[CANCER RESEARCH 55. 1261-1266. March 15. ITO] Malignant and Nonmalignant Brain Tissues Differ in Their Messenger RNA Expression Patterns for ERCC1 and ERCC2 Meenakshi D. Dabholkar, Mitchel S. Berger, Justine A. Vionnet, Charles Egwuagu, John R. Silber, Jing Jie Yu, and Eddie Reed1 Medical Ovarian Cancer Section, Clinical Pharmacology Branch, National Cancer Institute, Bethesda. Maryland 20892 ¡M.D. D., J. A. V., J. J. Y., E. R.¡:Department of Neurological Surgery, University of Washington Medical Center. Seattle, Washington 98195 ¡M.S. B.. J. K. SJ: and Laboratory of Immunologi. National E\e Institute. Belhesda. Maryland 20X92 ¡C.E.I ABSTRACT Studies in UV repair-deficient Chinese hamster ovary cells (5), clinical human ovarian cancer tissues (10), and human ovarian cancer Perturbation of the DNA repair process appears to be responsible for cells in tissue culture1 suggest that mRNA expression levels of the occurrence of a number of human diseases, which are usually associ ERCCÃŒmay represent the relative activity of the excision process ated with a propensity to develop internal malignancies and/or disorders within NER, if no other specific NER deficit exists. It is unclear of the central nervous system. We have been interested in the possibility that a subtle abnormality in DNA repair competency might be associated whether the selection of one gene from the helicase complex such as with the transformation of nonmalignant cells to the malignant state. To ERCC2 may represent activity of the helicase function of NER. study this question, we assayed malignant and nonmalignant brain tissues However, since ERCC2 appears to be an essential component of from 19 individuals for m UN Yexpression levels of the human DNA repair normal helicase function (6, 8), we have used ERCC2 as a tool to genes ERCC1, ERCC2, and XPAC and for differential splicing of the assess relative helicase activity within NER. ERCCÃŒand ERCC2 ERCC1 transcript. We separately compared expression levels of these are closely linked on chromosome 19ql3.2-13.3, along with DNA genes in the following situations: concordance of expression within ma ligase and XRCCÃŒ(11), suggesting a close linkage in nature of the lignant tissues; concordance of expression within nonmalignant tissues; four most basic functions in repairing DNA damage that occurs in concordance between malignant and nonmalignant tissues within individ the environment. uals of the cohort; and concordance of gene expression between two An abnormality in normal DNA repair appears to be responsible for nonmalignant tissue sites within a single individual. Linear regression analyses of mRNA values obtained suggested orderly concordance of these the occurrence of human nonpolyposis colon cancer, as recently three DNA repair genes in nonmalignant tissues within the patient cohort reported by Parsons et al. (12). Two recent reports have suggested that and an excellent concordance of these genes between two separate biopsy abnormalities of chromosome 19q are a common occurrence in human sites from the same individual. In contrast, malignant tissues showed brain cancer and may specifically involve region 19ql3.2-13.4 (13, disruption of concordance between the full-length ERCC1 transcript and 14). Concurrent with these studies, we have assessed the relationships ERCC2, which have excision and helicase functions, respectively. Further between two genes involved in DNA repair that are located in this more, within the same individuals, malignant tissues were discordant with area on chromosome 19, ERCCÃŒandERCC2. Our concern has been nonmalignant tissues for ERCC1 and ERCC2, although concordance for whether comparatively subtle abnormalities in DNA repair may con XPAC was preserved. These data suggest that one molecular characteristic tribute to the process of malignant transformation. of human malignancy may be the disruption of the normal relationship In this report, we have measured relative mRNA levels of ERCCI between the excision and the helicase functions of the nucleotide excision repair pathway. and ERCC2 in malignant and nonmalignant brain tissues taken from the same individuals. In nonmalignant brain tissues, mRNA levels of ERCCI and ERCC2 are tightly coordinated, particularly with respect INTRODUCTION to the full-length transcript of ERCCI, which is associated with NER2 is responsible for the repair of UV-induced DNA damage (1), efficient DNA repair. In malignant brain tissues, this coordination is completely lost. Nonmalignant brain tissues are similar in this respect DNA damage from polycyclic aromatic hydrocarbons, and DNA to four other situations where we have studied nonmalignant tissues damage induced by some chemotherapeutic agents such as cisplatin (15). Malignant brain tissues are dissimilar with nonmalignant tissues (2-5). The process is complex, with several models proposed in recent but very similar to malignant ovarian cancer tissue and chronic years (6, 7). There is agreement that the first step of NER (DNA lymphocytic leukemia, with respect to this type of analysis (16-18). damage recognition and excision) is rate limiting to the process (7). This dissimilarity between malignant brain tissues and nonmalignant There is also agreement that at least two protein complexes are brain tissues from the same individuals, as assessed by linear regres involved in the first step of NER. One of these complexes has helicase sion analysis, is strongly statistically significant. The possible biolog activity, may link DNA repair with DNA transcription, and has as one ical implications of this observation are discussed. component of the complex, ERCC2 (along with ERCC3 and other transcription proteins; Refs. 6 and 8). The complex that performs incision of the DNA strand is distinct from the helicase complex and MATERIALS AND METHODS has as one component, ERCC1 (along with ERCC4, ERCC11, XPF, XPAC, and possibly other proteins; Refs. 6 and 9). Procurement of Tissue. Malignant and/or nonmalignant tissues were ob tained from 19 patients undergoing surgery for suspected primary or recurrent Received 6/27/94; accepted 1/18/95. brain neoplasms at the University of Washington Medical Center (Seattle, The costs of publication of this article were defrayed in part by the payment of page WA). Table 1 shows information regarding the antitumor therapy received by charges. This article must therefore be hereby marked advertisement in accordance with each patient studied. For patients receiving only surgery as their anticancer 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Clinical Pharmacology therapy, this specimen was obtained at the initial surgical procedure. For Branch. National Cancer Institute. NIH. 9000 Rockville Pike, Building 10. Room 12N226, patients receiving radiation therapy and/or chemotherapy, this specimen was Bethesda, MD 20892. taken at the time of disease recurrence. Medications that were used to control - The abbreviations used are: NER. nucleotide excision repair; ERCC1. ERCC2. and edema and/or seizure activity are included under the category of "noncancer ERCC6. excision repair genes cross-complementing CHO mutant cell lines of comple mentation groups 1, 2. and ft, respectively; RT-PCR, reverse transcription-PCR: bp. base pair; XPAC. human xeroderma pigmcntosum A correcting gene. 3 E. Reed, unpublished observations. 1261 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1995 American Association for Cancer Research. EKCCl. XPAC, AND ERCC2 IN MALIGNANT BRAIN TISSUES Table 1 Tumor-related therapy received by palients prior lo the procurement of the RT-PCR-based analysis of ERCCI and ERCC2 expression were synthesized biopsy specimen studied in this report on a DNA synthesizer (Biosearch, Inc., San Rafael, CA) and purified by Patient polyacrylamide gel electrophoresis (25). Primers and probes for XPAC and no.therapy"1 Anti-cancer therapyDilantinTegretol, ß-actinPCR were synthesized by Lofstrand Laboratories (Gaithersburg, MD). Surgery2 RNA from the human T-lymphocytic cell line, H9 (26), was similarly pro Surgery3 phénobarbitalNoneDilantinDecadron,cessed and used as an internal control. Surgery4 Aliquots of 3 ¡¿\ofthe cDNA preparation from all samples were analyzed Surgery5 Surgery6 DilantinTegretol for alternative splicing of ERCCI. Primers flanking exon VIII, which is 72 SurgeryNon-cancer-related bases long, were used to detect the presence and relative proportions of Surgery for brain cancer full-length and alternatively spliced ERCCI mRNA in tissue samples (27). Two years prior to dx of brain cancer, patient was treated for breast cancer with DNA segments of 196 and 268 bp were obtained on amplification of the Cytoxan, Adriamycin, and 5-FU ERCCI cDNA sequence extending from bases 692 to 959 (27). RT-PCR was Surgery Decadron conducted for 30 cycles. Southern blots of amplified segments were hybridized Surgery Carbamazepine, with a probe 26 bases long extending from bases 764 to 789, 5' of exon VIII, levothyroxine, estrogen, which detects amplified products representing full-length ERCC1 mRNA (with lorazepam, nortriptyline 10 Surgery. XRT Dilantin, dexamethasone the 72-base long exon VIII) and an alternatively spliced species of ERCCI 11 Surgery Decadron, Dilantin, Tegretol, mRNA (without exon VIII). phenobarbitol Numerical values for the expression of the ERCCÃŒ,ERCC2, and XPAC 12 Surgery, XRT Dilantin, Proventil, Premarin, genes and of full-length and alternatively spliced ERCCI mRNA were ob Vanceril, phenobarbitol,