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1 Ontario Cancer Institute/Princess Margaret Hospital, University Health Network; and Departments of 2 Radiation Oncology, 3 Pathology, and 4 Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
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ABSTRACT |
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2- to 5-fold in comparison to normal PrEC cells. The XRCC1, DNA polymerase-ß and -
proteins were also elevated. There were no consistent differences in gene expression relating to the nonhomologous end-joining pathway. Despite increased expression of DNA repair genes, malignant prostate cancer cells had defective repair of DNA breaks, alkali-labile sites, and oxidative base damage. Furthermore, after ionizing radiation and mitomycin C treatment, chromosomal aberration assays confirmed that malignant prostate cells had defective DNA repair. This discordance between expression and function of DNA repair genes in malignant prostate cancer cells supports the hypothesis that prostate tumor progression may reflect aberrant DNA repair. Our findings support the development of novel treatment strategies designed to reinstate normal DNA repair in prostate cancer cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Defective DNA repair as a determinant of prostate cancer progression has not been extensively studied. Several groups have observed defective mismatch-repair in prostate cancer cell lines (8 , 9) . Other data support DNA polymorphisms in the Xrcc1, Ogg1, and DNA polymerase-ß genes (involved in base excision repair or DNA single-strand break repair) as risk factors for prostate cancer (8 , 10 , 11) . However, data are lacking concerning the homologous recombination and nonhomologous recombination (i.e., end-joining) pathways, which are involved in the repair of DNA double-strand breaks.
Nonhomologous end-joining repair requires little or no homology on the ends of the strands being joined and involves two main discrete repair protein complexes (1): the DNA-PK/XRCC4/LigIV complex and (2) the MRE11/RAD50 complex (12) . In homologous recombination, extensive homology is required between the region of the DNA double-strand break and the sister chromatid or homologous chromosome from which repair is directed. Homologous recombination involves the BRCA2, RAD51, RAD52, RAD54, RAD55–57, and RPA proteins and the RAD51 paralogs, XRCC2/3 and RAD51B/C/D. The homologous recombination pathway predominates in the late S/G2 phases of the cell cycle and provides relatively error-free repair. In contrast, the nonhomologous end-joining pathway predominates in the G1 phase of the cell cycle (13) . Recent data suggest an interplay between the two pathways dependent on cell type and initial versus late times after induction of DNA double-strand breaks (11) . Cells defective for either homologous recombination or nonhomologous end-joining show increased rates of mutagenesis and chromosomal instability, which could relate to the propensity for acquired genetic instability during prostate carcinogenesis and tumor progression. Consistent with a possible role for DNA double-strand break repair in prostate cancer progression, BRCA1 and BRCA2 mutations are associated with an increased risk of prostate cancer and development of an aggressive disease course (2 , 13, 14, 15) .
Our laboratory has shown previously that normal and malignant prostate cells preferentially respond to DNA damage by undergoing terminal growth arrest rather than apoptosis (16) . This may allow for attempted DNA repair during cell cycle arrests as a response to DNA damage. However, in malignant cells with aberrant cell cycle checkpoint control, defective DNA double-strand break repair could increase genetic instability as part of a "mutator" phenotype (17) . We hypothesized that one of the critical steps in prostate tumor progression may be the loss of the normal repair response to DNA damage and that specific defects in DNA double-strand break repair would be associated with prostate malignancy.
Herein, we show that malignant prostate cancer cells have increased expression of homologous recombination-related and base excision repair-related genes independent of p53 status, G1 cell cycle checkpoint control, and relative cell proliferation. However, despite expressing high levels of DNA repair proteins, malignant cells have a decreased capacity for DNA double-strand break, DNA single-strand break, and base excision repair and acquire discrete chromosomal aberrations after exposure to DNA-damaging agents. Our findings support inappropriate DNA repair as a potential determinant of prostate cancer progression.
