REPORT

In Vitro Evidence for Homologous Recombinational Repair in Resistance to Melphalan

Zhi-Min Wang, Zhong-Ping Chen, Zhi-Yuan Xu, Garyfallia Christodoulopoulos, Vanessa Bello, Gérard Mohr, Raquel Aloyz, Lawrence C. Panasci

Affiliations of authors: Z.-M. Wang, Z.-Y. Xu, G. Christodoulopoulos, V. Bello, G. Mohr, R. Aloyz, L. C. Panasci, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, PQ, Canada; Z.-P. Chen, Department of Neurosurgery/Neuro-Oncology, Cancer Center, Sun Yat-sen University of Medical Sciences, People's Republic of China, Guangzhou.

Correspondence to: Lawrence C. Panasci, M.D., Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, 3755 Côte Ste Catherine, Montreal, PQ, H3T 1E2 Canada (e-mail: lpanasci{at}hotmail.com).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The generation of DNA interstrand cross-links is thought to be important in the cytotoxicity of nitrogen mustard alkylating agents, such as melphalan, which have antitumor activity. Cell lines with mutations in recombinational repair pathways are hypersensitive to nitrogen mustards. Thus, resistance to melphalan may require accelerated DNA repair by either recombinational repair mechanisms involving Rad51-related proteins (including x-ray repair cross-complementing proteins Xrcc2, Xrcc3, and Rad52) or by nonhomologous endjoining involving DNA-dependent protein kinase (DNA-PK) and Ku proteins. We investigated the role of DNA repair in melphalan resistance in epithelial tumor cell lines. Methods: Melphalan cytotoxicity was determined in 14 epithelial tumor cell lines by use of the sulforhodamine assay. Homologous recombinational repair involving Rad51-related proteins was investigated by determining the levels of Rad51, Rad52, and Xrcc3 proteins and the density of nuclear melphalan-induced Rad51 foci, which represent sites of homologous recombinational repair. Nonhomologous endjoining was investigated by determining the levels of Ku70 and Ku86 proteins and DNA-PK activity. Linear regression analysis was used to analyze correlations between the various protein levels, DNA-PK activity, or Rad51 foci formation and melphalan cytotoxicity. All statistical tests were two-sided. Results: Melphalan resistance was correlated with Xrcc3 levels (r = .587; P = .027) and the density of melphalan-induced Rad51 foci (r = .848; P = .008). We found no correlation between melphalan resistance and Rad51, Rad52, or Ku protein levels or DNA-PK activity. Conclusion: Correlations of melphalan resistance in epithelial tumor cell lines with Xrcc3 protein levels and melphalan-induced Rad51 foci density suggest that homologous recombinational repair is involved in resistance to this nitrogen mustard.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In eukaryotes, two major DNA repair pathways are involved in the repair of double-strand breaks and possibly interstrand cross-links: homologous recombinational repair and nonhomologous DNA endjoining. Homologous recombination is implicated in the repair of double-strand breaks and interstrand cross-links and is important in maintaining chromosomal integrity (13). Nonhomologous DNA endjoining can repair double-strand breaks and may be implicated in the repair of interstrand cross-links. Nonhomologous DNA endjoining, unlike homologous recombinational repair, requires little or no homology for the repair of double-strand breaks because it ligates blunt ends of DNA, but it tends to be more error prone [reviewed in (4,5)].

The nitrogen mustards are an important group of alkylating agents, with activity against several human tumors in vivo, including breast and ovarian cancers (6). Cytotoxicity induced by nitrogen mustards is thought to be the consequence of DNA alkylation and the formation of DNA interstrand cross-links (6). Decreased formation and/or increased removal of DNA interstrand cross-links have been associated with resistance to nitrogen mustards (68).

Thus, identifying the mechanisms of interstrand cross-link repair, with the use of cell lines with mutations in DNA repair proteins, is important to understanding the mechanism by which tumors become resistant to nitrogen mustards. Several mammalian cell lines with mutations in DNA repair proteins, including ERCC-1, ERCC-4 (XPF [xeroderma pigmentosum complementation group F]), Xrcc2 (x-ray cross-complementing protein 2), Xrcc3, Rad54, Ku70, Ku86, and DNA-PKcs (DNA-dependent protein kinase [DNA-PK] catalytic subunit) are highly sensitive to nitrogen mustards (1,2,912). Moreover, cell lines with mutations in Xrcc2, Xrcc3, ERCC-1, or ERCC-4 (1,2,912) have been found to be severalfold times more sensitive to nitrogen mustards than wild-type cell lines. In addition, the cell lines lrs1 and lrs1SF, with mutations in Xrcc2 and Xrcc3, respectively, are hypersensitive (<=100-fold more sensitive than wild-type cell lines) to DNA cross-linking agents, such as mitomycin C, cisplatin, and nitrogen mustards (1). Hypersensitivity of these two cell lines is corrected by wild-type Xrcc2 and Xrcc3, respectively (1).

