Cancer Research Meeting Calendar Advances in Breast Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 66, 10302, November 1, 2006. doi: 10.1158/0008-5472.CAN-06-0638
© 2006 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.
Right arrow Articles by Liu, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by Liu, W.

Molecular Biology, Pathobiology, and Genetics

Truncating Variants in p53AIP1 Disrupting DNA Damage–Induced Apoptosis Are Associated with Prostate Cancer Risk

Xianshu Wang1, Fengwei Wang1, Ken Taniguchi1, Ratnam S. Seelan1, Liang Wang1, Katherine E. Zarfas2, Shannon K. McDonnell2, Chiping Qian1, Kaifeng Pan4, Youyong Lu4, Viji Shridhar1, Fergus J. Couch1, Donald J. Tindall3, Jennifer L. Beebe-Dimmer5, Kathleen A. Cooney5, William B. Isaacs6, Steven J. Jacobsen2, Daniel J. Schaid2, Stephen N. Thibodeau1 and Wanguo Liu1

Departments of 1 Laboratory Medicine and Pathology, 2 Health Sciences Research, and 3 Urology, Mayo Clinic/Mayo Clinical Medical College, Rochester, Minnesota; 4 Laboratory of Molecular Oncology, Peking University School of Oncology, Beijing Institute for Cancer Research, Beijing Cancer Hospital, Beijing, China; 5 Departments of Internal Medicine and Urology, University of Michigan, Medical School, Ann Arbor, Michigan; and 6 Brady Urological Institute, Johns Hopkins Medical Institution, Baltimore, Maryland

Requests for reprints: Wanguo Liu, Division of Experimental Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic/Mayo Medical School, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-266-0508; Fax: 507-266-5193; E-mail: liu.wanguo{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Germ line mutations in several genes (BRCA1, BRCA2, and CHEK2) whose products are involved in the DNA damage–signaling pathway have been implicated in prostate cancer risk. To identify additional genes in this pathway that might confer susceptibility to this cancer, we analyzed a recently identified DNA damage–response gene, p53AIP1 (a gene encoding for p53-regulated apoptosis-inducing protein 1), for genetic variants in prostate cancer. Five novel germ line variants were identified. The two truncating variants (Ser32Stop and Arg21insG) were found in 3% (4 of 132) of unselected prostate tumor samples. Genotyping of the two variants in an additional 393 men with sporadic prostate cancer showed a frequency of 3.1% (12 of 393) in contrast to 0.6% (2 of 327) in 327 unaffected men (Fisher's exact test, P = 0.018), with an odds ratio (OR) of 5.1 [95% confidence interval (95% CI), 1.1-23.0]. In addition, two of six tumors carrying the truncating variants were associated with loss of heterozygosity of the wild-type alleles, suggesting that p53AIP1 may act as a tumor suppressor. We also showed that the truncated p53AIP1 was unable to induce apoptosis and suppress cell growth in HeLa and COS-7 cells. These results suggest that loss-of-function variants in p53AIP1 associated with the risk of sporadic prostate cancer and further support the concept that the genetic defects in the DNA damage–response genes play an important role in the development of prostate cancer. (Cancer Res 2006; 66(21): 10302-7)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Many lines of evidence have shown that genetics play an important role in the development of prostate cancer (1). Studies in the last several years have suggested that a number of rare, highly penetrant loci may contribute to Mendelian inheritance of prostate cancer and that other genetic alterations contributing to the majority of non-Mendelian inheritance of prostate cancer are likely to be multiple low-penetrance alleles (2). These alleles could bear function-associated variants in the regulatory genes, such as the androgen receptor gene or in genes associated with certain signaling pathways that are involved in prostate tumorigenesis (3, 4). The DNA damage–signaling pathway is essential for the prevention of genomic instability, a common feature of all human cancers including prostate cancer. Germ line variants in several key components of this pathway (BRCA1, BRCA2, and NBS1) have been shown to associate with prostate cancer risk (5–7). We, along with others, recently reported that variants in CHEK2 have an increased risk for the male carrier to develop prostate cancer (8, 9). These studies suggest that integrity of the DNA damage–signaling pathway is crucial for prevention of neoplastic transformation in the prostate and that genes participating in the DNA damage–signaling pathway could be targets for mutations in prostate tumorigenesis.

