Expression in Normal Human Tissues of Five Nucleotide Excision Repair Genes Measured Simultaneously by Multiplex Reverse Transcription-Polymerase Chain Reaction1

Vol. 8, 801–807, September 1999 Cancer Epidemiology, Biomarkers & Prevention 801

Lie Cheng, Yongli Guan, Lei Li, Randy J. Legerski, expression of these NER genes in both normal and tumor Janine Einspahr, Jerry Bangert, David S. Alberts, and tissues. Qingyi Wei2 Departments of Epidemiology [L. C., Y. G., Q. W.] and Molecular Genetics Introduction [L. L., R. J. L.], The University of Texas M. D. Anderson Cancer Center, 3 Houston, Texas 77030, and Arizona Cancer Center, University of Arizona, NER is one of the primary pathways by which mammalian Tucson, Arizona 85724 [J. E., J. B., D. S. A.] cells remove DNA lesions caused by both endogenous and exogenous agents (1–4). A wide spectrum of structurally un- related lesions such as UV-induced photoproducts, bulky chem- Abstract ical adducts, and certain types of DNA cross-links are effi- DNA repair is central to the integrity of the human ciently removed by the NER pathway (5, 6). In the process of genome. Reduced DNA repair capacity has been linked to repair, the products of more than a dozen genes are involved in genetic susceptibility to cancer. An adequate expression damage recognition, incision, excision, elongation, and ligation level of DNA repair genes is essential for normal DNA and collectively restore the normal structure (1, 2, 6). There- repair activities. Although there is tissue specificity in the fore, efficient DNA repair plays a central role in the survival of expression, searching for a surrogate tissue is needed for cells upon exposure to carcinogens that cause damage to DNA molecular epidemiological studies. In this study, the and the fidelity of subsequent replication of the genome (7–11). relative expression levels of five selected human Consequences of defective NER are well illustrated in nucleotide excision repair (NER) genes (ERCC1, XPB/ several genetic disorders such as XP and CS (12). Although ERCC3, XPG/ERCC5, CSB/ERCC6, and XPC)in20 many genes participate in NER, there are overlaps in their different types of human normal tissue were functions. Cell-fusion studies have helped identify at least 11 simultaneously measured by a new multiplex reverse DNA-repair complementation groups (each group having only transcription (RT)-PCR assay using the expression level one defective gene) in cell lines established from patients with of the ␤-actin gene as an internal control. Transcripts of these diseases and a large collection of UV-sensitive rodent cell each of the five NER genes were detectable, but the levels mutants. A number of genes that correct defective human NER varied in these normal tissues. Both mitogen have been cloned and designated as ERCC genes (13–15). (phytohemagglutinin)-stimulated and unstimulated human Some of these ERCC genes are involved in the human NER peripheral lymphocytes showed similar expression disorders. For example, ERCC2, ERCC3, and ERCC5 are iden- patterns for the five NER genes. In general, the tical to XPD (16), XPB (17), and XPG (18, 19), respectively, expression levels of stimulated lymphocytes were also and mutations in these genes are involved in corresponding similar to most of the rapidly proliferating tissues, such groups of XP. ERCC6 is identical to CSB, and mutations in this as the skin, breast, intestine, liver, testis, ovary, placenta, gene are involved in CS (20). However, ERCC1 has not been or prostate, but was relatively higher than that of the found to be involved in any XP, CSB, or trichothiodystrophy slowly proliferating or nonproliferating tissues such as human syndromes (21), because defects in ERCC1 resulting adipose, brain, hippocampus, muscle, spleen, or lung. The from mutations or deletions of this gene cause early death data suggested that although the five NER genes were before the symptoms develop (22). expressed at different levels in the normal tissues It is technically feasible to measure the relative transcript examined, PHA-stimulated peripheral lymphocytes may levels of several genes at a time by a multiplex RT-PCR assay be used as a surrogate tissue for estimating expression (23). Physiologically, a high level of expression should be levels of these genes in proliferating tissues. This new correlated with enhanced DNA repair activities. Indeed, we multiplex RT-PCR assay may help detect aberrant have demonstrated that the level of ERCC1 expression corre- lates with NER activity (23). We also found that the expression levels of mismatch repair genes vary among individuals (24). To assess the variation in expression of multiple NER genes in Received 12/14/98; revised 6/17/99; accepted 6/24/99. different types of normal tissue, we conducted this study with The costs of publication of this article were defrayed in part by the payment of the following aims: (a) to design a multiplex RT-PCR assay for page charges. This article must therefore be hereby marked advertisement in measuring the relative mRNA expression of a panel of NER accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH Grants CA70334 and CA74851 and by National Cancer Institute Grant CA16672 to M. D. Anderson Cancer Center. 2 To whom requests for reprints should be addressed, at Department of Epide- miology, Box 189, The University of Texas M. D. Anderson Cancer Center, 3 The abbreviations used are: NER, nucleotide excision repair; XP, xeroderma Houston, TX 77030. Phone: (713) 792-3020; Fax: (713) 792-0807; E-mail: pigmentosum; CS, Cockayne’s syndrome; ERCC, excision repair cross-comple- [email protected]. menting; PHA, phytohemagglutinin; RT-PCR, reverse transcription-PCR.

