; Leu=human leukocytes. CYP2C9/359 in the analysed tumor samples are shown in Figure 3. In 23 tumor samples tested, 21 samples showed a Results complete Nsi I digestion indicating homozygous A1061 (Ile/Ile), and 2 samples showed heterozygous (Ile/Leu) PCR-RFLP test for CYP2C9/144 (Arg/Cys) in exon 3. The results (lanes 9 and 10). Both heterozygous samples were results of PCR-RFLP analysis for the mutated alleles of breast tumor tissues. CYP2C9 in the analyzed tumor samples are shown in Figure These results were confirmed by the Kpn I digestion. 1. The genotypes obtained from PCR-RFLP analysis were Only heterozygous samples were digested (lanes 9 and 10). as follows; lanes 1, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 20, 21, 22: Arg/Arg; lanes 2, 4, 12, 17, 18, 19, 23, Leu: Arg/Cys. PCR-RFLP test for CYP2C9/417 (Gly/Asp) in exon 8. The Breast and brain tumor samples differed in their results of the PCR-RFLP analysis for the mutated alleles of frequencies of Cys/Arg and Arg/Arg. Concerning breast CYP2C9/417 in the analyzed tumor samples are shown in tumors, only 2 out of 11 analyzed samples were Figure 4. All tumor samples gave a single 160-bp band in heterozygous, while 5 out of 12 brain tumors showed a the PCR-based test of the G1236 mutation, excluding the Cys/Arg genotype. presence of A1236 (Asp417). All tumor samples revealed None of the breast or brain tumors was positive for the homozygous Gly/Gly 417 alleles. Brain and breast tumors mutant allele Cys/Cys. were not different. The results concerning the different CYP2C9 alleles in PCR-RFLP test for CYP2C9/358 (Tyr/Cys) in exon 7. The brain and breast tumors are summarized in Table II. results of the PCR-RFLP analysis for the mutated alleles of Published frequencies of the *1, *2 and *3 alleles of CYP2C9 in the analyzed tumor samples are shown in Figure healthy material are listed in Table III and are compared to 2. All PCR probes were left undigested and were our results of tumor material (95% confidence interval homozygous for Tyr358 (wild-type). There were no (CI)). The frequency of the *2 allele in tumor material differences in breast and brain tumor samples. seemed to be slightly increased compared to the frequencies in healthy subjects, but the difference was not statistically PCR-RFLP test for CYP2C9/359 (Ile/Leu) in exon 7. The significant. The frequencies of the 2 other alleles (*1 and results of the PCR-RFLP analysis for the mutated alleles of *3) were not statistically significantly different.

301 ANTICANCER RESEARCH 26: 299-306 (2006)

Figure 3. PCR-RFLP strategy and analysis for substitution of the CYP2C9359 Ile/Leu. The results of the PCR-RFLP analysis for the presence of the wild-type and the mutant allele for 11 breast and 12 brain tumor samples are presented (10 breast tumors, 1 breast tumor cell line, 5 brain tumors, 7 glioma cell lines). The amplification products were digested with Nsi I and Kpn I, respectively. This strategy yielded a 131-bp band for mutant DNA (Leu/Leu) and wild-type DNA (Ile/Ile), respectively, 110 and 21 bp for the wild-type (Ile/Ile) and mutant DNA (Leu/Leu), respectively, and all bands with DNA from heterozygous individuals (Leu/Ile and Ile/Leu, respectively). K=undigested control; HL=human liver; BPL= base pair ladder; 1=breast tumor cell line, 2-11=breast tumor tissues; 12-18=malignant glioma cell lines, 19-23=brain tumor tissues; Leu=human leukocytes.

