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Glutathione S-Transferase Amplification is Associated with Cisplatin Resistance in Head and Neck Squamous Cell Carcinoma Cell Lines and Primary Tumors

¹ Department of Oncology, Lombardi Cancer Center, Washington, DC;
² Department of Otolaryngology, Medstar-Georgetown University Hospital, Washington, DC; and
³ Institute for Molecular and Human Genetics, Lombardi Cancer Center, Washington, DC

	ABSTRACT

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Purpose: The purpose is to evaluate the association of glutathione S-transferase (GST-) amplification and cisplatin resistance in head and neck cancer.

Experimental Design: An analysis of chromosomal abnormalities in 10 head and neck cancer cell lines by comparative genomic hybridization was performed. GST- amplification and expression were evaluated in head and neck cell lines and paraffin-embedded tissue by fluorescence in situ hybridization (FISH) and immunohistochemistry.

Results: Changes in the DNA copy number were seen in all 10 cell lines by comparative genomic hybridization. The most frequent chromosomal alterations were: gain at 3q; loss at 3p; gain at 8q; loss of 18q; gain at 20q; loss at 8p; and gain of 11q11-q13. Using FISH, 9 of 10 cell lines showed increased GST- copy number. GST- amplification was detected in 7 of 10 cell lines. Five were relatively cisplatin resistant, and 2 were relatively cisplatin sensitive (mean IC₅₀, 11.2 and 2.75 µM). Two relatively cisplatin-sensitive cell lines showed GST- gain and another relatively cisplatin-sensitive cell line had predominantly two copies of the gene. In 10 tumor specimens, 4 had two copies of GST-. All 4 had a complete response to neoadjuvant chemotherapy, 3 of whom are alive >50 months from treatment compared with 2 patients showing GST- amplification. Neither responded to chemotherapy, and both died of disease <9 months from diagnosis.

Conclusions: Using FISH, GST- amplification is a common event in head and neck squamous cell carcinoma and may be associated with cisplatin resistance and poor clinical outcomes in head and neck cancer patients treated with cisplatin-based therapy.

	Introduction

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CDDP⁴ is the most important chemotherapeutic agent involved in the treatment of HNCAs, and despite excellent responses in previously untreated patients (1) , patients treated for relapsed or recurrent disease usually have only a 20–30% response rate (2) . CDDP resistance in HNCAs may be mediated by a number of different mechanisms, including drug detoxification, up-regulation of DNA repair enzymes, and overexpression of antiapoptotic proteins such as bcl-2 and detoxifying enzymes such as GST- (3) . GST-, located on chromosome 11q13, is a member of a family of isozymes (, µ, , and ) that plays an important role in the detoxification of many xenobiotic substances through conjugation to glutathione [(GSH); Refs. 4 and 5 ]. Chromosome 11q13 amplification has been reported in a variety of malignancies including lung, esophagus and head and neck (6, 7, 8) . Amplification of 11q13 has been detected using a number of techniques, including CGH and FISH. Increased copy numbers of 11q13 have been seen in as much as 50% of HNCAs by CGH (9) .

In addition to the GST- gene, several other genes are located on 11q13, including the cyclin D1 gene (CCDN1), the gene containing the BCL-1 translocation breakpoint found in many B-cell lymphomas, the FGF-3 and FGF-4 genes, and the EMS-1 gene (10, 11, 12, 13) . GST- overexpression has been associated with increased resistance to various chemotherapeutic agents (5) . We have previously shown that in patients with advanced HNCA, elevated expression of GST- measured by immunohistochemistry has been associated with a poor response to CDDP-based chemotherapy and that GST- overexpression is relatively common, especially in relapsed tumors (14 , 15) . In contrast, GST- amplification has rarely been reported in HNCA (8 , 10, 11, 12, 13) . Gaffey et al. (10) examined GST- amplification by Southern blot analysis and found amplification in only 3% (2 of 64) of patients, whereas GST- expression was seen in 86% (55 of 64) of patients by immunohistochemistry. Using Southern blot analysis, Yellin et al. (11) did not see any amplification of GST- and found no correlation between expression and activity of GST- and CDDP sensitivity in 14 head and neck cell lines. Wang et al. (13) also used Southern blot analysis to examine expression and amplification of GST- in primary squamous cell carcinomas of the head and neck. Only 3 of 36 tumors (8%) showed GST- gene amplification.