Xrcc2 and Xrcc3 have a low level of sequence similarity to the human homologue of yeast Rad51 (HsRad51) and are members of the HsRad51-related proteins that are implicated in homologous recombination (1). In a yeast two-hybrid system, Xrcc3 was the only Rad51-related protein to interact with HsRad51, suggesting that these two proteins act together during homologous recombinational repair (1). Also, it appears that in vivo HsRad51 requires Xrcc3 to assemble on damaged chromosomes (13) during homologous pairing, a critical stage of homologous recombinational repair, and that expression of wild-type Xrcc2 and Xrcc3 in the lrs1 and lrs1SF cell lines, respectively, complements the high level of chromosomal aberrations found in these cell lines and restores chromosomal integrity (1). From studies of DNA interstrand cross-link removal in bacteria and yeast and the possibility that DNA double-strand breaks and interstrand cross-links are repaired in a similar fashion, a model was proposed in which nucleotide excision repair, via the ERCC-1/ERCC-4 (XPF) endonuclease, cuts the DNA on the 5' side of the interstrand cross-link and then recombinational repair enzymes further process the lesion error free [reviewed in (2,3)]. Various alkylating agents and ionizing radiation can induce Rad51 foci formation in cells; these foci are thought to represent sites of homologous recombinational repair (14,15).

The Rad51-related proteins are not involved in nonhomologous DNA endjoining. Instead, this repair mechanism involves an enzymatic complex consisting of multiple proteins, including Ku70 and Ku86, both of which form a dimer that binds the DNA double-strand end, DNA-PKcs, DNA ligase IV, and Xrcc4 (5). We have demonstrated that, in chronic lymphocytic leukemia, nitrogen mustard drug resistance is associated with enhanced DNA repair and, specifically, increased DNA-PK activity and increased Rad51 foci formation (1618). Increased nucleotide excision repair activity and, specifically, increased ERCC-1 protein levels were not associated with this nitrogen mustard drug resistance (19,20).

Because the nitrogen mustards are used to treat epithelial tumors, we investigated in this study whether proteins in these recombinational repair pathways (i.e., nonhomologous DNA endjoining and homologous recombination) were involved in nitrogen mustard drug resistance in epithelial tumor cell lines.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell Lines

Sixteen human tumor cell lines were used in this study: A-498, HT-29, ACHN, SF-295, 786–0, and CAKI-1 (National Cancer Institute, Bethesda, MD); T98-G (D. Yarosh, Applied Genetics Inc., New York, NY); SKMG-1 and SKMG-4 (G. Caincross, University of Western Ontario, Canada); SKI-1 (J. Shapiro, Borrow Neurological Institute, Phoenix, AZ); SKNSH (E. Shoubridge, Montreal Neurological Institute, Canada); MCF-7 (G. Batist, Jewish General Hospital, Montreal, Canada); MGR-3 and UW28 (F. Ali-Osman, The University of Texas M. D. Anderson Cancer Center, Houston); and MO59J and MO59K (cell lines and hybridomas; American Type Culture Collection, Manassas, VA). All cell lines were grown and maintained as monolayers in appropriate medium (i.e., McCoy's 5A supplemented with 10% fetal bovine serum [FBS], RPMI-1640 medium supplemented with 5% FBS, or Dulbecco's modified Eagle medium [DMEM] supplemented with 10% FBS) containing gentamycin (10 µg/mL) in a humidified 5% CO2 and 95% air atmosphere at 37 °C.

Sulforhodamine B (SRB) Cytotoxicity Assay

The cytotoxicity of melphalan, a nitrogen mustard analogue (Sigma Chemical-Aldrich Canada Ltd Co., Oakville, ON, Canada), or cisplatin (Jewish General Hospital, Montreal, PQ, Canada) was determined by use of a modified SRB colorimetric anticancer drug screening assay as described previously (21). The results represent the average and 95% confidence intervals (CIs) of at least three independent experiments.