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MATERIALS AND METHODS |
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-Modified Eagles Medium, respectively. PrEC (normal prostate epithelial cells) and PrSC (normal prostate stromal cells) were purchased from Clonetics Corporation (San Diego, CA) and maintained using suggested PrEGM and SCGM medium, respectively. The latter cell cultures have limited proliferative potential in culture and decrease in proliferation after passages 5 to 8 from frozen stock. Approximate doubling times for cell cultures under these conditions were as follows: PrEC, 48 to 72 hours; PrSC, 18 hours; LNCaP, 36 hours; PC-3, 24 hours; and DU-145, 18 hours (16)
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Asynchronous cultures were irradiated, or treated with mitomycin C, at 16 to 20 hours after plating to reduce the immediate effects of trypsinization. Cells were either mock irradiated or irradiated with 0 to 10 Gy under aerobic conditions using a 137Cs irradiator at 1 Gy/min at room temperature (16)
. Mitomycin C was prepared as a stock solution of 0.5 mg/mL in distilled water before each use.
Quantification of Gene Expression by RNase Protection Assays.
Asynchronously growing cells were harvested at 70% to 80% growth confluence from either nonirradiated cultures or at 0 to 24 hours after irradiation. RNase protection assays were carried out as per the manufacturer’s instructions (BD Sciences-PharMingen, San Diego, CA). Total RNA was first extracted using Trizol reagent (Life Technologies, Inc.). Antisense riboprobes (Rad50, Mre11, Rad52, Rad54, Rad51, Xrcc2, Xrcc3, Rad51B, Rad51C, Rad51D, L32 and Gapdh from DBSR1 set; Atm, Nbs1, Xrcc2, Xrcc3, Xrcc9, Ligase IV, Xrcc4, Ku70, DNA-PKcs, Ku86, L32, and Gapdh from DBSR2 set; and Bcl-x, p53, Gadd45, c-fos, p21WAF, Bax, Bcl-2, Mcl-1, L32, and Gapdh from HSTRESS set) were synthesized with multiprobe template sets and purified by MAXIscript (Ambion Inc., Houston, TX). Five micrograms of sample RNA were hybridized with 2,000 counts per minute of the synthesized multiriboprobe in a single tube with 10 mL hybridization buffer and digested with RNase centrifuged before acrylamide electrophoresis at 1200 to 1500V for 1 to 2 hours. The gel was then dried at 80C° for 1 hour, and the counts per minute values of the individual bands on the gel were counted using a Storm 840 PhosphorImager (Amersham Biosciences, Piscataway, NJ). The statement level of the Gapdh gene was used as an internal control for relative statement levels of other individual genes calculated as a ratio (i.e., the observed total volume for the target gene divided by the volume of Gapdh). Three different RNA extracts were analyzed in triplicate for each cell line. The results of three independent experiments are expressed as the mean value ±1 SEM. Statistical differences in gene expression were determined using nonparametric Mann-Whitney analyses.
Western Blot Analyses for Relative Expression of Homologous Recombination, Nonhomologous End-Joining, and Base Excision Repair Proteins.
Logarithmically growing cells were either mock-irradiated or irradiated and subsequently lysed on ice for 20 minutes with E7 lysis buffer (PBS, 0.1% SDS, and containing protease inhibitors; Roche Molecular Biochemicals, Indianapolis, IN) for Western blotting as described previously (16)
. SDS-PAGE was performed using 7% to 12% bis-acrylamide gels at room temperature. Each well was loaded with 20 µg of total protein plus loading buffer (final concentration 6% glycerol, 0.83% ß-mercaptoethanol, 1.71% Tris-HCl (pH 6.8), and 0.002% bromphenol blue). Samples were resolved by electrophoresis at 80 to 110 volts for 1.5 to 2.5 hours and then transferred onto nitrocellulose (Schleicher & Schuell Bioscience, Keene, NH). Prehybridization staining with Ponceau solution confirmed equal loading and transfer between running lanes. After transfer, membranes were rinsed with Tris-buffered saline Tween-20, incubated in appropriate secondary antibodies, and protein bands were detected using ECL Detection Reagent (Amersham BioScience) before film exposure. Relative expression was determined by densitometry (Molecular Dynamics Computing ImageQuant, Amersham Sciences) and standardized by -tubulin levels. Primary antibodies were used at dilutions ranging from 1:200 to 1:1,000 as suggested by the supplier and included the following: p21WAF, RAD51, RNase protection assay, and –tubulin (Oncogene Research Products, San Diego, CA); RAD50, NBS1 (p95), XRCC3, RAD51C, and RAD51D (Novus Biologicals, Inc., Littetown, CO), KU70, RAD52, RAD54, KU86, DNA polymerase-ß, and APE/REF1 (Santa Cruz Biotechnology, Santa Cruz, CA); XRCC1 (Abcam, Cambridge, United Kingdom); PARP (Biomol, Plymouth Meeting, PA); DNA polymerase- (BD Biosciences, San Diego, CA); MRE11 (Genetex, San Antonio, TX); and phospho-H3 (Upstate Technologies, Waltham, MA). Relative protein expression among PrSC, LNCaP, DU-145, and PC-3 cells was normalized to PrEC expression (e.g., arbitrary relative value of one). The Western blots shown are representative of at least two independent experiments.