The present study was undertaken to characterize chromosomal abnormalities in head and neck cell lines of known CDDP responsiveness using CGH. In addition, FISH analysis and immunohistochemistry were used to examine gene amplification and protein expression of GST- both in the cell lines, as well as in a group of pretreatment biopsy specimens from HNCA patients treated with CDDP and 5FU for advanced disease. The goal of the study was to correlate these findings with response to CDDP therapy and to identify any genetic similarities and differences between cell lines exhibiting varying degrees of sensitivity to CDDP.

	Materials and Methods

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Patient Selection.
Ten patients previously treated at Georgetown University Hospital with neoadjuvant CDDP/5FU for advanced (stages II, III, or IV) HNCA were selected for analysis. All patients received neoadjuvant chemotherapy for unresectable disease or organ preservation therapy after initial biopsy. Six of the patients were responsive to CDDP-based therapy and 4 were not. Patient characteristics are described in Table 2 .

View this table: [in this window] [in a new window]   Table 2 GST- evaluation by FISH, GST- expression, and clinical response to CDDP-based chemotherapy in primary tumors Immunohistochemical analysis was performed using a polyclonal antibody to GST- and FISH analysis using a GST- BAC clone. NED, no evidence of disease; DOD, dead of disease; CR, complete response; NR, no response).

GSH Assay.
Total intracellular GSH was determined using a modification of the enzymatic method of Tietze (17) . A standard curve was run using purified GSH (Sigma, St. Louis, MO). Total intracellular GSH was normalized to 25 µg of protein. All assays were carried out in triplicate.

Western Blot Assay.
Cell lines to be analyzed were grown to 80% confluence. Total protein was extracted using a cell lysis buffer and quantitated using a modification of the Bradford method, with BSA as a standard. Thirty µg of protein were applied per lane to a NuPAGE 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA). The blot was probed for the GST- protein using a commercially available mouse monoclonal antiserum (Novocastra, Peterborough, United Kingdom) at a 1: 500 dilution, followed by a goat antimouse antibody conjugated to horseradish peroxidase (1:20,000; Amersham, Buckinghamshire, United Kingdom).

CGH.
CGH was performed as described previously (18) . In brief, normal control DNA was prepared from peripheral blood lymphocytes of a normal donor, and test DNA was extracted from the cultured cell lines using standard protocols. Nick translation was performed to label the test DNA with biotin-16-dUTP (Roche, Indianapolis, IN) and control DNA with digoxigenin-11-dUTP (Roche). Five hundred ng of each of the labeled genomes were hybridized in the presence of excess Cot-1 DNA (50 µg; Invitrogen) to metaphase chromosomes prepared from a normal donor. The biotin-labeled tumor genome was visualized with avidin conjugated to FITC (Vector Laboratories, Inc., Burlingame, CA), and the digoxigenin-labeled control DNA was detected with a mouse antidigoxin antibody (Sigma-Aldrich, St. Louis MO) followed by detection with a goat antimouse antibody conjugated to tetramethylrhodamine isothiocyanate (Sigma-Aldrich). Chromosomes were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and embedded in antifading agent to reduce photobleaching. Gray scale images of the FITC-labeled tumor DNA, the tetramethylrhodamine isothiocyanate-labeled control DNA, and the DAPI counterstain from at least eight metaphases from each hybridization were acquired with a cooled charge-coupled device camera (CH250; Photometrics, Tucson, AZ) connected to a Leica DMRBE microscope equipped with fluorochrome-specific optical filters TR1, TR2, TR3 (Chroma Technology, Brattleboro, VT). Quantitative evaluation of the hybridization was done using commercially available software (Applied Imaging, Pittsburgh, PA). Average ratio profiles were computed as the mean value of at least eight ratio images to identify chromosomal copy number changes in all cases.

