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Clinical Cancer Research Vol. 9, 4034-4042, September 15, 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

The Androgen Receptor Gene is Preferentially Hypermethylated in Follicular Non-Hodgkin’s Lymphomas

Hongyu Yang, Chuan-Mu Chen, Pearlly Yan, Tim H-M. Huang, Huidong Shi, Mattias Burger, Inko Nimmrich, Sabine Maier, Kurt Berlin and Charles W. Caldwell1

Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, Missouri 65203 [H. Y., P. Y., T. H-M. H., H. S., C. W. C.]; Department of Life Sciences and Institute of Biomedicine, National Chung Hsing University, 402 Taichung, Taiwan [C-M. C.]; and Epigenomics, AG, Berlin D-10435, Germany [M. B., I. N., S. M., K. B.]

ABSTRACT

This investigation examined promoter DNA methylation of the androgen receptor (AR) gene in non-Hodgkin’s lymphoma (NHL) representing different stages of B-cell differentiation. Steroid hormones are important endocrine messengers with a broad range of physiological functions, including regulation of B-cell lymphopoiesis. Some of these effects are mediated via specific receptors such as AR that can act as a ligand-dependent transcription factor for other genes. DNA was isolated from 76 NHL specimens representing pregerminal center, germinal center, and postgerminal center states of differentiation. Initial methylation data were obtained from oligonucleotide microarrays and was confirmed and extended using methylation-specific PCR. Methylation of the AR gene promoter was present in a nonrandom pattern. Those tumors derived from pregerminal center or postgerminal center stages showed virtually no methylation and expressed AR mRNA. Cases of germinal center origin, mainly follicular lymphomas and some diffuse large B-cell lymphomas, showed hypermethylation. Studies with NHL cell lines revealed that demethylation or reversal of histone deacetylation partially restored AR expression but reversal of both simultaneously provided a synergistic release from suppression. Promoter methylation of AR occurs in a differentiation stage-selective manner; those cases arising in the germinal center are preferentially methylated. Full re-expression of AR requires both demethylation and reacetylation, a finding that may affect treatment decisions.

INTRODUCTION

The molecular pathophysiology of NHL2 is characterized by distinct genetic pathways that selectively associate with different clinicopathological categories of the disease and shows relationships to putative normal stages of B-cell differentiation (Fig. 1)Citation . After production in the bone marrow and migration of naïve, mature B cells in peripheral blood, additional differentiation in secondary lymphoid tissues can be divided into pregerminal center, germinal center, and postgerminal center phases. (1 , 2) During the pregerminal center phase, selection and recombination of V(D)J genes occurs, as well as the insertion of nontemplated nucleotides within the V(D)J region. Later, in the germinal center phase, somatic hypermutations occur during V(D)J rearrangement, with or without antigen exposure. Those B cells not undergoing productive rearrangements and receiving appropriate survival signals are destined to undergo apoptotic cell death. Those cells successfully transiting the germinal center phase, then enter a postgerminal center phase whereby the cells have acquired memory and cease additional somatic hypermutations.



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  		Fig. 1. Hypothetical model of the relationship between types of NHL and stages of normal B-cell differentiation.

 
In this model, MCL most closely emulates pregerminal center mantle zone B cells that have not yet undergone somatic hypermutations (1) . Approximately half of B-CLL/SLL cases are comprised of naive (unmutated), pregerminal center B cells (3) , but the remaining 50% of cases have undergone and then ceased additional somatic hypermutations and are actually postgerminal center memory B cells (mutated). FLI and FLII cases, comprised of various proportions of centroblasts and centrocytes, are similar in their composition to cells in reactive secondary follicles or germinal centers (1) . They typically have mutations of immunoglobulin variable (V) region genes and demonstrate ongoing somatic hypermutations characteristic of normal germinal center B cells. Cases of DLBCL are comprised of two main types, a germinal center type comprised of centroblasts usually expressing CD10 and a postgerminal center type resembling memory B cells. Both LPL and MM are postgerminal center in their biology, both having undergone the GC reaction and isotype switching but without continuing somatic hypermutations. Thus, the NHL cases studied represent a spectrum of pregerminal center, germinal center, and postgerminal center cell types.

Steroid hormones are important endocrine messengers with a broad range of physiological functions, including regulation of B-cell lymphopoiesis (4, 5, 6) . The effects of androgens are mediated via a specific AR belonging to the nuclear receptor family and acting as a ligand-dependent transcription factor for other genes containing AREs in their promoter or upstream regions (5) . In its inactive form, the receptor is complexed to heat shock proteins and located mainly in the cytoplasm. Upon ligand binding and activation, the receptor enters the nucleus, binds to its cognate ARE(s) as a homodimer, and stimulates gene transcription.