Table 1 Primers used to amplify the five NER genes and ␤-actin

Position of PCR product size (bp)/Optimal Gene Primer (21- or 22-Mer) 1st basea annealing temperature (°C)b ␤-actin 621/59.6 5Ј-ACACTGTGCCCATCTACGAGG-3Ј (sense) 2147 5Ј-AGGGGCCGGACTCGTCATACT-3Ј (antisense) 2954 XPG/ERCC5 462/59.5 5Ј-AATCGAAGGCAGGCCCGTGGG-3Ј (sense) 1141 5Ј-ATTCGGGAGCCCAGGTGCGTC-3Ј (antisense) 1582 CSB/ERCC6 383/57.4 5Ј-TTGAGCTGCAGGGTTTGGGTG-3Ј (sense) 348 5Ј-TGCATCCTCCTCCAGACTGGC-3Ј (antisense) 710 ERCC1 273/61.0 5Ј-CCCTGGGAATTTGGCGACGTAA-3Ј (sense) 500 5Ј-CTCCAGGTACCGCCCAGCTTCC-3Ј (antisense) 751 XPC 215/55.4 5Ј-CCAGAGCAGGCGAAGACAAGA-3Ј (sense) 348 5Ј-AAGCGGGCTGGGATGATGGAC-3Ј (antisense) 542 XPB/ERCC3 171/58.7 5Ј-CCAGGAAGCGGCACTATGAGG-3Ј (sense) 136 5Ј-GGTCGTCCTTCAGCGGCATTT-3Ј (antisense) 286 a From GenBank database: accession no. M13194 for ERCC1, M31899 for ERCC3, L20046 for ERCC5, L04791 for ERCC6, X65024 for XPC, and M10277 for ␤-actin. b Chosen to prevent primer mismatches during PCR.