Discussion

The aims of our study were i) to detect CYP2C9 allelic variants in tumor material, which might have an impact on anticancer pro-drug activation, and ii) to compare allele frequencies with frequencies of healthy people. There are some studies on the connections between CYP polymorphism and cancer risk. A modest association between the frequency of CYP2C9*2 alleles and lung cancer risk has been described in a North-American population. However in the same study, the frequency for CYP2C9*3 variant alleles was similar among lung cancer patients and healthy subjects Figure 4. PCR-RFLP strategy and analysis for substitution of the CYP2C9417 Gly/Asp. The results of the PCR-RFLP analysis for the (30). Another study was carried out, and no association presence of the wild-type and the mutant allele for 11 breast and 12 brain between either CYP2C9*2 or CYP2C9*3 alleles and lung tumor samples are presented (10 breast tumors, 1 breast tumor cell line, 5 cancer risk was identified (31). Concerning colorectal cancer, brain tumors, 7 glioma cell lines). The amplification products were Martinez et al. (32) found an association between CYP2C9 digested with Hph I. This strategy yielded a 160-bp band for wild-type gene polymorphism and colorectal cancer risk. Individuals DNA (Gly/Gly), 90 and 70 bp for the mutant DNA (Asp/Asp), and all bands with DNA from heterozygous individuals (Gly/Asp). lacking the *2 or *3 alleles were at increased risk, while K=undigested control; HL=human liver; BPL= base pair ladder; another study (33) failed to detect any association between 1=breast tumor cell line, 2-11=breast tumor tissues; 12-18=malignant CYP2C9 polymorphisms and colorectal cancer. However, little glioma cell lines, 19-23=brain tumor tissues; Leu=human leukocytes. is known about allelic variants of CYP2C9 in tumor material, although the knowledge about intratumoral activation of anticancer pro-drugs is increasing. Nothing is known about polymorphic alleles in tumors of the breast and brain. For this linked to a genetic polymorphism in the enzymes purpose, we examined 11 breast (10 breast tumors, 1 breast responsible for their metabolism. The consequence of this tumor cell line) and 12 brain tumors (5 brain tumors, 7 brain variation may be of significance with respect to either the tumor cell lines) with regard to allelic variants of CYP2C9, by efficacy or toxicity of the drug. One of the most extensively RFLP-PCR methods. studied classes of enzymes is the cytochrome P450 (CYP) A large number of drugs display interindividual variation superfamily, with the CYP2D6 polymorphisms being the in their metabolic profiles, which, in some cases, has been best characterized (34). Many other polymorphisms have

302 Knüpfer et al: CYP2C9 Polymorphisms in Human Tumors

Table II. CYP2C9 RFLP results in brain and breast tumors.

AS 144 AS 358 AS 359 AS 417

No. Arg/Arg Arg/Cys Cys/Cys Tyr/Tyr Tyr/Cys Cys/Cys Ile/Ile Ile/Leu Leu/Leu Gly/Gly Gly/Asp Asp/Asp

Brain 12 7 5 0 12 0 0 12 0 0 12 0 0

Breast 11 9 2 0 11 0 0 9 2 0 11 0 0

Total 23 16 7 0 23 0 0 21 2 0 23 0 0

been described in CYP enzymes, although the functional Table III. Allele frequencies. consequences are less clear. Concerning the CYP2C subfamily, 3 of the 4 different Author *1 Arg144 *2 Cys144 *3 Leu359 members have been shown to be expressed polymorphically. Sullivan-Klose et al. (26) n.a. 0.08 0.06 Regarding CYP2C9, 4 important genetic polymorphisms in Steward et al. (37) n.a. 0.07 n.a. different exons, namely Arg(*1)144Cys(*2), Tyr358Cys, Yasar et al. (36) 0.819 0.107 0.074 Ile359Leu(*3) and Gly417Asp, exist in humans. PCR Stubbins et al. (35) 0.79 0.125 0.085 analysis of 100 Caucasians showed that the most Scordo et al. (39) 0.796 0.112 0.092 Burian et al. (38) 0.81 0.14 0.05 predominant CYP2C9 sequence is CYP2C9*1/*1 Our results 0.848 0.152 0.043 (Arg/Arg144, Tyr/Tyr358, Ile/Ile359, Gly/Gly417) (35). We found wild-type homozygotes in 7 out of 12 analyzed brain n.a., not analyzed tumors and 7 out of 11 analyzed breast tumor samples, healthy controls respectively, indicating that 60.9% of the analyzed tumor (published data were pooled) *1: 95% CI 0.79-0.83 *2: 95% CI 0.1-0.12 samples are homozygous wild-type. Our results are in *3: 95% CI 0.06-0.09 agreement with studies of healthy tissues from Caucasians. cancer tissue *1: 95% CI 0.71-0.94 The frequency of the wild-type allele (*1) in the present *2: 95% CI 0.06-0.29 study was 0.848. Our results are in agreement with the *3: 95% CI 0.0053-0.1484 results of frequencies of Caucasians described by other authors (Table III), ranging from 0.79 (35) to 0.819 (36), indicating that the frequencies of the *1 allele is not different in tumor material and healthy non-transformed The purpose of the studies by Chang et al. (6) was to tissue. examine the influence of naturally occurring allelic None of the tumor samples was homozygous Cys/Cys144. variations of CYP2C9 on the capacity of these enzymes to This result is in agreement with other published data. Yasar activate cyclophosphamide and ifosfamide. The CYP2C9*2 et al. (36) detected 0.5% of individuals with *2/*2 alleles. mutation at residue 144 had little effect compared to the The study of Steward et al. (37) revealed no homozygous wild-type activation capacity with regard to the anticancer individual. agents. Whether this is also true for the tumorally expressed Concerning the heterozygous individuals at residue 144, CYP2C9*2 remains to be elucidated. our results seem to deviate from the results of others. We Concerning CYP2C9*3 (Arg/Arg144, Tyr/Tyr358, found 30.4% to be heterozygous (*1/*2), with a *2 allele Ile/Leu359, Gly/Gly417), no brain tumor showed this allele, frequency of 0.152, while 6 other authors found lower whereas 2 of the breast tumors were heterozygous, indicating frequencies ranging form 0.07 (37) to 0.14 (38), indicating that 8.7% of the analyzed tumor samples were heterozygous. that the frequency of the *2 allele may be slightly increased None of the tumor samples was positive for Leu/Leu359. The in tumor material. This result might have an important frequency of the *3 allele is 0.043, which seems to be in impact on tumoral drug metabolism and additionally on an agreement with the results of others ranging from 0.05 (38) association of *2 to cancer. However, comparing our results to 0.092 (39) (Table III), indicating that tumor material is not with published frequencies in healthy material, we could not different from healthy tissue with regard to this allele. establish a significant difference. We are aware of the fact With regard to the metabolism of the oxazaphosphorines, that more tumor samples have to be screened to validate Chang et al. (6) demonstrated that the Ile359 to Leu mutation our results. decreased catalytic efficiency toward cyclophosphamide by