In normal cells, DNA methylation usually occurs by adding a methyl group to the fifth carbon position of cytosine within a 5'-CG-3' palindrome (CpG with p denoting the phosphate group) during DNA replication (7 , 8) . Across the genome, ~80% of CpG dinucleotides are heavily methylated but some remain unmethylated in specific GC-rich regions (G+C content: >50–60%), called CpG islands (9) . These 0.2–1-kb long DNA sequences are frequently located in the promoters and first exon regions of 40–50% of all genes (8) . The rest may be located in the intronic or other exonic regions of the genes or in regions containing no genes.

In cancer cells, patterns of DNA methylation are altered (7 , 10 , 11) . Promoter (including the first exon) CpG island hypermethylation is a frequent epigenetic event in many types of cancer, including NHLs (12, 13, 14) . This epigenetic mutation can result in gene silencing via alteration of local chromatin structure preventing normal interaction of the promoters with the transcriptional machinery (15) . If this occurs in genes critical to growth inhibition, the silencing event could promote tumor progression (7) .

Although androgens are involved in normal B-cell differentiation, the AR has not been studied extensively in NHL. McDonald et al. (16) reported promoter DNA methylation in 16 of 19 cases, mostly of follicular lymphomas. On the basis of data derived from microarray-based discovery experiments and the known relationships of B cells and NHL to sex steroid manipulation (16) , we additionally examined DNA methylation of AR as a potential epigenetic modifier in selected B-cell lines and subtypes of NHL that represent pregerminal center, germinal center, and postgerminal center stages of differentiation.

MATERIALS AND METHODS

Tissue Samples.
Tissue samples were obtained from patients after diagnostic evaluation for suspected lymphoma at the Ellis Fischel Cancer Center (Columbia, MO) in compliance with our local Institutional Review Board. DNA was isolated from a total of 76 specimens; 14 from patients with B-CLL/SLL, 14 from FLI, 12 FLII, 14 MCL, 14 DLBCL, 4 LPL, and 4 MM were examined. All cases of B-CLL/SLL had peripheral blood and bone marrow involvement, and thus were technically categorized as CLL. These are all referred to as B-CLL/SLL in this article. On the basis of CD38 expression, 8 of 14 (57.2%) cases were pregerminal center tumors, and the remaining 6 (42.8%) were postgerminal center. For the DLBCL group, 6 of 14 (42.8%) were CD10+, a finding usually associated with DLBCL of a germinal center type. All specimens contained a minimum of 80% neoplastic cells, as determined by flow cytometry (data not shown). Total RNA was isolated using RNeasy Total RNA System (Qiagen, Valencia, CA). Enrichment of B cells from nonneoplastic lymph nodes was accomplished by immunoseparation into CD19+ B cells and CD19-lymph node cells. Cell suspensions (~1 x 107/ml in RPMI 1640) were incubated with CD19-conjugated immunomagnetic beads (Dynal, Lake Success, NY) at 4°C for 30 min, washed twice in PBS containing FCS (0.5%), and released with Detach-a-Bead (Dynal). This method routinely yields >95% purity and >85% recovery in our laboratory (data not shown).

Bisulfite Treatment and PCR Amplification for Microarray Hybridizations.
Bisulfite treatment of genomic DNA was performed with minor modifications as described previously (17) . For the PCR amplification of the bisulfite-treated sense strand of the genes used for class prediction and discovery, the primers were designed according to the guidelines of Clark et al. (18) , ensuring preferred amplification of DNA fragments with complete bisulfite conversion. Note that primers were designed to hybridize to DNA fragments containing no CpG dinucleotides, thus allowing unbiased amplification of methylated and unmethylated alleles. For the methylation profiling of lymphoma samples, CpG sites from regulatory regions of the genes listed in Table 1Citation were analyzed. Seven or eight fragments were amplified in parallel using multiplex PCR. The following protocol was used for multiplex PCR reactions: 10 ng of bisulfite-treated DNA as template, 2 pmol of each primer (seven or eight primer pairs), 1.6 mM deoxynucleoside triphosphates, and 1 unit of Taq polymerase (HotStarTaq; Qiagen, Hilden, Germany) in reaction buffer supplied with the enzyme with 3.5 mM MgCl2 were incubated in 25 µl of total volume. After activation of the enzyme (15 min, 96°C), the incubation times and temperatures were 95°C for 1 min followed by 39 cycles (95°C for 1 min, 55°C for 45 s, and 65°C for 75 s) and 65°C for 10 min.