genes; (b) to describe the expression patterns or differences in in T-25 flasks at 37°C in a 5% CO2 atmosphere in the standard various types of tissue; and (c) to see whether the expression in medium RPMI 1640 (Life Technologies, Inc., Grand Island, lymphocytes is comparable with that in other target organ NY) supplemented with 15% fetal bovine serum (Life Tech- tissues so that the lymphocytes can be used as a potential nologies, Inc.) without antibiotics. surrogate tissue. We report here that we simultaneously ana- Multiplex RT-PCR. To amplify the five NER genes, we used lyzed the relative expression of ERCC1, XPB/ERCC3, XPG/ a modification of our previously described multiplex RT-PCR ERCC5, CSB/ERCC6, and XPC in 20 types of selected human technique (23). To select the primers, we used GenBank Se- normal tissue by using a new multiplex RT-PCR assay. quence Data Library mRNA or “cDNA” or genomic DNA sequences for ERCC1 (GenBank accession no. M13194), XPB/ Materials and Methods ERCC3 (M31899), XPG/ERCC5 (L20046), CSB/ERCC6 Tissue Samples. cDNAs from 18 types of human normal tis- (L04791), XPC (X65024), and ␤-actin (M10277), which we sue were purchased from Invitrogen (Invitrogen Corp., San accessed through the Genetic Computer Group Genetic Data- Diego, CA). The other two types of tissue were primary lym- base (version 8.0). The optimal primers were selected with phocytes and skin. Additional cDNAs were synthesized from Oligo Software (version 3.4; National Biosciences, Inc., Plym- total RNA extracted from PHA-stimulated peripheral blood outh, MN). The primers for the PCR were designed to minimize lymphocytes of 12 healthy subjects and snap-frozen skin biopsy base-pairing interaction between the primers and to have sim- samples (3 mm in diameter) from four healthy donors. All of ilar annealing temperatures to optimize the reaction. All of the the tissues used were histopathologically confirmed to be nor- primers were commercially synthesized by Life Technologies, mal. Total RNA was extracted by using Tri-Reagent (Molecular Inc. (Table 1). The genes were chosen because they were Research Center, Cincinnati, OH) as described previously (25). relatively easier to be coamplified in our hands than other genes The cDNA was synthesized by reverse transcriptase of 5 ␮gof we tested (data not shown). For instance, we were unable to the extracted total RNA in 20 ␮l of first-strand synthesis cDNA design an effective pair of primers for coamplification of XPD/ reaction mixture. Each reaction contained 0.5 ␮g of random ERCC2; therefore, we did not include this NER gene in the primers (Promega Biotech, Madison, WI), 200 units of Molony study. To optimize the multiplex PCR reaction, the primers murine leukemia virus reverse transcriptase (United States Bio- were chosen to prevent nonspecific annealing and primer chemical, Cleveland, OH), 4 ␮lof5ϫ RT buffer [250 mM dimerization and to have similar annealing temperatures. ␮ ␮ ϫ Tris-HCl (pH 8.3), 375 mM KCl, 50 mM DTT, 15 mM MgCl2 Each 40 l of PCR contained 3 l of RT mixture, 1 PCR (United States Biochemical), 0.5 mM each deoxynucleotide buffer [500 mM KCl, 100 mM Tris-HCl (pH 9.0), 1% Triton

triphosphate, and 20 units of RNasin (Promega Biotech)] and X-100, and 2.5 mM MgCl2], 0.1 mM each deoxynucleotide diethylpyrocarbonate-treated water. The reaction mixture was triphosphate, 2 unit of Taq polymerase (Promega Biotech), 75 then incubated at room temperature for 10 min and then at 42°C pM ␤-actin primers, 35 pM ERCC1 primers, 50 pM XPB/ERCC3 for 45 min, heated to 95°C for 10 min, and then quick-chilled primers, 50 pM XPG/ERCC5 primers, 50 pM CSB/ERCC6 prim- on ice. ers, and 25 pM XPC primers. The reaction mixtures were heated Cell Lines. Four EBV-immortalized human lymphoblastoid at 95°C for 5 min. Amplification was performed in sequential cell lines from the Human Genetic Mutant Cell Repositories cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 45 s. After (Camden, NJ) were used: three apparently normal cell lines 29 cycles of amplification (chosen from the PCR kinetic anal- (GM00892B, GM03798, and GM00131A) and two XP cell ysis; see “Results”), all samples were incubated for an addi- lines, GM02246b (XP-C) and GM02345a (XP-A), with defi- tional 10 min at 72°C. The ␤-actin PCR fragment was used to cient nucleotide excision repair. All of the cells were cultured monitor genomic DNA contamination. Because all genes were