P53AIP1 is a downstream target of p53 and is induced by DNA damage (10). This gene has three transcripts ({alpha}, ß, and {gamma}). The {alpha} and ß forms of p53AIP1 are localized to the mitochondria and induce apoptosis through dissipation of mitochondria membrane potential. The expression of p53AIP1 and p53AIP1-induced apoptosis are closely correlated with phosphorylation of p53 at Ser46, indicating that p53AIP1 plays an important role in mediating p53-dependent apoptosis. Moreover, p53AIP1 interacts with bcl-2, an inhibitor of apoptosis, in mitochondria (11). These findings suggest that p53AIP1 is crucial for regulation of p53-dependent DNA damage–signaling pathways and disruption of the function of p53AIP1 in p53-mediated apoptosis could play an important role in cancer development. Because the frequency of p53 mutations in prostate cancer is much lower than in other cancers, p53AIP1 could be another candidate mutation target in prostate cancer.

In this study, we reported the identification of novel truncating variants in the p53AIP1 gene in prostate cancer and showed that the truncating variant carriers had increased risk of developing prostate cancer. We also presented the results of functional analysis of the p53AIP1 truncating variants in cultured mammalian cells to further support the genetic evidence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Tumors and patients. The 132 prostate tumor specimens used in this study were unselected and collected at the Mayo Clinic (Rochester, MN) between 1997 and 1998. All of the specimens were obtained by surgery, quickly frozen in liquid nitrogen, and transferred to –80°C freezers until further analysis. DNA was extracted from cryocut slices of frozen tissues with Easy-DNA kits (Invitrogen, Carlsbad, CA).

The group of patients with sporadic prostate cancer (n = 393) has been previously described in our other gene-association studies, with a reduction of seven cases because of availability at the time the study was conducted (8, 12). These patients were selected from respondents to a family history survey that reported no family history of prostate cancer (13, 14). The diagnosis of prostate cancer was confirmed by pathology reports. Prostate-specific antigen (PSA) values at diagnosis were available for 317 men, with a median value of 7.1 and with 76% having values of ≥4. The median age of diagnosis for this group was 65.0 years (range 46.0-79.0 years).

The familial cases from Mayo Clinic, University of Michigan, and Johns Hopkins University, were ascertained as described elsewhere (15–17). The familial prostate cancer families were defined as those that had occurrence of prostate cancer in a minimum of three men over at least two generations. The familial cases participated in this study were independent affected men from these families. The mean age of diagnosis for Mayo cases was 66.5 years, for Michigan cases 64.1 years, and for Johns Hopkins cases 64.3 years.

The unaffected control group (n = 327, all male) has also been described in our previous publications, although four controls have been dropped from the original group of 331 due to the occurrence of prostate cancer (8, 12). These control subjects were recruited from a sampling frame of the local population provided by the Rochester Epidemiology Project (18): 475 men were randomly selected for a clinical urologic examination (19). This clinical examination included digital rectal examination and transrectal ultrasound of the prostate, abdominal ultrasound for postvoid residual urine volume, measurement of serum levels of PSA and creatinine, focused urologic physical examination, and cryopreservation of serum for subsequent sex hormone assays. Any patient with an abnormal digital rectal examination, elevated serum PSA level, or suspicious lesion on transrectal ultrasound was evaluated for prostatic malignancy. Those men who were found to be without prostate cancer on the basis of this extensive workup at baseline or at any of the follow-up examinations through 1994 were used for the control population. The median age of these men in the control group was 59.6 years (range 49.0-89.0 years). All of the participants in this study gave full informed consent and were approved by the Mayo Institutional Review Board.