Single-Cell Gel Electrophoresis (Comet Assay) to Detect DNA Strand Breaks and Base Damage.
Repair of DNA double-strand breaks, DNA-single-strand breaks, DNA base damage, and repair of formamidopyrimidine-DNA glycosylase-sensitive sites was quantitated using the Comet assay as previously described (18
, 19
; www.cometassay.com). For the neutral comet assay, 10 and 25 Gy doses were used; for the alkaline comet and formamidopyrimidine-DNA glycosylase-comet assays, a dose of 6 Gy was used. For the determination of DNA double-strand breaks by the neutral comet assay, 105 cells were admixed with 100 µL of 0.7% of low melting-point agarose (Sigma-Aldrich, St. Louis, MI) at 45°C and spread on regular glass microscope slides precoated with 1% normal melting agarose. Slides were then treated overnight with lysis solution [2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Trizma base, 10% DMSO, and 1% Triton X-100 (pH 9.0)] at pH 9.0. The slides were then placed in 1x Tris-buffer boric acid-EDTA and electrophoresed for 15 minutes at 32V (current 25mA). After electrophoresis, the slides were dried and stored until scoring. The slides were finally stained with ethidium bromide (2 ng/mL) and the comets scored under a Zeiss fluorescence microscope coupled to KOMET 5.0 software (Kinetic Imaging, Durham, NC). The alkaline comet assay detects alkali-labile sites in the DNA, which are a global assessment of DNA single-strand breaks, DNA double-strand breaks, and DNA base damage. In brief, cells were suspended in 0.5% low melting-point agarose and spread on glass microscope slides precoated with 1% normal agarose. After immersion in lysis solution (2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Trizma base, 10% DMSO, and 1% Triton X-100) at 4°C for 1 hour to remove cellular proteins, the slides were immersed in electrophoresis buffer [300 mmol/L NaOH, 1 mmol/L EDTA (pH >13)] for unwinding DNA. Finally, single cells were subjected to electrophoresis (25 V; 300 mA) for a total of 20 minutes. Neutralized and dehydrated slides were finally stained with ethidium bromide (2 ng/mL; Sigma-Aldrich) before scoring.
Repair of DNA base damage was scored by treating the DNA with a lesion-specific glycosylase (formamidopyrimidine-DNA glycosylase). This enzyme recognizes oxidative damage as specific DNA base modifications including 8-oxo-7,8-dihydro-2'-deoxyguanosine, 7-methyl-guanine, 5-OH- cytosine, and 5-OH-uracil. Cells were suspended in 0.5% low-melting point agarose and spread on glass microscope slides precoated with 1% normal agarose. Slides were immersed in lysis solution (2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Trizma base, 10% DMSO, and 1% Triton X-100) at 4°C for a minimum period of 1 hour and then equilibrated with enzyme reaction buffer (HEPES 9.5g, KCl 7.5 g, and EDTA 0.5 mmol/L). Bovine serum albumin fraction V was then added [0.2 per mL (pH 8.0)] for 10 minutes on ice. The slides were finally treated with formamidopyrimidine-DNA glycosylase enzyme (obtained from Dr. Karel Angelis, Institute of Experimental Botany, Praha, Czech Republic; 1:100 dilution in enzyme reaction buffer) at 37°C for 30 minutes in a moist box. The enzyme treatment was terminated by dipping the slides in denaturing solution [0.3 mol/L NaOH and 5 mmol/L EDTA (pH 12.1)] for 20 minutes, followed by neutralization in 1x Tris-buffer-boric acid-EDTA for 5 minutes and, finally, electrophoresis. The ratio of DNA migration between the enzyme treated and buffer control slides gives an estimate of the formamidopyrimidine-DNA glycosylase sensitive sites in the sample.