amplified in the same test tube, using only one control for DNA contamination was sufficient. The 621-bp ␤-actin fragment spans exon 3 through exon 5, containing intron 4 (95 bp) and intron 5 (112 bp), which should not be present in the cDNA. If the cDNA was contaminated with genomic DNA, the PCR product would be a 828-bp (i.e., 621 ϩ 95 ϩ 112 bp) band, which would be distinguished by agarose gel electrophoresis. The assays were performed three times for the purchased cDNA samples and performed in triplicate for the skin biopsies and lymphocytes. The PCR products were separated by 2% agarose gel electrophoresis, stained with 0.5 ␮g/ml ethidium bromide, and visualized with UV light. To confirm that the PCR products were copies of the target sequences, each target gene was amplified separately as well as in the multiplex reaction. The sizes of the multiplex RT-PCR products were determined by electrophoresis with size marker ␾X174RF DNA/HaeIII (Life Technologies, Inc.). Then, each product was purified with a Centricon Concentrator (Amicon, Beverly, MA) according to the manufacturer’s instructions. The sequence of Fig. 1. Multiplex RT-PCR amplification of six genes in lymphoblastoid cell each PCR product was then confirmed by direct sequencing lines. A, lymphoblastoid cell line (GM00131). M, molecular marker (øX174RF with an automated Model 373A Sequencer (Applied Biosys- DNA/HaeIII); Lane 1, XPG/ERCC5 coamplified with ␤-actin only; Lane 2, CSB/ERCC6 coamplified with ␤-actin only; Lane 3, ERCC1 coamplified with tems, San Francisco, CA). ␤-actin only; Lane 4, XPC coamplified with ␤-actin only; Lane 5, XPB/ERCC3 To quantify the relative levels of gene expression, the PCR coamplified with ␤-actin only; Lane 6, all of the five NER genes coamplified with products were electrophoresed, stained with ethidium bromide, ␤-actin. B: Lane 1, unstimulated lymphocytes; Lane 2, PHA-stimulated lympho- and scanned with a Digital Imaging System (Model IS-1000; cytes; Lane 3, GM00892b; Lane 4, GM03798; Lane 5, GM00131a; Lane 6, Alpha Innotech Co., San Leandro, CA), and the areas of the GM02246b (XP-C); Lane 7, GM02345a (XP-A). peaks were calculated in arbitrary units. The expression level of the internal standard (␤-actin) in each reaction was used as the baseline expression (100%) of that sample, and the relative 26th, 27th, 28th, 29th, 30th, and 31st. A representative value (percentage of baseline) was calculated for each of the experiment was shown in Fig. 2, in which the relationship target genes amplified in the same reaction. between the number of cycles and the product yield was approximately linear between 25 and 30 cycles, but PCR Statistical Analysis. The relative expression levels of five amplification was saturated at the 30th cycle. Therefore, we NER genes in tissues, the means, and SDs were calculated by arbitrarily chose 29 cycles as the cycle number for later using the SAS statistical software package (version 6.11; SAS experiments, as described previously (23). Institute, Inc., Cary, NC). Dose-Response Curve for the Relative Quantification by Multiplex RT-PCR. To determine the relationship between Results the initial amount of template and the yield of the PCR product Amplification of Six Genes in a Single Reaction. Amplifi- of each gene, a serial dilution of cDNA containing reverse cation of a specific fragment of a target gene is often compro- transcriptase mixture of lymphoblastoid cell line GM00131 mised by nonspecific reactions resulting from sequence homol- (0.3125, 0.625, 1.25, 2.5, 5, and 10 ␮l) was prepared and ogy between targeted and stand-by sequences in the genome. amplified under the conditions described above. The dose- As a result, multiple unidentifiable bands, in addition to the amplification curve for each gene indicated that 0.3125–10 ␮l expected band, are generated by PCR. This is particularly true of cDNA reaction mixture allowed the amplification of all when multiple pairs of primers are used in a single PCR. We genes simultaneously within an approximately linear range have demonstrated previously that in our hands, targeted se- (Fig. 3). On the basis of these results, it is clear that by quences of five mismatch repair genes can be coamplified with optimizing concentrations of primers used in the multiplex ␤-actin gene in a single reaction (23). In this study, optimal RT-PCR, we achieved similar efficiencies of amplification for conditions were achieved in the multiplex RT-PCR of five NER all six NER genes. An approximately linear (dose-dependent) genes by comparing the PCR results of a single pair with those amplification of the six genes was also achieved with 29 am- of multiple pairs of primers used in the PCRs. As shown in Fig. plification cycles, avoiding reaching the plateau of amplifica- 1A, each gene was amplified with the ␤-actin gene only (Lanes tion. Given fixed 29 cycles and the amount of cDNA in a single 1–5), and all five NER genes were also amplified with the PCR, the relative expression levels of the NER genes were then ␤-actin gene in a single PCR (Lane 6). In addition, the expres- calculated by comparison with the level of the housekeeping sion of five genes was detectable and similar in several lym- gene, ␤-actin. It was noted that the relative expression level of phoblastoid cell lines (Fig. 1B), except for low expression of each NER gene did not have a dose response as did the absolute XPCC in XP-C cells. These results indicated that the primers level, i.e., they appear to be independent of the amount of designed and the PCR conditions were optimal, and the mul- cDNA used (Fig. 3). This feature is important for such relative tiplex RT-PCR generated the same bands as did a single pair of quantification, because the measurement will be less likely primers in a single reaction. biased by the variation in the amount of cDNA used for PCR Kinetics of Multiplex PCR. To determine the linear range of amplification. simultaneous amplification of six genes, we performed a NER Gene Expression in Human Normal Tissues. The ex- PCR kinetic analysis. The same amount of cDNA was ali- pression levels of the five NER genes from 18 Invitrogen quoted into each of seven tubes, one of which was then tissues plus the skin and PHA-stimulated peripheral blood T removed from the cycle at each of the following cycles: 25th, lymphocytes are listed in Table 2. The means and SDs were