Preparation of Cell Extracts

Protein extracts for western blot analyses were prepared as described previously (20). The cells were homogenized at 4 °C in RIPA buffer (i.e., phosphate-buffered saline [PBS], 1% Nonidet P-40 [NP-40], 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) containing protease inhibitors (protease inhibitor Cocktail Tablets®; Roche Molecular Biochemicals, Indianapolis, IN). The cell debris was pelleted by centrifugation at 12000g for 10 minutes at 4 °C, and the supernatants containing the protein extracts were frozen at -70 °C until analysis.

Whole-cell extracts were prepared for the DNA-PK activity assay as described previously (17), with slight modifications. Briefly, cell pellets were quickly thawed and suspended in extraction buffer (1 x 108 cells/100 µL) containing 10 mM NaF, 20 mM HEPES (pH 7.8), 450 mM NaCl, 25% glycerol, 0.2 mM EDTA, and 0.5 mM dithiothreitol (DTT) in the presence of proteinase inhibitors (0.5 mM phenylmethysulfonyl fluoride, 0.5 µg/mL aprotinin, 0.5 µg/mL leupeptin, and 1.5 µg/mL pepstatin), then frozen in liquid nitrogen and thawed at 30 °C three times. After centrifugation at 12000g for 30 minutes at 4 °C, the pellets were discarded and the supernatants stored at –70 °C until analysis.

Western Blot Analyses

Ku70, Ku86, HsRad51, HsRad52, Xrcc3, and {alpha}-tubulin protein levels in the 14 human tumor cell lines were determined by western blot analysis as described previously (21). Briefly, whole-cell extracts were separated by 10% SDS–polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), blocked in Blotto solution (i.e., 5% nonfat milk in PBS containing 0.2% Tween 20 [PBST]) at room temperature for 2 hours, and incubated with the primary antibody diluted in Blotto for 1 hour at room temperature. The antibodies were used at a final concentration of 0.5 µg/mL to detect Ku70 or Ku86 (NeoMarkers; Medicorp, Montreal, Canada), HsRad51 (H-92; Santa Cruz Biotechnology, Santa Cruz, CA), HsRad52 (C-17; Santa Cruz Biotechnology), and {alpha}-tubulin (N-353; AmershamPharmacia Biotech, Buckinghamshire, U.K.). The Xrcc3 antibody (from P. Sung, The University of Texas at San Antonio) is a rabbit polyclonal anti-Xrcc3 antiserum that was used at a 1 : 2500 dilution. The membranes were washed three times with PBST and incubated for 1 hour at room temperature in 1 : 5000 dilutions of specific anti-mouse (NA 931; AmershamPharmacia Biotech) or anti-rabbit (SC-2004; Santa Cruz Biotechnology) horseradish peroxidase-conjugated secondary antibodies in Blotto. The proteins were visualized after four 10-minute washes in PBST by the enhanced chemiluminescence system (AmershamPharmacia Biotech) and subsequent exposure of the membrane to film (Eastman Kodak Co., Rochester, NY). Band intensities were quantified by densitometry with the use of the Scion Image program (National Institutes of Health Image version 1.6 [Bethesda, MD], with an HP ScanJet 5100C Scanner [Hewlett-Packard Co., Greeley, CO]). Protein levels were normalized against {alpha}-tubulin levels. Three different protein extracts were analyzed in triplicate for each cell line. The results of the three separate experiments are expressed as the average values and 95% CIs.