Mutation detection and loss of heterozygosity analysis. The primers used to amplify three p53AIP1 exons are p53AIP1e2F/R (5'-AAATGAGGAGAAGCCAAGTT-3' and 5'-CGGCACCACGGTGAGA-3'), p53AIP1e3F/R (5'-AACCATCCAAGAGACGG-3' and 5'-ATCACTTAATTCTATCACGG-3'), and p53AIP1e4F/R (5'-AAGGACTCCATACGTTTTGC-3' and 5'-GCTGGAGCCATTTCTCGAC-3'). PCR, denaturing high-performance liquid chromatography analysis, and direct sequencing were done as previously described (8, 20). Loss of heterozygosity (LOH) analysis was done on six pairs of tumor and matched normal tissues. Basically, flash-frozen prostate tumor tissues embedded in optimum cutting temperature were sliced at 10 µm onto microscope slices (Gold Seal Products, Portsmouth, NH) and H&E stained. After dehydrating, slides were placed on a PixCell II LCM stage (Arcturus Engineering, Mountain View, CA). A CapSure LCM cap was placed on the area of interest on the tissue. A low-power IR laser was pulsed at 65 mW for 1.2 ms to activate the transfer film resulting in adherence of the desired cells to the cap. After collecting 2,000 to 4,000 cells, the CapSure LCM film was placed into a sterile 0.6 mL centrifuge tube for DNA isolation using Easy-DNA kits (Invitrogen). Exon 2 of p53AIP1 was PCR amplified from the genomic DNA isolated from tumor and matched normal tissue and then directly sequenced.

Plasmid construction and transfection. Wild-type (wt) and mutant (mut) p53AIP1 cDNAs were PCR amplified with primers containing hemagglutinin (HA) sequence tags and cloned into PCI (Promega, Madison, WI) or IRES-enhanced green fluorescent protein (IRES-EGFP; Clontech, Mountain View, CA) vectors. HeLa, COS-7, Saos-2, and T98G cells were grown in DMEM/10% FCS supplemented with antibiotics. Transient transfections were done when cells reached 60% to 80% confluence either in culture dishes or on slides with Superfect (Qiagen, Valencia, CA) or FuGENE 6 (Roche, Indianapolis, IN).

Immunoprecipitation, Western blotting, and immunostaining. These procedures were followed as previously described (21). Briefly, to immunoprecipitate the ectopically expressed protein, whole cell lysates were obtained using Beach lysis buffer. The beads conjugated with anti-HA mouse monoclonal antibody (clone 12CA5, Roche) were mixed with cell lysates to pull down the recombinant protein. The precipitated protein was resolved on 15% SDS-PAGE gels and transferred onto polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The blot was then stained with 12CA5 and visualized by ECL (Amersham Biosciences, Piscataway, NJ). For the immunostaining of wt- and mut-HA-p53AIP1 protein, 12CA5 and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) were used for the primary and secondary staining. To label the mitochondria with mitotracker CMXRos (Invitrogen), cells were first incubated with 100 nmol/L fluorescent probe in the culture medium for 30 minutes in the incubator just before fixation. The indirect immunofluorescence was visualized and recorded on a Carl Zeiss Confocal Laser Scanning Microscope LSM510.

Colony formation analysis and terminal deoxyribonucleotide transferase–mediated nick-end labeling assay. Wt- and mut-p53AIP1 expression constructs in IRES-EGFP (Clontech) vectors were transfected into COS-7 cells. EGFP-positive cells were sorted 48 hours after transfection and seeded in 3.5 cm dishes with equal numbers. Colonies of cells were fixed in ethanol and stained with crystal violet in ~10 days. The plates were photographed and the number of colonies was counted. Terminal deoxyribonucleotide transferase–mediated nick-end labeling (TUNEL) staining was done on T98G cells transfected with wt- and mut-p53AIP1 expression constructs in PCI vectors. After immunostaining of p53AIP1 by 12CA5 antibody, the nuclear DNA double-strand breaks were labeled with TMR-red–labeled nucleotides catalyzed by terminal deoxynucleotidyl transferase (In situ Cell Death Detection kit, TMR red; Roche) and analyzed by fluorescence microscopy.