Fig. 3. Dose-amplification curve of 29 cycles for five NER genes and ␤-actin. The expression level was detected in the cDNA-containing mixture ranging from Fig. 2. Amplification kinetics of the six genes in a normal cell line (GM00131) 0.3125 to 10 ␮l for a normal cell line (GM00131) by multiplex RT-PCR assay. from cycles 25 to 31. Each band in the gel (inset, top) is quantified by densi- The amplification of all of the five NER genes was approximately linear. Lane M, tometry using arbitrary units. All genes were amplified in an approximately linear molecular marker (␾X174RF DNA/HaeIII); Lane 1, water only; Lanes 2–7, manner between cycles 25 and 30. 0.3125, 0.625, 1.25, 2.5, 5, and 10 ␮l of cDNA reaction mixture, respectively. Each band in the gel (inset, bottom) is quantified by densitometry using arbitrary units.

calculated from three repeated PCR assays performed on the same cDNA samples, which provided information on intraassay Discussion (individual) variation. The mean expression of PHA-stimulated We have developed a new multiplex RT-PCR assay for simul- T lymphocytes from 12 individual blood samples and the mean taneous amplification of five NER genes, ERCC1, XPB/ expression of four skin biopsies were used for comparison, ERCC3, XPG/ERCC5, CSB/ERCC6, and XPC. We found that which provided information on interassay (individual) varia- the expression of these five NER genes was detectable but tion. In general, the interassay variation, particularly for lym- variable in the 20 types of normal human tissue including phocytes, was larger than intraassay variation. All 18 Invitrogen lymphocytes. These results provided evidence that the genes tissues plus skin had detectable expression levels for all five were actively transcribed in all of the tissues tested, which is NER genes. Fig. 4 shows the results of one experiment with 13 consistent with the notion that the NER pathway is central to Invitrogen tissues, plus peripheral blood T lymphocytes. It mammalian cells (1, 2, 26, 27). In addition, the expression level appears that PHA-stimulated and unstimulated lymphocytes in lymphocytes may be used as a surrogate measurement for had comparable expression levels of the five NER genes. Most that in other tissues, if the expression level is genetically de- tissues had similar patterns of expression levels, whereas the termined. brain and spleen had relatively low expression levels, which Because the output of the RT-PCR assay depends on the may be due to the quality of the cDNA. Compared with the number of amplification cycles and starting copies of the tem- expression in PHA-stimulated T lymphocytes, ERCC1 had a plates in the cDNA sample, the arbitrary selection of 29 cycles similar or higher expression and XPB/ERCC3 had a lower and the unmeasured starting amount of the target gene tran- expression in most of the tissues tested. NER gene expression script may render the quantification inaccurate. Therefore, the measurement may be considered as semiquantitative, and the was relatively higher in most of the proliferating tissues (such assay may be used to approximate changes in gene expression as skin, breast, intestine, liver, testis, ovary, placenta, and relative to ␤-actin. prostate) than in most of the nonproliferating or slowly prolif- Because the Invitrogen tissues were obtained from differ- erating tissues (adipose tissue, brain, hippocampus, muscle, ent individuals, the variation of the five NER genes in the spleen, and lung; Table 2). The Invitrogen cDNA samples were tissues examined in this study may be due to either interindi- obtained from individuals of different ages and sex (Table 2), vidual variation or intertissue variation. The inter- and intrain- and statistical comparisons of these expression levels across dividual variations need to be investigated in future studies different tissues suggested significant differences. However, using the tissues from the same individuals. PHA-stimulated T the comparisons of expression levels between different tissues lymphocytes were chosen in this study for comparison because from different subjects as well as multiple tests with an arbi- studies have shown that although unstimulated lymphocytes do trary reference may not be valid and, therefore, were not pre- have detectable levels of DNA repair enzymes (28–31), they sented. have limited NER activity (32). We have confirmed that the

Table 2 Relative expression of the five NER genes in selected human normal tissues