DNA-PK Activity Assay

The DNA-PK pulldown kinase assay was performed as described previously (17,22). The human glioma cell lines MO59J (without detectable DNA-PK activity) and MO59K (with detectable DNA-PK activity) were used as negative and positive controls, respectively, to validate the technique (17). DNA-cellulose beads (Sigma, Ontario, Canada) were equilibrated by two 5-minute hydrations in an equal volume of Z' 1X buffer (i.e., 25 mM HEPES–KOH [pH 7.8], 50 mM KCl, 10 mM MgCl2, 20% glycerol, 0.1% NP-40, and 1 mM DTT) at 4 °C, each followed by centrifugation at 2096g for 5 minutes at 4 °C. The equilibrated beads were incubated with cellular protein extract (40 µg protein extract, Z'0.5X buffer) in a total volume of 20 µL per sample for 1 hour at 4 °C under constant agitation. Each sample was washed twice with 500 µL of Z'0.5X buffer followed by centrifugation at 2096g for 5 minutes at 4 °C. The supernatant was removed completely after the last wash, and the pellet (containing pulled-down DNA-PK) was incubated with 20 µL of reaction mix (i.e., 10 µCi [3000 Ci/mmol] {gamma}-[32P]adenosine triphosphate [ATP], 0.1 mM ATP, 0.2 mM peptide, and Z'0.5X buffer) for 30 minutes at 30 °C, with frequent, gentle agitation. Each sample was assayed in the presence of the substrate peptide EPPLSQEAFADLLKK, which is part of the p53 protein that is phosphorylated by DNA-PK, and the mutated peptide EPPLSEQAFADLLKK, which serves as a negative control. The reaction was stopped by centrifugation at 21 466g for 5 minutes at room temperature. Fifteen microliters of supernatant (containing peptide and {gamma}-[32P]ATP) was removed, taking caution to avoid the beads, diluted with an equal volume of 30% acetic acid, and then vortex mixed. This supernatant (5 µL) was spotted on a 1-cm2 precut filter paper (Chromatography Paper P81; Whatman, Maidstone, U.K.), air-dried thoroughly, and washed with 15% acetic acid for 5 minutes under constant agitation. The acetic acid was aspirated, and the wash step was repeated four times. The filter was air-dried thoroughly and placed in 10 mL Ecolite (ICN Biomedicals Inc., Costa Mesa, CA), and the radioactivity was measured with a Liquid Scintillation Analyzer (Packard, Mississauga, Ontario, Canada).

Results are expressed as counts per minute (cpm) of wild-type peptide minus cpm of mutated peptide per microgram of protein. The results represent the average and 95% CIs of three independent experiments.

Induction of HsRad51 Foci Formation After In Vitro Drug Treatment

Tumor cells were seeded at a density of 0.5 x 105 cells/mL in complete culture medium onto glass coverslips in six-well plates and allowed to adhere for 16 or 36 hours for each experiment. To induce the DNA interstrand cross-links in the tumor cells and to activate the repair process, we added melphalan to the medium at a final concentration of 1.8 or 5.5 µM. Control cells were kept in complete medium containing melphalan diluent solution without drug. The choice of drug concentrations was based on the mean LD50 (lethal dose required to kill 50% of the cell population) of sensitive and resistant cell lines, respectively, as determined by the SRB assay. Sensitive cell lines have an LD50 of less than 3 µM, while resistant cell lines have an LD50 of greater than 3.5 µM. Rad51 foci density was assessed by immunocytochemistry as described below.

Immunofluorescent Staining With Anti-HsRad51 Protein Antibodies

Four and 24 hours after treatment, the cells were washed twice with ice-cold PBS and fixed with a mixture of 80% methanol/20% acetone (vol/vol) that was precooled at –80 °C and added directly to the cells, which were then placed at –20 °C for 15 minutes. After fixation, the cells were washed for four 10-minute periods with PBS/CM (PBS at 0.1 and CaCl2 at 1 µM MgCl2) and then incubated with blocking solution containing 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) for 30 minutes at room temperature. The cells were incubated overnight at 4 °C or for 1 hour at room temperature with anti-human Rad51 rabbit antibody (1 : 100 dilution, H-92; Santa Cruz Biotechnology). The anti-human Rad51 antibody used is specific, since the antibody recognizes only one 31-kd band on a western blot. The cells were washed for several 10-minute periods in PBS/CM at room temperature and then incubated for 1 hour with 1 : 50 dilution of fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin (Santa Cruz Biotechnology) diluted in blocking solution. After four 10-minute washes with PBS/CM, the nuclei were counterstained with propidium iodide (Sigma Chemical Co., St. Louis, MO) at a concentration of 2 µg/mL for 5 minutes, washed twice with PBS/CM, and mounted in antifade solution (i.e., 2.3% 1,4-diazabicyclo[2.2.2.]octane, 0.1 M Tris–HCl [pH 8.0], and 90% glycerol).