For all of the comet assays, the comet parameter, "Olive Tail Moment" (i.e., % DNA x distance of center of gravity of DNA) was used as the indicator of DNA damage (19) . One hundred consecutive cells were scored at random from the middle of each slide for two to three independent experiments and the final result expressed as the (mean of the median Olive Tail Moment values) ± SEM of the medians. For graphical purposes in the formamidopyrimidine-DNA glycosylase assay, the difference between the Olive Tail Moment for formamidopyrimidine-DNA glycosylase-treated slides versus the Olive Tail Moment of same population treated with buffer alone were plotted as the final end point of formamidopyrimidine-DNA glycosylase-sensitive sites. Statistical differences in Olive Tail Moment values were determined using nonparametric Mann-Whitney analyses.
Chromosomal Aberration Assays.
The frequency of spontaneous, ionizing radiation-induced (4 Gy) or mitomycin C-induced (40 ng/mL for 24 hours) chromosomal aberrations were determined in exponentially growing cell cultures of PrSC, LNCaP, DU-145, and PC-3. The cells were harvested by trypsinization 24 hours after treatment and incubation with 1 µg of Colcemid/mL for 2 hours to collect metaphase spreads for analysis. The cells were fixed, after treatment with hypotonic solution (0.6% sodium citrate), in EtOH-glacial acetic acid (3:1). Air-dried preparations were made and slides were stained 4', 6-diamidino-2-phenylindole/Vectashield antifade mixture (Vector-Laboratories, Burlingame, CA). For chromosomal aberrations, 25 mitotic cells were analyzed for each treatment per cell line.
Immunohistochemistry of Prostate Xenografts.
For xenograft studies, 1 x 106 of PC-3 or DU-145 cells were injected i.m. into the gastronemius muscle (calf of hind leg) of Balb/c-nu mice. Tumors were mock-irradiated or irradiated at a weight of 0.5 g with 20 Gy using a 250KvP X-ray unit without anesthesia. For irradiation, the mouse was lightly restrained in a lucite holder box with lead shielding, such that only the tumor-bearing hind leg was within the irradiation volume. Tumors were then excised and placed immediately in formalin for subsequent fixation and immunostaining using primary RAD51 antibodies (Ab-1, Oncogene Research Products, San Diego, CA) and secondary horseradish peroxidase antibodies for immunodetection. All of the studies were in ethical compliance with the PMH-UHN Animal Care Committee.
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RESULTS |
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PrSC, PrEC, and LNCaP cells express two wild-type p53 alleles and have an intact G1 cell cycle arrest checkpoint with increased p21WAF protein expression after irradiation. PC-3 cells are devoid of p53 protein expression due to chromosome 17p hemizygosity and a mutation in the remaining allele at codon 138 that results in a premature stop codon at position 169. DU-145 cells express high levels of trans-dominant, mutant (MT) p53 proteins due to missense mutations at codons 223 and 274. Both latter cell lines lack a G1 checkpoint and do not show increased p21WAF protein expression after DNA damage (15)
. Using the RNase protection assay analyses at 4 hours after 10 Gy, we observed increased expression of p21WAF, Gadd45, Bax, and Bcl-2 genes in the WTp53-expressing cells (i.e., PrSC, PrEC, and LNCaP), whereas similar increases in gene expression were not observed for the null-p53 and MTp53-expressing, PC-3 and DU-145 cells (see Fig. 1A and B
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inset and data not shown). We conclude that the RNase protection assay is a sensitive indicator of gene expression under the conditions of DNA damage and repair within our panel of prostate cell cultures.
Expression and Functional Assessment of DNA Double-Strand Break and DNA Single-Strand Break Repair Proteins in Prostate Cultures.