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  		Table 1 A listing of the genes examined on the microarray chip

 
Microarray Hybridizations.
Fig. 2Citation outlines the oligonucleotide microarray strategy for DNA methylation analysis. The PCR-amplified products contain pools of DNA fragments with bisulfite-converted nucleotides because of their differential methylation status. As shown, the unmethylated allele of a given DNA sequence is expected to have unmethylated cytosines converted to thymine, whereas this CpG sequence remains unchanged in the methylated allele. All PCR products performed on an individual sample were mixed and hybridized to glass slides carrying a pair of immobilized oligonucleotides for each CpG position.



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  		Fig. 2. Schematic outline for analysis of DNA methylation using the oligonucleotide microarray. Genomic DNA is bisulfite treated and amplified by PCR for a specific CpG island region of interest (or combinations of primer sets). The amplified product is labeled with Cy5 fluorescent dye and hybridized to oligonucleotide probes attached to a glass slide surface. At left, an oligonucleotide probe is designed to form a perfect match with a target DNA containing the unmethylated (TG) allele. At right, a probe is designed to form a perfect match with the methylated (CG) DNA target. The proportion of intensities CG/(CG+TG) reflects the proportion of methylated alleles relative to the total number of alleles.

 
Oligonucleotides with a C6-amino modification at the 5'-end were spotted with 4-fold redundancy on activated glass slides (19 , 20) . For each analyzed CpG position, two oligonucleotides, N(2–16)-CG-N(2–16) and N (2–16)-TG-N(2–16), reflecting the methylated and nonmethylated status of the CpG dinucleotides, were spotted and immobilized on the glass array. Hybridization conditions were selected to allow the detection of the single nucleotide differences between the TG and CG variants. The microarrays contained 156 CpG sites from 38 genes (Table 1)Citation and were hybridized with Cy5-labeled PCR fragments as described previously (19 , 20) . Subsequently, the fluorescent images of the hybridized slides were obtained using a GenePix 4000 microarray scanner (Axon Instruments, Union City, CA). Hybridization experiments were repeated at least six times for each sample. For each hybridization experiment, both CG and TG detection oligonucleotides of each CpG position the median spot intensities are taken. The methylation proportion was calculated as follows: CG/(CG+TG). The mean methylation proportion overall six repetitions was used for additional analysis.

Cell Lines and Treatments.
Four NHL cell lines were studied for methylation and gene expression. The germinal center cell line RL is a follicular lymphoma derived from a male patient with the t(14,18) gene rearrangement (21) , and the CD77+ Burkitt lymphoma-derived Daudi cell line is often used as a model of germinal center function (22) . The postgerminal center cell line DB is a DLBCL cell line that has undergone isotype switching (21) , and Raji cells are of germinal center derivation (23) . All four of these cell lines expressed surface CD10 (data not shown), thus suggesting a germinal center relationship. These were obtained from the American Type Culture Collection and all were maintained in RPMI 1640. Cells were harvested and RNA, and DNA were extracted for reverse transcriptase-PCR expression analysis and for MSP.

For some experiments, RL cells were treated during the log phase of growth with TSA, 5-Aza, or both. Cells were seeded at 5 x 106/ml in RPMI 1640 with 10% calf serum. Control cell received no additives, whereas 5-Aza was added at 2.5 µM for 60 h of incubation, TSA (100 ng/ml) was added for 12 h, and the culture that received both was first incubated for 48 h with 5-Aza followed by an additional 12 h with TSA included in the culture. Cells were harvested and RNA and DNA were extracted for reverse transcriptase-PCR expression analysis and for MSP.

MSP.
For MSP, 2 µg of genomic DNA were treated with Na-bisulfite according to the manufacture’s recommendations (CpGenome; Intergen, Purchase, NY), and 100 ng of bisulfite-treated DNA were used for each assay. The following primers designed for the AR promoter region were; unmethylated (5'-TAGTGTGGTGGTTTTGAAGTTGTTGTTT-3' and 5'-CAAACTAACAACACTAAACCAACAAAAACA-3') and methylated (5'-GCGGCGGTTTCGAAGTCGTCGTTC-3' and 5'-CGCTAAACCGACGAAAACGAAAAAACG-3').

Semiquantitative Reverse Transcriptase-PCR.
First strand cDNA was synthesized using SuperscriptII reverse transcriptase (Invitrogen, Carlsbad, CA). Reverse transcriptase-PCR was conducted (30 cycles) using primers 5'-CCTGGCTTCCGCAACTTACAC-3' and 5'-GATTTTTCAGCCCATCCACTGG-3' for AR cDNA amplification. The levels of AR mRNA were normalized with the level of ß-actin mRNA as described previously (24) .