FISH.
A GST--specific BAC clone was obtained from BACPAC Resources (Oakland, CA) for use as a FISH probe. BAC clone DNA was prepared and labeled with biotin-11-dUTP (Roche) using nick translation as described previously (19) . Biotin-labeled DNA was detected with Fluorescein-Avidin DCS (Vector Laboratories, Inc.). Correct chromosomal location of the BAC clone was confirmed using standard FISH mapping. Interphase nuclei and metaphases prepared from the cell lines and one 4-µm paraffin section prepared from each of the primary tumors were evaluated by FISH using the BAC FISH probe as described earlier (20) . Using standard protocols, FISH analysis was carried out by a blinded observer. One hundred nonoverlapping interphase nuclei were counted for each case using a Leica DMRBE microscope. Detection of two FISH signals was considered as normal copy number, three signals as gain, and 4 signals as high-level gain or amplification. To characterize a tumor as having gain or amplification, at least 30% of the nuclei counted must fall into one of the above categories.

Immunohistochemistry.
Paraffin blocks were obtained from the Department of Pathology at Georgetown University. GST- immunohistochemistry using a polyclonal antibody (Novocastra) has been described previously (14 , 15) . A positive control tissue was included in each set of sections stained: normal kidney known to be positive for GST- was used. In negative controls, PBS with 1% BSA and 1% sodium azide replaced the primary antibody solution. Immunohistochemical staining was assessed by at least two independent observers, each of whom was blinded to patient identity and outcome. Criteria for positive staining for GST- expression have been described previously (14 , 15) .

Statistical Analysis.
Total cellular GSH, GST- protein expression, and GST- gene amplification were compared with the IC₅₀ for the 10 head and neck cell lines. Statistical significance was calculated using GraphPad Prism Statistical Software (version 3.02, San Diego, CA).

	Results

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Cytotoxicity Assays and GSH Levels in Head and Neck Cell Lines.
Total GSH expression and CDDP sensitivity data are shown in Table 1 . There was variable CDDP sensitivity among the 10 cell lines, with a 70 fold difference between the most sensitive (PCI 13) and the most resistant (SCC 25CP) cell lines. In addition, cell lines showing relatively more resistance to CDDP (such as SCC 25CP and JSQ-3) were associated with higher GSH levels than more sensitive cell lines (such as PCI-13 and HN22A). The average GSH level of the more sensitive cell lines was 19.64 nmol/mg protein, compared with an average of 89.42 nmol/mg protein for the relatively more resistant cell lines.

View this table: [in this window] [in a new window]   Table 1 Cisplatin response along with GST- FISH analysis, CGH at 11q13, GST- protein levels and total intracellular glutathione levels in cell lines Using FISH analysis, a designation of * gain means that 30% or greater of the cell nuclei counted contained three gene copies; ** amplification represents at least four gene copies. Cell lines SCC 25-1, SCC25CP, JSQ-3, SCC 9, and FaDu are relatively resistant to cisplatin (bold; IC50 6) compared with cell lines HN 17B, HN 22A, SCC 4, SCC 25-2, and PCI 13 (IC50 < 6). Relative GST- expression is determined based on assigning the lowest GST- expressing cell line (PCI 13) a value of 1 relative to expression of the control protein ß-actin.

CGH, GST- FISH Analysis, and GST- Western Blot for Head and Neck Cell Lines.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Karyogram of the DNA copy number changes observed in the 10 HNCA cell lines. Bars on the left side of the chromosome ideogram indicate losses; bars on the right side indicate gains. Heavy solid bars on the right side indicate high-level copy number increases (amplifications). For each chromosome, the bars have been grouped to summarize the results of the more cisplatin-sensitive lines (S = Sensitive) and the more cisplatin-resistant lines (R = Resistant).