The initial data relating to the Atm/p53 stress response provided confidence in the use of RNase protection assay for quantification of gene expression relating to the nonhomologous (nonhomolo-gous end-joining) and homologous recombination pathways of DNA double-strand break repair. We observed a significant and differential increase (approximately 2–3 fold) in homologous recombination associated genes (i.e., Rad51, Rad54, Xrcc3, and Rad52) in malignant prostate cell cultures at both the RNA and protein levels when compared with PrEC cells (see Fig. 2
). Gene expression relating to the MRN complex (Mre11/Rad50/Nbs1-p95) was not differentially expressed. We also found little evidence for increased basal mRNA or protein expression of the nonhomologous end-joining-related Ku70, Ku80, DNA-PKcs, XRCC4, or Ligase IV genes (see Fig. 3A
and data not shown). We observed elevated levels of the DNA single-strand break repair protein XRCC1 in malignant cultures (Fig. 3A)
. In contrast to a previous report (20)
, homologous recombination and nonhomologous end-joining gene expression at both the RNA and protein levels was invariant after irradiation (Fig. 2A
and data not shown). We conclude that the endogenous expression of the homologous recombination-related Rad51, Rad52, Rad54, Xrcc3, and Xrcc1 genes are increased at RNA and/or protein levels in malignant prostate cell lines.
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. Despite similar amounts of initial DNA damage, the malignant cultures had significantly decreased capacity in repairing ionizing radiation-induced DNA damage. These data suggest that despite high levels of homologous recombination-related and XRCC1 proteins, malignant prostate cells are defective in the rejoining of DNA double-strand breaks and alkali-labile sites (the latter reflecting DNA single-strand breaks and DNA base damage).
Base Excision Repair in Malignant Prostate Cultures.
As the alkaline Comet assay also scores abasic sites, we used the formamidopyrimidine-DNA glycosylase Comet assay to directly determine whether the malignant cultures had relatively increased spontaneous and ionizing radiation-induced levels of oxidative damage. Treating DNA with formamidopyrimidine-DNA glycosylase unmasks nonrepaired oxidative damage as 8-oxo-7,8-dihydro-2'-deoxyguanosine, 7-methyl-guanine, 5-OH-cytosine, or 5-OH-uracil DNA lesions (19)
. The results shown in Fig. 4A
are consistent with a decreased capacity for base excision repair of these lesions in the malignant cultures. This defect was not related to decreased levels of the base excision repair-related p53, APE/REF1, or OGG1 proteins. In fact, the malignant cells lines had increased levels of DNA-polymerase-ß and -, two key enzymes involved in short-patch and long-patch base excision repair (ref. 21
; Fig. 4B and C
). We also assessed whether increased expression of homologous recombination-related or base excision repair-related proteins could be secondary to cell cycle bias given that these proteins are optimally expressed in S and G2 phases of the cell cycle (13)
. In comparing the cell culture doubling times in vitro with mitotic and S-phase biomarkers (phosphorylated-histone 3 and RNase protection assay proteins, respectively), we did not observe any correlation between increased DNA repair protein expression and cell proliferation indices among the five cultures (Fig. 4B)
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show representative structural cytogenetic aberrations, which are quantified in Table 1
for four of the five cultures. The protracted doubling time of PrEC cells precluded their assessment in this assay. All three malignant cell lines show an increased incidence of a variety of aberrations associated with aberrant homologous recombination occurring in the S and G2 phases of the cell cycle phases including: chromatid breaks; double minutes; tri-, quad-, and complex-radial chromosomes; abnormal telomeric associations; and centromere fissions. Furthermore, nonhomologous end-joining–associated defects occurring within the G1 cell cycle phase were also observed as increased chromosomal breaks and di- or tricentric chromosomes in malignant cultures. These data support our hypothesis that defects in DNA double-strand break repair can be observed in malignant cultures and augment our findings using the Comet assay.