RESULTS

Microarray-based Discovery in Gene Methylation.
Fig. 3Citation illustrates comparative DNA methylation data from a single locus of AR interrogation from both genders (top panel) and also separated into male-only cases (bottom panel) to examine any bias because of the influence of methylation-associated X chromosomal inactivation in the analyzed gene set (16) .



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  		Fig. 3. Ranked matrix evaluation of a single AR gene locus. The methylation proportion relative to the mean of the group is expressed for each individual case as red (increased methylation compared with the mean), green (decreased methylation compared with the mean), and no difference as black.

 
On the basis of visual patterns that suggested preferential methylation in the FLI/FLII group and some cases of DLBCL, we chose to reexamine differences between the aggregated FLI/FLII/DLBCL group and the combined B-CLL/SLL and MCL group. When examined separately, there was no difference in methylation patterns between FLI and FLII (P > 0.05) or between DLBCL and the combined FLI/FLII groups (P > 0.05). For the four AR loci tested in all cases using the microarray, a two-sample t test with equal variance produced Ps as shown in Table 2Citation . Thus, locus 1, illustrated in Fig. 3Citation , demonstrated the strongest statistical separation of groups and suggested preferential methylation within the aggregated group of FLI/FLII/DLBCL.


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  		Table 2 AR methylation comparison between FLI/FLII/DLBCL group and the B-CLL/SLL/MCL group

Using a 2-sample t-test with equal variance for each of 4 loci, Ps are provided in all cases and also for male only cases.

 
Because AR is located on the X chromosome, healthy females are expected to have half of their copies methylated because of X chromosomal inactivation, whereas healthy males should only have unmethylated alleles (16) . This gender-based differential methylation was confirmed by the observation of both methylated and unmethylated alleles by MSP of nonmalignant female CD19+ B cells from lymph nodes but only an unmethylated band in male CD19+ B cells from lymph nodes (Fig. 4)Citation . On the basis of the known sensitivity of the MSP method, which can readily detect ~1:1000 positive cells, we conclude there is no detectable AR methylation in males, but both methylated and unmethylated alleles are present in females. We also tested gender separation on the basis of AR methylation across all NHL classes (female, n = 18; male, n = 49) specifically with the same four loci of AR and three loci from ELK1 and found statistical separation at Ps shown in Table 3Citation .



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  		Fig. 4. MSP for female cases of B-CLL/SLL and for FLI/FLII. PCR products from the MSP procedure are shown for the promoter region, where M represents the methylated AR CpG island allele and U represents the unmethylated AR CpG island allele. The top row includes cases of B-CLL/SLL, whereas the middle row are cases of FLI/FLII. Shown below is the MSP pattern for nonmalignant CD19+ B cells from lymph nodes of female and male cases of nonspecific reactive hyperplasia.

 

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  		Table 3 Methylation comparison of two X-chromosome-related genes between genders across all cases of FLI/FLII/DLBCL/B-CLL/SLL/MCL

Using a two-sample t test with equal variance for each gene locus, Ps are provided. For females, n = 18, and males, n = 49.

 
This clearly shows the ability to separate female and male cases specifically using AR methylation patterns for the first three AR loci tested. These results are similar to those reported by Model et al. (25) across female and male cases of acute leukemias and, thus, support the validity of the present measurements. Additional analysis of ELK1, another X-located gene from the microarray, also shows the gender separation across all three loci tested and further confirms the validity of our microarray measurements (Table 3)Citation .

When only female cases of B-CLL/SLL (n = 4) were compared with FLI/FLII/DLBCL (n = 4), the first locus of AR differed with P = 0.00379, whereas the other three loci all had P > 0.05. This may be a limitation of the few cases examined. Fig. 4Citation shows female cases of B-CLL/SLL and FLI/FLII methylation patterns. Virtually all cases demonstrate both methylated and unmethylated bands as expected in normal female cells, but additionally, the cases of FLI/FLII demonstrate stronger methylated:unmethylated band densities compared those of B-CLL/SLL. Thus, in this group, there is promoter DNA methylation beyond that expected from the X chromosomal inactivation process. A comparison of the ratios of methylated to unmethylated alleles in B-CLL/SLL (where virtually no methylation of AR occurs in male patients) to cases of FLI/FLII demonstrates an increase in methylation in the latter. In fact, it would be quite surprising if tumor-related DNA methylation were gender specific. Nevertheless, this pattern differs from that of male patients with B-CLL/SLL and FLI/FLII (Fig. 5)Citation .