% relative expression level (mean Ϯ SD)a Subject’s age (y)/sex XPG/ERCC5 CSB/ERCC6 ERCC1 XPC XPB/ERCC3 Stimulated T lymphocyteb 55.5c 68.3 Ϯ 15.1 64.7 Ϯ 16.5 68.3 Ϯ 12.5 53.0 Ϯ 19.6 52.9 Ϯ 19.9 Rapidly proliferative tissues Skind 82.3 Ϯ 3.3 71.5 Ϯ 7.6 103.5 Ϯ 3.4 73.9 Ϯ 5.2 88.2 Ϯ 11.7 Testis 24/M 55.3 Ϯ 6.8 26.7 Ϯ 4.4 117.6 Ϯ 10.3 21.7 Ϯ 1.9 34.5 Ϯ 3.2 Ovary 30/F 89.0 Ϯ 4.4 90.2 Ϯ 11.0 104.1 Ϯ 10.7 90.1 Ϯ 7.9 71.9 Ϯ 12.4 Stomach 24/M 83.3 Ϯ 5.8 33.1 Ϯ 12.7 105.5 Ϯ 15.6 65.1 Ϯ 11.3 37.9 Ϯ 11.6 Prostate 26/M 62.5 Ϯ 8.5 56.2 Ϯ 12.6 94.5 Ϯ 13.9 39.6 Ϯ 8.0 40.8 Ϯ 6.3 Placenta 30/F 56.9 Ϯ 9.5 66.3 Ϯ 13.7 89.6 Ϯ 20.3 50.7 Ϯ 14.8 40.7 Ϯ 10.0 Breast 34/F 69.4 Ϯ 8.5 62.6 Ϯ 7.1 86.2 Ϯ 8.2 47.8 Ϯ 10.8 37.6 Ϯ 7.1 Liver 26/M 93.4 Ϯ 2.4 66.8 Ϯ 8.9 84.1 Ϯ 5.5 65.2 Ϯ 2.0 34.6 Ϯ 6.4 Kidney 64/M 77.4 Ϯ 1.7 54.0 Ϯ 5.2 80.7 Ϯ 8.6 46.2 Ϯ 8.6 28.5 Ϯ 9.4 Colon 26/M 72.5 Ϯ 10.5 31.3 Ϯ 4.5 74.0 Ϯ 4.0 34.3 Ϯ 4.6 26.6 Ϯ 7.1 Intestine 64/M 76.4 Ϯ 6.3 42.5 Ϯ 7.8 74.0 Ϯ 10.0 44.7 Ϯ 9.3 24.2 Ϯ 10.9 Slowly proliferative tissues Muscle 26/M 35.7 Ϯ 8.0 16.3 Ϯ 3.6 101.4 Ϯ 5.4 15.8 Ϯ 2.0 20.4 Ϯ 8.0 Heart 64/F 50.9 Ϯ 5.7 40.2 Ϯ 2.8 99.2 Ϯ 11.7 23.1 Ϯ 6.8 18.8 Ϯ 3.4 Hippocampus 28/M 33.6 Ϯ 6.3 47.9 Ϯ 5.0 87.2 Ϯ 9.3 16.9 Ϯ 2.5 18.0 Ϯ 5.4 Brain 64/M 26.8 Ϯ 5.0 11.4 Ϯ 3.4 77.4 Ϯ 17.7 9.8 Ϯ 3.7 12.2 Ϯ 4.1 Spleen 24/M 37.1 Ϯ 17.2 17.8 Ϯ 10.0 75.1 Ϯ 14.4 14.3 Ϯ 5.8 13.9 Ϯ 2.6 Bladder 24/F 48.4 Ϯ 7.7 37.4 Ϯ 1.8 73.6 Ϯ 16.3 34.4 Ϯ 11.4 35.4 Ϯ 7.6 Lung 64/M 35.9 Ϯ 5.3 36.9 Ϯ 7.5 65.1 Ϯ 4.2 34.8 Ϯ 2.4 29.5 Ϯ 7.5 Adipose 24/M 23.4 Ϯ 1.8 17.4 Ϯ 5.1 61.5 Ϯ 6.6 24.8 Ϯ 9.0 15.7 Ϯ 4.1 a The expression level of ␤-actin was used as the baseline (100%) for normalization, and the values were calculated from three independent experiments on the same cDNA samples. b Average expression levels of PHA-stimulated lymphocytes of 12 normal blood donors used as the reference group. c Average age of six male and six female blood donors. d Average expression levels of normal skin biopsies from four donors.

groups are not (34–37). The higher expression levels of NER genes observed in most of the proliferating tissues may reflect these tissues’ greater need for proofreading of newly replicated DNA strands and repair of replication errors as a result of rapid cell proliferation in these tissues. Variation in gene expression among individuals may result from inherited germ-line mutations or deletions (38), methylation (39), or polymorphisms (40), which may affect gene transcription and stability of transcripts. Because humans are constantly exposed to