Confocal Microscopy

Changes in HsRad51 nuclear density were determined by confocal microscopy as described previously (18). FITC and propidium iodide labeling were distinguished with the x60 Nikon Plan Apochromat objective of a dual channel Bio-Rad 600 laser scanning confocal microscope equipped with a krypton/argon laser and the corresponding dichromic reflectors (Bio-Rad Laboratories, Hercules, CA). To generate a composite image of the complete cell depth for the double-labeled cells, we collected optical sections at 1-µm increments that encompassed a major portion of the FITC and propidium iodide immunofluorescent labeling. The images were projected with the use of the Bio-Rad COMOS software (Bio-Rad Laboratories) in which the most intense image value for each pixel was presented.

Cells were imaged under conditions of equivalent pinhole and black level settings. The gain was adjusted for each image to ensure that the pixel values in each section were not maximal and, therefore, not saturating. Fluorescence signals were amplified and fed into a multiparameter image analysis program. The images were analyzed by use of the image analysis system, Northern Eclipse (Empix Imaging, Mississagua, ON, Canada). The images were colored electronically (green for FITC and red for propidium iodide labeling). The images were then merged electronically with yellow staining representing the merging of the red and green labeling, indicating colocalization (Rad51 in the nucleus). To quantify the total Rad51 foci density, we determined the ratio of the average yellow pixel intensity (fluorescent total intensity, FTI) relative to the total yellow (fluorescent total area, FTOA)-stained nuclear area in five randomly selected nuclei for each treatment. The calculated value represents the mean and 95% CIs of three independent experiments. The relative increase in the ratio of yellow fluorescence intensity to the total yellow fluorescent area in the melphalan-treated cells was compared with that of the untreated control cells in the same experiment. Confocal images were printed with the use of a Polaroid TX 1500 video printer (Polaroid, Etobicoke, Ontario, Canada).

Statistical Analysis

Linear regression analysis (Linear Regression Analysis Function, ToolPak for Microsoft Excel 97 software; Microsoft, Mississauga, Ontario, Canada) was used to analyze the correlations between melphalan cytotoxicity and the protein levels or HsRad51 foci density. All statistical tests were two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To understand the mechanisms responsible for resistance to nitrogen mustards, such as melphalan, we assessed the sensitivity of 14 epithelial tumor cell lines to the drug. The sensitivity to melphalan in each of the 14 tumor cell lines is shown in Table 1Go. Cell lines with an LD50 of less than 3 µM were considered to be sensitive to melphalan, whereas those with an LD50 of greater than 3.5 µM were considered to be resistant. The 14 cell lines were statistically significantly cross-resistant to cisplatin by linear regression analysis (r = .801; P<.001, data not shown), indicating that the resistance was not specific to nitrogen mustards.


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Table 1. Panel of 14 human epithelial tumor cell lines were screened for melphalan cytotoxicity, Ku70, Ku86, Rad51, Rad52, and Xrcc3 protein levels, and DNA-PK activity*
 
Resistance to melphalan is thought to involve alterations in the formation of or removal of DNA interstrand cross-links. Because proteins responsible for nonhomologous endjoining and homologous recombinational repair are possibly implicated in the repair of DNA interstrand cross-links, we assessed the expression of a variety of DNA repair proteins in the 14 epithelial tumor cell lines. We first assessed the expression of Ku70 and Ku86 by western blot analysis and the activity of DNA PK by the DNA-PK pulldown kinase assay (Table 1Go). We found no correlation between either Ku70 protein levels (r = .159; P = .59), Ku86 protein levels (r = .192; P = .51), or DNA-PK activity (r = .00033; P = .99) and melphalan resistance in the 14 cell lines. Therefore, it is unlikely that nonhomologous endjoining DNA repair plays a predominant role in melphalan resistance in these tumor cell lines.

We next assessed the expression of proteins involved in homologous recombinational repair. The expression of HsRad51, HsRad52, and Xrcc3 was analyzed by western blot analysis (Table 1Go). We found no correlation between the HsRad51 (r = .316; P = .271) or HsRad52 (r = .023; P = .913) protein levels and melphalan resistance in the 14 cell lines. We did find a statistically significant correlation between Xrcc3 protein levels and melphalan resistance (r = .587; P = .027), suggesting that there is a relationship between the expression level of Xrcc3 and melphalan cytotoxicity in these 14 cell lines (Fig. 1Go, A and B). Because Xrcc3 is a component of the Rad51-related protein family, this result suggested that homologous recombination might be important in melphalan drug resistance.