GST-

GST-

GST-

View larger version (46K): [in this window] [in a new window]   Fig. 2. FISH analysis using a GST- probe of cell lines PCI 13 (Fig. 2 A) and SSC25/CP (Fig. 2 B). Both figures show metaphase (left) and interphase (right) results. FISH analysis of the PCI 13 line (relatively cisplatin sensitive) shows two copies of the gene. SCC25/CP is a squamous cell carcinoma cell line that has been developed to be relatively resistant to cisplatin. FISH analysis shows amplification of the gene in this line.

GST- FISH and Immunohistochemical Expression in Clinical Biopsy Specimens.
Of the 10 patients treated with CDDP and 5-FU, 6 had a complete response to CDDP therapy and 4 were nonresponders. FISH analysis using a GST--specific probe showed increased copy number in 6 of the 10 specimens, whereas the remaining 4 had two copies of the gene. Of the 6 patients with increased copy number of the GST- gene, 2 showed clear amplification. Neither responded to chemotherapy and both died of disease < 9 months from diagnosis. Four patients had a gain of signal, with >30% of the cells showing three signals. Two of those had a complete response to therapy and are alive >50 months from treatment; the other 2 did not respond to therapy and died an average of 9.5 months after diagnosis. All 4 patients with two copies of the GST- gene had a complete response to neoadjuvant chemotherapy. Three of those 4 patients are alive >50 months from treatment. One patient relapsed and died > 2 years from his initial therapy. Of note, all 4 patients in the nonresponder group showed gain or amplification of GST- by FISH, whereas only 2 of 6 patients in the complete responder group showed gain and none showed amplification (Table 2 , ², P < 0.05).

Four of 4 patients in the nonresponder group with gain or amplification showed increased GST- expression, with an intensity score of 2 compared with 1 of 2 patients exhibiting gain in the complete responder group. Examples of GST- FISH and immunohistochemistry in complete responders and nonresponders are shown in Fig. 3 .

View larger version (159K): [in this window] [in a new window]   Fig. 3. A, GST- FISH evaluation of a tumor sample from a patient (case 4-cisplatin responsive) with stage IV tongue cancer showing mostly two copies of the gene. B, GST- immunohistochemistry study of a tumor from a patient (case 5-cisplatin responsive) with a stage IV hypopharyngeal cancer. Note the lack of detectable cytoplasmic GST- expression (x100). C, GST- FISH analysis of a tumor from a patient (case 7-cisplatin unresponsive) with a stage IV tongue cancer showing mostly amplification. D, GST- immunohistochemistry of the tumor from the same patient showing increased cytoplasmic GST- expression in tumor cells (x100).

	Discussion

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In the present study, we investigated the relationship of chromosomal abnormalities, GST- amplification, and GST- expression to CDDP sensitivity. We used CGH to detect chromosomal aberrations in 10 head and neck cell lines of known CDDP response. We found that 6 of 10 HNCA cell lines (60%) showed increased copy number at 11q13, the locus where GST- maps. Increased copy numbers of 11q13 occurs frequently in HNCAs and may be associated with a more aggressive phenotype (9 , 21) . Similar to other studies, several cell lines also showed gains on 3q, 5p, 8q, and 20q and losses on 3p, 8p, 9p, and 18q (22) . We observed a similar pattern of chromosomal changes in all of the cell lines were regardless of CDDP sensitivity, except for the gain at 9q, which occurred in 4 of the 5 more sensitive cell lines versus 1 of the 5 more resistant cell lines, and the loss of 3p, which occurred in all 5 of the more sensitive cell lines and in only 2 of 5 more resistant ones.