environmental toxicants and endogenous metabolites that cause DNA damage, increases in cellular DNA repair activity in response to DNA damage should result from Fig. 4. Expression in normal human tissues of the five NER genes, measured by a concomitant increase in gene transcription and translation multiplex RT-PCR. S-Lymphocytes, PHA-stimulated lymphocytes; U-Lympho- activities. Therefore, apparently normal individuals with low cytes, unstimulated lymphocytes. levels of expression of DNA repair genes may have low DNA repair capacity, which may result in genetic alterations involved in carcinogenesis. We showed recently that low expression of expression (mRNA) of proliferating cell nuclear antigen, a cell hMLH1 and hMSH6 in peripheral lymphocytes is associated cycle-dependent and essential element of DNA repair, was with an increased risk of head and neck cancer (24). In addition, detectable in PHA-stimulated lymphocytes but not in unstimu- 4 aberrant expression of hMLH1 and hMSH2 in tumor tissues is lated lymphocytes. This is consistent with the report that also associated with tumor progression (41). Therefore, this proliferating cell nuclear antigen is only expressed in prolifer- new multiplex RT-PCR assay described here may be a useful ating cells and is required for normal NER activity (33). tool for evaluating the role of NER expression in the develop- The relative expression levels of ERCC1 were consistently ment of cancer and tumor progression. higher (but not for XPB/ERCC3, XPG/ERCC5, CSB/ERCC6, or It is difficult, if not impossible, to study simultaneously the XPC) in most Invitrogen tissues and the skin. This may reflect expression of several NER genes by Northern blot hybridiza- the fact that ERCC1 plays an important role in NER. ERCC1 is tion analysis or the RNase protection assay, because the amount involved in the repair of a wide range of DNA damage. Cells of tissue available is often small and the expression of the NER of complementation group 1 (which lack ERCC1 and ERCC4) genes in some tissues is relatively low. Therefore, a more are hypersensitive to many bifunctional cross-linking DNA- sensitive method is necessary, particularly in studies where damaging agents, whereas the cells of other complementary blood is the only accessible tissue. We have demonstrated that the multiplex RT-PCR assay provides a fast and sensitive technique to simultaneously detect the levels of specific tran- 4 Unpublished data. scripts of several NER genes in the tissues tested. The ability of