303 ANTICANCER RESEARCH 26: 299-306 (2006) increasing the apparent Km, whereas the same mutation 6 Chang TK, Yu L, Goldstein JA and Waxman DJ: Identification reduced the efficiency of P450 towards ifosfamide by of the polymorphically expressed CYP2C19 and the wild-type decreasing the Vmax. Furthermore, the authors concluded CYP2C9-ILE359 allele as low-Km catalysts of cyclophosphamide and ifosfamide activation. Pharmacogenetics 7: 211-221, 1997. that the finding that CYP2C9-Ile359 was more efficient than 7 Gray IC, Nobile C, Muresu R, Ford S and Spurr NK: A 2.4- CYP2C9-Leu359 in cyclophosphamide 4-hydroxylation and megabase physical map spanning the CYP2C gene cluster on ifosfamide 4-hydroxylation is consistent with the proposal that 10q24. Genomics 28: 328-332, 1995. the CYP2C9 amino acid 359 is part of a putative substrate 8 Goldstein JA and de Morais SM: Biochemistry and molecular recognition site (40). biology of the human CYP2C subfamily. Pharmacogenetics 4: The studies of Chang et al. (6) demonstrated that 285-299, 1994. substitution of CYP2C9-Gly417 by Asp resulted in a 2-fold 9 Shimada T, Yamazaki H, Mimura M, Inui Y and Guengerich lower catalytic efficiency for cyclophosphamide metabolism, FP: Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and but a 3-fold higher efficiency for ifosfamide metabolism, toxic chemicals: studies with liver microsomes of 30 Japanese whereas the mutation at CYP2C9 residue 358 had no effect. and 30 Caucasians. J Pharmacol Exp Ther 270: 414-423, 1994. These 2 mutations, namely Tyr358Cys and Gly417Asp 10 Krishna DR and Klotz U: Extrahepatic metabolism of drugs in alleles of CYP2C9, were analyzed by our group. None of the humans. Clin Pharmacokinet 26: 144-160, 1994. examined tumors were heterozygous for Tyr/Cys, 11 McLemore TL, Adelberg S, Liu MC, McMahon NA, Yu SJ, homozygous for Cys/Cys, heterozygous for Gly/Asp or Hubbard WC, Czerwinski M, Wood TG, Storeng R, Lubet RA, homozygous for Asp/Asp. These findings are consistent with et al: Expression of CYP1A1 gene in patients with lung cancer: evidence for cigarette smoke-induced gene expression in normal the results of others. Several authors have found no cases lung tissue and for altered gene regulation in primary of such alleles in either the Japanese or the Caucasian pulmonary carcinomas. J Natl Cancer Inst 182: 1333-1339, 1990. samples examined (29, 41, 42, 43). 12 Kivisto KT, Fritz P, Linder A, Friedel G, Beaune P and In summary, our data indicated that the same allelic Kroemer HK: Immunohistochemical localization of cytochrome variants of CYP2C9 occur in tumor material and healthy P450 3A in human pulmonary carcinomas and normal bronchial tissue. Compared to healthy tissue, tumor material seems to tissue. Histochem Cell Biol 103: 25-29, 1995. reveal a higher incidence of the *2 allele, which might have 13 Kivisto KT, Linder A, Friedel G, Beaune P, Belloc C, Kroemer an impact on anticancer pro-drug activation. In addition, the HK and Fritz P: Immunohistochemical localization of cytochrome P450 2E1 in human pulmonary carcinoma and results showed that brain and breast tumors seem to differ normal bronchial tissue. Virchows Arch 426: 243-247, 1995. in their frequency of heterozygotes at residues 144 and 359. 14 Massaad L, de Waziers I, Ribrag V, Janot F, Beaune PH, However, larger sets of individuals must be recruited to Morizet J, Gouyette A and Chabot GG: Comparison of mouse detect possible differences in the frequency of CYP2C9 allele and human colon tumors with regard to phase I and phase II variants between healthy and tumor material. drug-metabolizing enzyme systems. 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