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). Similar to a previous report in pancreatic cancer (23)
, we observed intratumoral heterogeneity with respect to endogenous RAD51 expression. Both cytoplasmic and nuclear RAD51 expression was observed in PC-3 and DU-145 xenograft histologic sections. However, in contrast to our data in which RAD51 expression in vitro was invariant after DNA damage, nuclear RAD51 expression increased in the PC-3 or DU-145 xenografts irradiated under in vivo conditions. Taken together, our data suggest that RAD51 expression in vivo can be additionally modified by intratumoral biology and physiology with the three-dimensional tumor architecture. |
DISCUSSION |
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There have been previous reports of elevated levels of Rad51 mRNA or RAD51 protein expression within human tumor cell lines (29 , 30) . However, to our knowledge, this is the first direct comparison of DNA double-strand break gene expression within normal and malignant cells from the same histopathologic type linked to a functional assessment of DNA double-strand break repair. Altered stoichiometry of repair proteins or a disconnect between cell cycle checkpoint control and DNA repair may be the basis for the observed discordance between repair protein expression and function (13 , 31) . Altered homologous recombination-related protein expression might indirectly lead to altered nonhomologous end-joining activity given the interplay between the two pathways during DNA double-strand break repair (32 , 33) . Maintenance of survival in malignant cells, despite high levels of DNA double-strand breaks and chromosomal aberrations after DNA damage, is probably secondary to loss of potentially deleterious accentric fragments or other chromosomal abnormalities within micronuclei at 48 to 72 hours after irradiation (34 , 35) .
In isogenic systems, the relationship among RAD51 expression, homologous recombination, and induction of chromosomal aberrations remains controversial (29 , 36, 37, 38) . This may relate to variability in genetic background or the plasmid homologous recombination reporter substrates used for study (22) . Transfection studies with forced overexpression of RAD51 have led to observations of both increased and decreased frequencies of homologous recombination, arguing for RAD51 acting as either a promoter or repressor of genetic instability and tumor progression (37 , 39) . Other transfection studies suggested that RAD51, XRCC3, and XRCC1 protein overexpression leads to increased p21WAF expression and a decreased apoptotic response with resulting radioresistance (23 , 40 , 41) . However, our data do not support such a direct correlation. Our malignant cultures, which overexpress homologous recombination-related proteins have varying G1 checkpoint control and p21WAF expression, and all five of the cultures are resistant to apoptosis (16) . The increased DNA repair protein expression observed in the malignant cell lines was independent of p53 status, G1-checkpoint control, androgen responsiveness, clonogenic radiation cell survival, cell proliferation, and susceptibility for radiation-induced apoptosis (16 , 20 , 42) .
It had also been hypothesized that RAD51 overexpression might abrogate RAD51-p53 interactions and override the G1 checkpoint leading to aneuploidy and high levels of homologous recombination (37 , 43) . The decreased levels of MMS- and ionizing radiation-induced homologous recombination-related chromosomal aberrations in the G1 checkpoint-proficient LNCaP cultures, relative to the G1 checkpoint-deficient DU-145 and PC-3 cultures, would support this hypothesis. Additionally, our observed overexpression of the Rad51, Xrcc3, Rad52, and Rad54 genes at both the RNA and protein level suggests that loss of control of homologous recombination expression in malignant cells is operational at the transcriptional level and may be secondary to altered activity of transcription factors in cancer cells. Rad51 gene overexpression can be mediated by Bcr/Abl and STAT5-dependent transcription in addition to inhibition of caspase-3-dependent RAD51 protein cleavage (44 , 45) . Similar mechanisms may exist for other homologous recombination-related genes in prostate cancer. Indeed, preliminary data in our laboratory suggests that endogenous BRCA1 and BRCA2 protein expression are also elevated in the three malignant cell lines.5
Our results implicate DNA repair, and particularly DNA double-strand break repair and homologous recombination, as a potential factor in prostate tumor progression. Importantly, our data may have implications for both prostate cancer diagnosis and therapy. Profiling of DNA repair protein expression may be useful to discover new biomarkers of genetic instability, malignant progression, and aggressive tumor phenotypes (46 , 47) . We would also hypothesize that prostate intraepithelial neoplasia (PIN) may have altered frequencies of homologous recombination and defective DNA double-strand break repair when compared with nonmalignant epithelium. Unfortunately, the paucity of cell lines for the study of prostatic intraepithelial neoplasia or early stage prostate cancer prevents us from making this direct link in vitro (48) .