Cytochrome c release by immunofluorescence staining. For cytochrome c immunostaining, COS-7 cells were cultured on slides and transfected with wt- and mut-p53AIP1 expression constructs in IRES-EGFP vectors. After 48 hours, cells were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 for 10 minutes at room temperature. Cells were then stained with primary mouse monoclonal anticytochrome c antibody (clone 6H2.B4; BD, Franklin Lakes, NJ; 1:100) followed by secondary antibody Alexa Fluor 568 goat anti-mouse IgG (1:2,000).

Statistical analysis. The association of p53AIP1 truncating mutations (Ser32Stop and Arg21insG) with prostate cancer was evaluated using Armitage's test for trend in the number of variant alleles (22).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
In this study, we first screened the p53AIP1 gene for sequence variants in 132 primary prostate tumor specimens. The entire coding and exon/intron junction sequences of this gene were analyzed. Three nonsynonymous variants (C20T, Ala7Val; A304G, Arg102Glu; and C313T, Pro105Ser) and two truncating variants (C95A, Ser32Stop and 64insG, Arg21insG) were identified (Fig. 1 ). The two truncating variants were present in 3% (4 of 132) of the tumor samples (Table 1 ). The three nonsynonymous variants were present in 4.5% (Ala7Val), 40.9% (Arg102Glu), and 40.2% (Pro105Ser) of prostate tumor specimens, respectively. All five variants identified in p53AIP1 are germ line variants because they were present in both tumor and matched normal tissues. To further evaluate the significance of the two p53AIP1 truncating variants in prostate cancer susceptibility, we assessed their frequencies in an additional 393 men with sporadic prostate cancer and in 327 population-based controls. The two truncating variants were detected in 12 (3.1%) sporadic prostate cancer cases and in two (0.6%) controls (Table 1), suggesting that men who carry a germ line p53AIP1 truncating variant have a 5-fold increased risk of developing prostate cancer (OR, 5.1; 95% CI, 1.1-23.0; P = 0.018).


Figure 1
View larger version (27K):
[in this window]
[in a new window]
  		Figure 1. Genetic analysis of the p53AIP1 gene. Identification of two novel p53AIP1 truncating variants (B, C) and three nonsynonymous variants (A, D, E) in prostate tumor specimens. Top, electropherograms displaying the wild-type sequence. Bottom, electropherograms displaying the sequence variants. Arrows, location of the sequence mismatches. Indication of LOH in two prostate cancer specimens carrying p53AIP1 truncating variants (F). LCM was used to isolate malignant cells from the two prostate tumor specimens. Arrows, loss of the wild-type allele.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Frequencies of p53AIP1 mutations in patients with prostate cancer, nonprostate cancers, and unaffected control subjects

 
We also examined the p53AIP1 variants in other types of tumors by genotyping the two truncating variants and the C20T (Ala7Val) variant in 403 tumor specimens including 127 breast cancer, 110 ovarian cancer, 72 gastric cancer, and 94 neuroblastomas (Table 1). Although the C20T variant was detected in these cancers with a similar frequency as detected in prostate cancer, we did not detect the two truncating variants in any of these nonprostate tumors, suggesting that the p53AIP1 truncating variants are probably specific for prostate cancer. However, analysis of additional tumor types and more samples for each type will be required to determine whether the p53AIP1 truncating variants are unique to prostate cancer.