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Fig. 1. A ) Expression of Xrcc3 protein levels and {alpha}-tubulin in 14 human epithelial tumor cell lines. Protein levels were assessed by western blot analysis as described previously (20).. B) Melphalan cytotoxicity versus Xrcc3 protein levels for 14 human epithelial tumor cell lines in a linear regression analysis. The x-axis represents melphalan LD50 (lethal dose required to kill 50% of the cell population) concentration; the y-axis represents Xrcc3 protein levels relative to {alpha}-tubulin levels determined by western blot analysis as described previously (20). Solid line represents the linear regression line; line equation is y = 0.068x + 0.083; correlation coefficient is r = .587; and P = .027.

 
To evaluate the role of homologous recombinational repair in the processing of melphalan-induced DNA interstrand cross-links, we examined HsRad51 foci formation. We chose eight tumor cell lines with different sensitivities to melphalan, ranging from highly sensitive (MCF-7 and SKI-1), to moderately sensitive (SKMG-1 and SKMG-4), to resistant (T98G, HT-29, SKNSH, and A498) phenotypes to examine the complete spectrum of drug resistance in the HsRad51 foci assay. Each cell line was incubated with 1.8 or 5.5 µM melphalan for 4 and 24 hours to allow repair of drug-induced DNA damage, and the cells were fixed and immunostained with anti-HsRad51 antibodies. The antibody localized to subnuclear foci in both untreated control and drug-treated cells (Fig. 2Go). A small number of foci (typically fewer than five) were detected in untreated control cells (Fig. 2Go). These results are qualitatively similar to previous observations made in CHO cells, human fibroblasts, and lymphocytes (14,15).



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Fig. 2. Identification of HsRad51 foci in untreated and melphalan-treated cell lines. The melphalan-sensitive SKI-1 cell line was cultured without melphalan (A) or with 5.5 µM melphalan for 4 hours (B), and the melphalan-resistant A498 cell line was cultured without melphalan (C) or with 5.5 µM melphalan for 4 hours (D). The cells were fixed and immunostained with an anti-human Rad51 antibody and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The cells were counterstained with propidium iodide. Representative confocal fluorescent images were collected and analyzed with the use of the Northern Eclipse image analysis system. Green images, representing FITC-stained anti-Rad51 antibody, were electronically merged with the red images, representing the propidium iodide nuclear counterstain, to generate yellow foci, representing colocalized nuclear HsRad51.

 
The density of Rad51 foci was determined by the ratio of the FTI and FTOA obtained with the HsRad51 antibody. There was no statistically significant difference in the density of Rad51 foci between untreated melphalan-sensitive cell lines (MCF-7, SKI-1, SKMG-1, and SKMG-4) and resistant cell lines (T98G, HT-29, SKNSH, and A498) (P = .37 as assessed by Student's t test). Moreover, the correlation between melphalan resistance and basal (untreated cells) Rad51 foci density was also not statistically significant (r = .28; P = .51). After the cells were incubated with 1.8 or 5.5 µM melphalan for 4 or 24 hours, both the sensitive and resistant cells demonstrated an increase in the density of HsRad51 foci (Fig. 2Go). Because the dramatic change in the pattern of HsRad51 staining induced by melphalan treatment was not associated with corresponding changes in FTOA Rad51 signal detected by immunofluorescence, we also carried out western blot analysis to determine the steady-state level of HsRad51 protein before and after melphalan treatment. No statistically significant difference in HsRad51 protein levels was observed between treated and untreated cell lines, suggesting that melphalan-induced formation of Rad51 foci is not a consequence of increased HsRad51 protein levels after treatment (data not shown).

Because our results suggest that in vitro melphalan treatment activates homologous recombinational repair pathways, we next examined whether the increase in the density of HsRad51 foci after melphalan treatment correlates with melphalan resistance in the eight tumor cell lines that differ in their sensitivity to the drug. We found a statistically significant correlation between melphalan resistance and the relative increase in density of HsRad51 foci in the eight cell lines incubated with 5.5 µM melphalan for 4 hours (r = .848; P = .008) (Fig. 3Go) or incubated with 1.8 µM melphalan (r = .844; P = .008) or with 5.5 µM melphalan (r = .739; P = .036) for 24 hours. Thus, in these tumor cell lines, HsRad51 foci formation is induced by melphalan treatment and represents enhanced DNA repair in melphalan-resistant epithelial tumor cell lines. We found no correlation between Xrcc3 protein levels and cisplatin cytotoxicity (r = .347; P = .400), but there was a statistically significant correlation between cisplatin cytotoxicity and Rad51 foci density induced by 5.5 µM melphalan for 4 hours and both drug concentrations for the 24-hour treatment (r>=.894; P<=.003 for all three determinations).