Prostate tissue arrays will be useful to confirm that our in vitro findings are operational in vivo and that loss of checkpoint control and DNA repair activity is correlated to tumor progression similar to that reported for breast cancer (49)
. However, the data presented in Fig. 5B
suggest that interpretation of tissue arrays may be difficult without added information pertaining to cell cycle phase, oxygenation, nutritional status, or clonal variation, which may affect the intratumoral heterogeneity of protein expression. For example, the relative increase in RAD51 expression after irradiation in vivo may be secondary to the arrest of cells in the G2 phase of the cell cycle, which has higher levels of RAD51 expression (50)
, or due to microenvironmental factors. Indeed, in a separate study, we have observed that RAD51 expression can be altered under conditions of intratumoral hypoxia (51)
. Additional experiments with defined doses and time points after irradiation in vivo using prostate xenografts may clarify these issues and glean more information regarding RAD51 function within solid tumors.
We speculate that the use of agents that either augment DNA repair in premalignant prostate epithelium or inhibit the hyper-recombinogenic phenotype of malignant cells may be valuable in prostate cancer treatment. For example, the antioxidant selenium has been implicated as a chemoprotective agent for high-risk prostate cancer and has been shown recently to increase DNA repair and chemoprotect normal cells in response to DNA damaging agents (52) . Additionally, molecular-targeted agents, which decrease aberrant Rad51 or other DNA repair gene expression, such as STI571-Gleevec (45 , 53) or small interfering RNA approaches (54) , may be potentially novel strategies to sensitize prostate cancer cells to radiotherapy or chemotherapy (55 , 56) .
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FOOTNOTES |
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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.
Note: R. Fan and T. S. Kumaravel contributed equally to this work; T. S. Kumaravel is currently at the Genetic and Molecular Toxicology, Covance Labs Limited, Otley Road, Harrogate HG3 2XQ, United Kingdom. Supplementary data for this article can be found at Cancer Research Online (http://cancerres.aacrjournals.org).
Requests for reprints: Robert G. Bristow, Radiation Medicine Program, Princess Margaret Hospital (UHN), 610 University Avenue, Room 5–923, Toronto, Ontario, Canada M5G2M9. Phone: 416-946-2129; Fax: 416-946-4586; E-mail: rob.bristow{at}rmp.uhn.on.ca
5 R. Fan, F. Jalali, and R. G. Bristow, unpublished observations. 
Received 5/10/04. Revised 8/17/04. Accepted 9/28/04.
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S. Vijayakumar, D. C. Hall, X. T. Reveles, D. A. Troyer, I. M. Thompson, D. Garcia, R. Xiang, R. J. Leach, T. L. Johnson-Pais, and S. L. Naylor Detection of Recurrent Copy Number Loss at Yp11.2 Involving TSPY Gene Cluster in Prostate Cancer Using Array-Based Comparative Genomic Hybridization. Cancer Res., April 15, 2006; 66(8): 4055 - 4064. [Abstract] [Full Text] [PDF]
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R. S. Bindra, S. L. Gibson, A. Meng, U. Westermark, M. Jasin, A. J. Pierce, R. G. Bristow, M. K. Classon, and P. M. Glazer Hypoxia-Induced Down-regulation of BRCA1 Expression by E2Fs Cancer Res., December 15, 2005; 65(24): 11597 - 11604. [Abstract] [Full Text] [PDF]
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T. Shiraishi, T. Yoshida, S. Nakata, M. Horinaka, M. Wakada, Y. Mizutani, T. Miki, and T. Sakai Tunicamycin Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Human Prostate Cancer Cells Cancer Res., July 15, 2005; 65(14): 6364 - 6370. [Abstract] [Full Text] [PDF]
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 G. L. Mayeur, W.-J. Kung, A. Martinez, C. Izumiya, D. J. Chen, and H.-J. Kung Ku Is a Novel Transcriptional Recycling Coactivator of the Androgen Receptor in Prostate Cancer Cells J. Biol. Chem., March 18, 2005; 280(11): 10827 - 10833. [Abstract] [Full Text] [PDF]
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