p53AIP1 is localized in mitochondria, leading to apoptosis through disruption of the membrane potential (dissipation of mitochondrial {Delta}{Psi}m; ref. 10). To determine whether the truncating variants detected in prostate cancer would disrupt the normal function of p53AIP1, we generated expression constructs containing both {alpha} and ß forms of wt- and mut-p53AIP1 all with HA-tags (designated as FLAG-p53AIP1{alpha}-HA, p53AIP1ß-HA, p53AIP1{alpha}-Arg21insG-HA, p53AIP1ß-Arg21insG-HA, and p53AIP1{alpha}-Ser32Stop-HA) for transient expression in HeLa cells. Both wild-type p53AIP1 and Arg21insG mutant proteins were detected (Fig. 2A ), except for Ser32Stop, which is likely too small (only 31 amino acids) to be stably expressed in the cells. Subsequent coimmunostaining of these recombinant proteins and mitochondria revealed that {alpha} forms of both wt-p53AIP1 and Arg21insG mutant were localized to the mitochondria (Fig. 2B). However, unlike wt-p53AIP1, the Arg21insG mutant was unable to disrupt the mitochondrial membrane potential as indicated by clear mitotracker CMXRos labeling of mitochondria. These results indicate that the mut-p53AIP1 without COOH terminus is able to translocate to mitochondria but unable to disrupt mitochondrial membrane potential.


Figure 2
View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. Ser32Stop and Arg21insG of p53AIP1 are loss-of-function variants in programmed cell death. A, ectopic expression of wt- and mut-p53AIP1 in HeLa cells. The expression vector alone was used as the negative control. B, mitochondrial localization of mut-p53AIP1 Arg21insG and membrane potential. Top, HeLa cells transfected with wt-p53AIP1{alpha} and stained with 12CA5 (anti-HA) antibody (green), mitotracker CMXRos (red), and Hoechst (blue). The fourth image is a merged picture to show disrupted mitochondrial membrane potential. Bottom, representative HeLa cells overexpressing mut-p53AIP1{alpha}-Arg21insG (green). The fourth merged image shows colocalization of the mutant protein (green) and the mitotracker (red), indicating that the membrane potential is preserved. Similar results were obtained when ß-form of mut-p53AIP1-Arg21insG was expressed in HeLa cells (data not shown).

 
Because disruption of the mitochondrial membrane potential followed by cytochrome c release was suggested as the mechanism of p53AIP1-induced apoptosis (11), we did TUNEL assays on T98G cells transfected with {alpha} form of wt- or mut-p53AIP1. As shown in Fig. 3A , the cells expressing wt protein produced 3- to 4-fold more dead cells shown with positive TMR red staining than those expressing mutant protein. We then transfected COS-7 cells with expression constructs of wt-p53AIP1 and Arg21insG in IRES-EGFP vectors that can express our target protein and EGFP simultaneously and immunostained these cells with anti-cytochrome c antibody. As shown in Fig. 3B, the cytochrome c staining in mitochondria is retained in mut-p53AIP1–expressing cells (EGFP-positive cells; Fig. 3B, top), but not in wt-p53AIP1 transfectants (EGFP-positive cells; Fig. 3B, bottom). These results show that mut-p53AIP1 is unable to induce cytochrome c release in mitochondria and therefore unable to trigger programmed cell death.