One of the mechanisms by which chemotherapy resistance may occur is by gene amplification or the overexpression of gene products (such as GST-) that provide a tumor cell with survival advantage relative to normal cells. In the past, other studies of 11q13 amplification in HNCA have focused on the amplification of the cyclin D1 gene (CCND1) and other genes more centromeric than GST-. CCND1 amplification and cyclin D1 overexpression are commonly accepted to be associated with tumor progression, higher tumor grade, increased rates of recurrence, and decreased survival in HNCA studies(23 , 24) . The role of GST- gene amplification is less clear. We have previously reported that expression of GST- correlates with prognosis and response to therapy in HNCA patients (14 , 15) . In an analysis of >50 patients with advanced HNCA treated with CDDP-based neoadjuvant therapy for unresectable disease or organ preservation therapy (14 , 15) , high GST- expression was associated with poor overall survival. GST- expression was also associated with a lack of response to therapy, with high GST--expressing tumors having a response rate of 19% compared with 87% for low GST--expressing tumors (relative risk = 4.7, P = 0.001). Although convincing evidence for chromosomal gain at 11q13 has been demonstrated by several groups using different approaches, the few available studies looking specifically at GST- amplification (none of which used FISH) have concluded that this is a rare event and failed to show any important clinical correlation between amplification of the gene and/or protein expression (8 , 10, 11, 12, 13) .

In the present study, we found that 9 of 10 cell lines showed either GST- gain (2 cell lines) or frank amplification (7 cell lines) by FISH. This correlated well with the patient data presented here, with 60% of the specimens examined showing either gain or amplification using the same GST--specific BAC clone. Given the frequency with which 11q13 amplification has been seen in HNCA, it is unclear why previous studies have failed to show a higher incidence of GST- amplification. One reason for the discrepancy between these results and those of previous studies may involve the different techniques used to detect amplification. Previous studies assessing 11q13 amplification and GST- amplification in HNCA have used Southern blot analysis, which may be inadequate in its ability to detect subtle differences in gene amplification secondary to stromal contamination in tissue samples. In contrast, FISH is a very sensitive and rapid method for detecting genetic abnormalities associated with malignancy and has been shown to detect gene amplification even when Southern blotting has not (25) . Using FISH, we have been able to effectively detect frequent variances in the GST- gene copy number at a cell by cell level.

Although squamous cell carcinoma of the head and neck is histologically the most common type of cancer of the upper aerodigestive tract, it is very clear clinically that squamous cell carcinomas behave differently depending on the primary site. It is reasonable to assume that some of these differences may be mediated by specific genetic alterations and may be detectable by CGH. Site-specific genetic abnormalities have been identified for the oral cavity, pharynx, and larynx and are associated with higher tumor grade and nodal spread (22) . We did not see any specific subsite associated with GST- amplification or increased expression in our pilot study group, although this may be a result of sample size. Additional investigation is warranted.

Among the head and neck cell lines, there was not a consistent correlation between GST- amplification and expression as detected by Western blot analysis (Table 1) . This finding is similar to what has been reported by other groups for HNCA (11 , 14) . Reasons for this are unclear. Enzyme activity may be important in mediating resistance in cell lines showing gain or amplification. In addition, recent data suggest that subcellular localization of GST- may play a role in mediating CDDP sensitivity (26) . We saw a better correlation in clinical tumors between GST- gain or amplification and GST- protein expression measured by immunohistochemistry. However, this correlation was also weak and may only be an indirect indicator of GST- activity.

In conclusion, GST- amplification is a frequent event in HNCA cell lines and tumors when studied using FISH and is associated with increased expression in tumors poorly responsive to CDDP-based chemotherapy. Total cellular GSH expression and total cellular GST- expression are not predictive of CDDP sensitivity in head and neck cell lines. However, given the frequency of GST- amplification detected by FISH, additional investigation of potential mechanisms is warranted.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Kevin J. Cullen, Lombardi Cancer Center, 3910 Reservoir Roads, NW, Room 501, Washington, DC 20057. E-mail: cullenk{at}georgetown.edu

⁴ The abbreviations used are: CDDP, cisplatin; HNCA, head and neck cancer; GST-, glutathione S-transferase ; GSH, glutathione; CGH, comparative genomic hybridization; FISH, fluorescence in situ hybridization; BAC, bacterial artificial chromosome; 5FU, 5-fluorouracil.

Received 7/ 3/03. Revised 10/10/03. Accepted 10/15/03.

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