this assay to provide a measure of the relative levels of gene 8. Francis, A. A., Snyder, R. D., Dunn, W. C., and Regan, J. D. Classification of expression should help characterize variation in gene expres- chemical agents as to their ability to induce long- or short-patch DNA repair in sion in humans, and the assay may be a useful tool in molecular human cells. Mutat. Res., 83: 159–169, 1981. 9. Mitchell, D. L., Haipek, C. A., and Clarkson, J. M. (6-4) photoproducts are epidemiology studies (24). removed from the DNA of UV-irradiated mammalian cells more efficiently than Because they are accessible and easy to collect, peripheral cyclobutane pyrimidine dimers. Mutat. Res., 143: 109–112, 1985. blood lymphocytes are commonly used as surrogates for target 10. Claver, J. E. DNA damage and repair in normal, xeroderma pigmentosum and tissues to monitor molecular events, such as the level of DNA XP revertant cells analyzed by gel electrophoresis: excision of cyclobutane adducts (42, 43), that may be associated with development of dimers from the whole genome is not necessary for cell survival. Carcinogenesis cancer. Our data have demonstrated that in all of the tissues we (Lond.), 10: 1691–1696, 1989. 11. Nakane, H. S., Takeuchi, S., Yuba, S., Sajo, M., Nakatsu, Y., Murai, H., examined, including PHA-stimulated T lymphocytes, the ex- Nakane, Y., Ishikawa, T., Hirota, S., Kitamura, Y., Kato, Y., Tsunoda, Y., pression levels of the five NER genes were measurable. Al- Miyauchi, H., Horio, T., Tokunaga, T., Matsunaga, T., Nikaido, O., Nishimune, though the tissues tested came from different individuals, the Y., Okada, Y., and Tanka, K. 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Identification believe that the expression levels of the five NER genes in of the eleventh complementation group of UV-sensitive excision repair-defective PHA-stimulated peripheral T lymphocytes may provide a rea- rodent mutants. Cancer Res., 52: 6690–6691, 1992. 15. Collins, A. R. Mutant rodent cell lines sensitive to ultraviolet light, ionizing sonable estimate for that of target tissues that are not accessible radiation and cross linking agents: a comprehensive survey of genetic and for clinical trials or epidemiological studies. biochemical characteristics. Mutat. Res., 293: 99–118, 1993. Although in vivo gene expression can be modulated by 16. Flejter, W. L., McDaniel, L. D., Johns, D., Friedberg, E. C., and Schultz, many factors including exposure to carcinogens, diet, and med- R. A. 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Lie Cheng, Yongli Guan, Lei Li, et al.

Cancer Epidemiol Biomarkers Prev 1999;8:801-807.

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