Figure 3
View larger version (39K):
[in this window]
[in a new window]
  		Figure 3. Function of wt- and mut-p53AIP1 in apoptosis and cell growth. A, TUNEL assays of wt- and mut-p53AIP1{alpha} Arg21insG in T98G cells. Mut-p53AIP1 was tagged with a COOH-terminal HA epitope and wt-p53AIP1 with both an NH2-terminal FLAG and a COOH-terminal HA epitope. The cells were immunostained with anti-HA (12CA5) antibody and then labeled with TMR red fluorescence. Expression-positive and tunnel-positive cells are indicated by FITC green fluorescence and TMR red fluorescence, respectively. Images were taken on a Carl-Zeiss Axiovert II fluorescence microscope. B, p53AIP1 mutant loses the ability to induce cytochrome c release from mitochondria in COS-7 cells. Fixation and staining of the cells were done 24 hours after transient transfection of wt- and mut-p53AIP1 in IRES-EGFP vectors as described in Materials and Methods. The transfected cells were those with green fluorescence of EGFP. Red fluorescence, cytochrome c staining. Top, cytochrome c release in COS-7 cells transfected with wt-p53AIP1 expression construct. Arrows, diffused cytochrome c staining. Bottom, cytochrome c staining in COS-7 cells transfected with mut-p53AIP1construct. Arrows, cytochrome c retained in mitochondria. C and D, effects of wt- and mut-p53AIP1 on cell growth as shown by the colonies in culture dishes after crystal violet staining. COS-7 cells were transfected with the indicated p53AIP1 expression constructs and sorted by flow cytometry. Control cells were transfected with IRES-EGFP vector alone to serve as a negative indicator of cell growth suppression. Expression of wt-p53AIP1 in COS-7 cells was used as a positive control. The colonies for each construct were counted and expressed as percentage of colony formation efficiency. Columns, average mean of three experiments.

 
We did colony formation assays to further evaluate the role of mutant p53AIP1 in cell growth. COS-7 cells were transfected with expression constructs of wt-p53AIP1, mut-p53AIP1 of Arg21insG, and Ser32Stop in IRES-EGFP vectors, and the EGFP-positive cells were enriched by flow cytometry and continuously cultured for 10 days. As expected, expression of wt-p53AIP1 in COS-7 cells strongly suppressed cell growth indicated by significantly less and smaller colonies than vector control. In contrast, the number and size of colonies in the dishes for mut-p53AIP1 (Arg21insG or Ser32Stop) are comparable with the vector control (Fig. 3C and D). These data showed that truncated p53AIP1 lost the ability to suppress cell growth.

In this study, we provide evidence, for the first time, that p53AIP1 is mutated in prostate cancer and the men carrying the p53AIP1 truncating variants have increased risk of developing prostate cancer. Although we estimate the risk to be high, with an OR of 5.1, the rarity of the variants translates to a wide confidence interval for this risk, 1.1 to 23.0. Larger studies are required to refine this risk estimate. Other than the two truncating variants, we also identified three nonsynonymous variants in this gene. The Ala7Val variant is present in both cases and controls with a similar frequency. The ectopically expressed Ala7Val variant localizes to mitochondria and dissipates mitochondrial membrane potential (data not shown). Thus, it is very likely a nonsynonymous polymorphism. Two other variants at the very COOH terminus of p53AIP1 showed very high frequencies in prostate cancer patients. However, the roles of these two variants in prostate cancer susceptibility remain to be elucidated.

The functional analyses of the two p53AIP1 truncating variants provide some insights into the possible mechanism underpinning the association of these variants with increased risk of prostate cancers. Our results show that Ser32Stop and Arg21insG are loss-of-function variants in terms of inducing apoptosis. These deleterious variants may decrease the capacity of prostatic cells to undergo apoptosis after DNA damage and, hence, these damaged cells with accumulating somatic mutations may eventually survive and develop into tumor clones. On the other hand, we analyzed six available tumors carrying the p53AIP1 truncating variants and loss of wt-p53AIP1 alleles was observed in two of them (Fig. 1F), indicating that p53AIP1 may function as a tumor suppressor. Collectively, these results from our combination of genetic and functional studies suggest that p53AIP1 is a prostate cancer susceptibility gene and truncating variants in this gene severely impair its function to induce apoptosis, probably causing tumorigenesis in patients with prostate cancer.