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Fig. 3. Linear regression analysis of melphalan cytotoxicity versus the relative increase in the density of Rad51 foci in eight tumor cell lines treated with 5.5 µM melphalan for 4 hours. Rad51 foci were identified by immunohistochemistry by use of an anti-human Rad51 antibody. The x-axis represents melphalan LD50 (lethal dose required to kill 50% of the cell population) concentration; the y-axis represents the relative increase in the density of HsRad51 foci expressed by the cells calculated as the ratio of fluorescent total intensity (FTI) and the fluorescent total area (FTOA) in treated cells compared with untreated cells. Solid line represents the linear regression line; line equation is y = 0.043x + 0.948; correlation coefficient is r = .848; and P = .008.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our previous investigations (1620) have implicated both nonhomologous endjoining and homologous recombinational repair in resistance to nitrogen mustard in chronic lymphocytic leukemia. Specifically, we identified that the activity of DNA-PK, a component in nonhomologous endjoining, is low in the chlorambucil-(hyper)sensitive B lymphocytes of most untreated chronic lymphocytic leukemia patients (17). Furthermore, homologous recombinational repair, as represented by chlorambucil-stimulated Rad51 foci, correlated with chlorambucil cytotoxicity in drug-resistant B lymphocytes from chronic lymphocytic leukemia patients (18).

The present study was done to determine the role of nonhomologous endjoining and homologous recombinational repair in nitrogen mustard drug resistance in epithelial tumor cell lines. The cytotoxicity of melphalan, which is used in the treatment of breast and ovarian cancers, was determined for 14 epithelial tumor cell lines. Unlike the results with chronic lymphocytic leukemia (17,18), only homologous recombinational repair appears to be important in melphalan resistance in epithelial cells. There was no correlation between DNA-PK activity (or Ku protein levels) and melphalan resistance, suggesting that nonhomologous endjoining is not important in melphalan resistance in epithelial cell lines. By contrast, there was a correlation between melphalan resistance and the density of Rad51 foci after melphalan treatment, which is indicative of homologous recombinational repair. A similar correlation was found between cisplatin cytotoxicity and the density of melphalan-induced Rad51 foci, suggesting that Rad51-related homologous recombination may be involved in the mechanism of both cisplatin and melphalan resistance. Although there was no correlation between melphalan resistance and baseline HsRad51 protein levels or baseline HsRad51 foci, there was a correlation between baseline Xrcc3 protein levels and melphalan resistance in the 14 cell lines. Although the correlation was statistically significant, it was not overwhelming; thus, the result is only suggestive that Xrcc3 plays a role in nitrogen mustard drug resistance. Moreover, there are discrepancies between melphalan resistance and Xrcc3 levels as shown by the UW28 cell line, which expresses a low level of Xrcc3 protein and is moderately resistant to melphalan.

A mutation in Xrcc3 renders the Irs1SF cell line defective in maintaining chromosome integrity by homologous recombinational repair mechanisms. Furthermore, Irs1SF cells are defective in inducing Rad51 foci in response to either alkylating agents or radiation (1,13). However, introduction of wild-type Xrcc3 restores the ability to induce Rad51 foci (1,13), suggesting that Xrcc3 may have a role in HsRad51 foci formation, homologous recombinational repair in response to DNA-damaging agents, and, thus, in melphalan drug resistance. Confirmation of this hypothesis awaits experiments in which overexpression of Xrcc3 in cell lines with low endogenous Xrcc3 protein levels increases resistance to melphalan.

Our results may ultimately have clinical relevance. For example, modulating Xrcc3 function by either biochemical or gene therapeutic approaches may sensitize cancer cells to melphalan treatment or because only Xrcc3 protein levels correlated with melphalan resistance, immunohistochemical analysis of Xrcc3 in primary tumor specimens may be useful in determining the nitrogen mustard resistance of epithelial tumors.


    NOTES
 
Supported by the Leukemia and Lymphoma Society and by a private donation from Helen Rosenbloom Lang. L. C. Panasci is a recipient of the Gertrude and Stanley Vinberg Clinical Scientist Award.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received February 9, 2001; revised July 17, 2001; accepted August 1, 2001.


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