To explore the possibility that the two truncating variants of p53AIP1 might play a role in familial prostate cancer, we analyzed a total of 981 affected men from 426 families with prostate cancer, including 160 families collected at Mayo Clinic, 142 at Johns Hopkins, and 124 at the University of Michigan (15–17). The association of p53AIP1 truncating mutations with familial prostate cancer was evaluated using a test for trend in the number of variant alleles, analogous to Armitage's test for trend in proportions, yet with the appropriate variance to account for correlated family data (23). The frequencies of the two truncating variants among familial cases were higher than that in controls, with frequencies of 1.2%, 0.8%, and 2.7% detected among the familial prostate cancer cases from Mayo Clinic, Johns Hopkins, and University of Michigan, respectively (Table 2 ). None of these frequencies was significantly different than the frequency observed in controls (0.6%). However, all familial cases show a slightly increased risk (OR > 1) compared with controls. The frequency of the variant carriers was not statistically different among the three groups of familial cases (P = 0.19), nor when contrasted with the sporadic cases (P = 0.10), although the power to detect differences is weak because of the rarity of the variant. A test of differences among the ORs contrasting familial cases versus controls was not statistically significant (P = 0.5), and the Mantel-Haenszel stratified OR comparing all familial cases to controls was 2.5 (95% CI, 0.98-6.62). Other genes may play a more prominent role for prostate cancer susceptibility in the familial cases.


View this table:
[in this window]
[in a new window]
  		Table 2. P53AIP1 germ line mutations in men with prostate cancer and in unaffected men

 
The results from this study and our previous study of CHEK2 in prostate cancer suggest that the deleterious variants in the DNA damage–response pathway genes are low-risk factors for prostate cancer (8). The "minor" contribution of these variants most likely reflects the extensive heterogeneity, both genetically and etiologically, that is known to characterize the factors that affect risk for prostate cancer (2). This heterogeneity aspect most likely explains the lack of association with familial prostate cancer cases. That familial cases are not associated emphasizes that, as of yet, unidentified risk factors are accounting for the observed familial clustering of prostate cancer. Presumably, the familial cases are enriched in these unidentified factors to the extent that the effect of P53AIP1 in this group is diluted, resulting in a nonsignificant level. However, it is important to point out that the risk contribution of the rare variants in each of the DNA damage–response genes is relatively low; the cumulative risk contribution of these variants in the same pathway could be high in the population if they are independent. Thus, identification of this type of variants in more DNA damage–response genes in prostate cancer will improve our understanding of the etiology of this disease and also potentially identify men at increased risk of developing prostate cancer in whom prevention strategies might be targeted.


   Acknowledgments
 
Grant support: NIH Prostate Specialized Programs of Research Excellence Career Development Award CA91956-01K-5B7170 (W. Liu), Department of Army Prostate Cancer Idea Development Award W81XWH-04-1-0212 (W. Liu), and NIH grant CA72818 (S.N. Thibodeau).

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.

We thank Drs. Scott H. Kaufmann and Junjie Chen (Mayo Clinic) for their helpful discussions.

Received 2/20/06. Revised 7/28/06. Accepted 8/14/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Schaid DJ. The complex genetic epidemiology of prostate cancer. Hum Mol Genet 2004;13(Spec No 1):R103–21.[Abstract/Free Full Text]
  2. Nwosu V, Carpten J, Trent JM, Sheridan R. Heterogeneity of genetic alterations in prostate cancer: evidence of the complex nature of the disease. Hum Mol Genet 2001;10:2313–8.[Abstract/Free Full Text]
  3. Mononen N, Syrjakoski K, Matikainen M, et al. Two percent of Finnish prostate cancer patients have a germ-line mutation in the hormone-binding domain of the androgen receptor gene. Cancer Res 2000;60:6479–81.[Abstract/Free Full Text]
  4. Ikonen T, Matikainen M, Mononen N, et al. Association of E-cadherin germline alterations with prostate cancer. Clin Cancer Res 2001;7:3465–71.[Abstract/Free Full Text]
  5. Ford D, Easton DF, Bishop DT, Narod SA, Goldgar DE. Breast Cancer Linkage Consortium. Risks of cancer in BRCA1-mutation carriers. Lancet 1994;343:692–5.[CrossRef][Medline]
  6. Edwards SM, Kote-Jarai Z, Meitz J, et al. The Cancer Research UK/British Prostate Group UK Familial Prostate Cancer Study Collaborators; British Association of Urological Surgeons Section of Oncology. Two percent of men with early-onset prostate cancer harbor germline mutations in the BRCA2 gene. Am J Hum Genet 2003;72:1–12.[CrossRef][Medline]
  7. Cybulski C, Gorski B, Debniak T, et al. NBS1 is a prostate cancer susceptibility gene. Cancer Res 2004;64:1215–9.[Abstract/Free Full Text]
  8. Dong X, Wang L, Taniguchi K, et al. Mutations in CHEK2 associated with prostate cancer risk. Am J Hum Genet 2003;72:270–80.[CrossRef][Medline]
  9. Cybulski C, Huzarski T, Gorski B, et al. A novel founder CHEK2 mutation is associated with increased prostate cancer risk. Cancer Res 2004;64:2677–9.[Abstract/Free Full Text]
  10. Oda K, Arakawa H, Tanaka T, et al. p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 2000;102:849–62.[CrossRef][Medline]
  11. Matsuda K, Yoshida K, Taya Y, Nakamura K, Nakamura Y, Arakawa H. p53AIP1 regulates the mitochondrial apoptotic pathway. Cancer Res 2002;62:2883–9.[Abstract/Free Full Text]
  12. Wang L, McDonnell SK, Elkins DA, et al. Analysis of the RNASEL genes in familial and sporadic prostate cancer. Am J Hum Genet 2002;71:116–23.[CrossRef][Medline]
  13. Schaid DJ, McDonnell SK, Blute ML, Thibodeau SN. Evidence for autosomal dominant inheritance of prostate cancer. Am J Hum Genet 1998;62:1425–38.[CrossRef][Medline]
  14. Wang L, McDonnell SK, Cunningham JM, et al. No association of germline alteration of MSR1 with prostate cancer risk. Nat Genet 2003;35:128–9.[CrossRef][Medline]
  15. Berry R, Schroeder JJ, French AJ, et al. Evidence for a prostate cancer-susceptibility locus on chromosome 20. Am J Hum Genet 2000;67:82–91.[CrossRef][Medline]
  16. Lange EM, Chen H, Brierley K, et al. Linkage analysis of 153 prostate cancer families over a 30-cM region containing the putative susceptibility locus HPCX. Clin Cancer Res 1999;5:4013–20.[Abstract/Free Full Text]
  17. Xu J, Zheng SL, Chang B, et al. Linkage of prostate cancer susceptibility loci to chromosome 1. Hum Genet 2001;108:335–45.[CrossRef][Medline]
  18. Melton LJ. 3rd History of the Rochester epidemiology project. Mayo Clin Proc 1996;71:266–74.[Abstract]
  19. Oesterling JE, Jacobsen SJ, Chute CG, et al. Serum prostate-specific antigen in a community-based population of healthy men: establishment of age-specific reference ranges. JAMA 1993;270:860–4.[Abstract/Free Full Text]
  20. Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN, James CD. Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res 1998;26:1396–400.[Abstract/Free Full Text]
  21. Wang X, Babu JR, Harden JM, et al. The mitotic checkpoint protein hBUB3 and the mRNA export factor hRAE1 interact with GLE2p-binding sequence (GLEBS)-containing proteins. J Biol Chem 2001;276:26559–67.[Abstract/Free Full Text]
  22. Sasieni P. From genotypes to genes: doubling the sample size. Biometrics 1997;53:1253–61.[CrossRef][Medline]
  23. Slager S, Schaid D. Evaluation of candidate genes in case-control studies: a statistical method to account for related subjects. Am J Hum Genet 2001;68:1457–62.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.
Right arrow Articles by Liu, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by Liu, W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online