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  		Fig. 5. Examples of MSP results for various classes of NHL. Upper Panel, shows a schematic of the AR gene promoter and first exon regions. The vertical bars below, labeled CGI, illustrate the relative positions and densities of CpG islands within these regions. The illustration labeled MSP shows the regions of the promoter amplified in the analysis. Below this, PCR products from the MSP procedure are shown for the promoter region, where M represents the methylated AR CpG island allele and U represents the unmethylated AR CpG island allele. The pregerminal center group includes: Lane 1, molecular marker; Lanes 2–9, cases of B-CLL/SLL; Lanes 10–17, cases of MCL; Lanes 18 and 19, positive control; and Lanes 20 and 21, H20-negative control. The germinal center group includes: Lane 1, molecular marker; Lanes 2–9, cases of FLI/FLII; Lanes 10–17, cases of DLBCL; Lanes 18 and 19, positive control; and Lanes 20 and 21, H20-negative control. The postgerminal center group includes: Lane 1, molecular marker; Lanes 2–7, cases of LPL; Lanes 9–13, cases of MCL; Lanes 14 and 15, positive control; and Lanes 16 and 17, H20-negative control.

 
AR Gene Methylation of Pregerminal Center, Germinal Center, and Postgerminal Center Stages of B-Cell Differentiation.
Beyond the initial discovery-based methylation analysis of the AR gene, we extended a focused study by MSP analysis of additional de novo cases of NHL using only male patients representing pregerminal center, germinal center, and postgerminal center tumors. This gender bias was purposeful to better understand AR methylation in situations where no methylation should be present.

Some of the samples examined by MSP were cases that were previously examined by microarray (for confirmation). As examples, Fig. 5Citation illustrates MSP patterns from NHL cases of pregerminal center (MCL and B-CLL/SLL) germinal center (FLI, FLII, and DLBCL) or postgerminal center B cells (LPL and MM). Cases of DLBCL are included as germinal center cases, although it is recognized that some are likely of postgerminal center stage (2) . In fact, 42.8% of our cases of DLBCL showed AR methylation, but the number of cases is too low for statistical validation. Virtually all cases of FLI (10 of 10 male, 4 of 4 female) and FLII (5 of 6 male, 6 of 6 female) and 35.7% of the DLBCL cases (5 of the 6 CD10+ cases) revealed methylation within the promoter region. Thus, there was concordance with the microarray methylation data, but in addition, we demonstrated lack of promoter methylation of AR in the additional de novo cases of NHL of pregerminal center and postgerminal center stages of differentiation.

Relationship of Methylation to Gene Silencing in NHL Cell Lines.
In an analogous manner, we examined promoter DNA methylation of four NHL cell lines and demonstrate that all show evidence of promoter DNA methylation (Fig. 6A)Citation . We also investigated whether AR gene expression might be altered in conjunction with promoter CpG island methylation from these same NHL cell lines (Figs. 6B)Citation . Semiquantitative reverse transcriptase-PCR was performed using ß-actin as an internal control. Lymph node cells from 1 male subject were included as an unmethylated control. As shown, methylation was present in all cell lines, but reciprocal down-regulation or ablation of mRNA expression was variable. For instance, although RL (follicular lymphoma) was methylated and had no detectable mRNA, Daudi showed methylation but also strong mRNA expression. It is known that methylation works in concert with acetylation with respect to AR expression (26) , so this is not totally unexpected.



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  		Fig. 6. Methylation and gene expression in NHL cell lines. A shows the methylated (M) and unmethylated (U) alleles as determined by MSP in the four NHL cell lines indicated, as well as normal male lymph node tissue (L. N.), the positive control, and the negative control with water. B shows the expression of AR mRNA in all 4 cell lines by reverse transcriptase-PCR (top portion) compared with expression of ß-actin (bottom portion). For all cases, the left lane contains a molecular ladder for comparison of product sizes.

 
Reversal of Suppression by 5-AZA and TSA.
Methylation-related gene silencing can frequently be reversed by 5-Aza, alone or in combination with inhibition of HDAC activity. As a model system, we used the cell line RL, a t(14,18)+ follicular lymphoma that has undergone somatic hypermutations from a male patient (21) . Untreated RL cells were methylated in the promoter of AR and did not express appreciable AR mRNA (Fig. 6)Citation . When treated with the demethylating agent 5-Aza, the cells regained mRNA expression. Incubation with the HDAC inhibitor TSA alone also produced detectable, although not maximal, mRNA production. In contrast to individual treatments, when both were used simultaneously, a marked increase in mRNA was evident, suggesting a synergistic response (Fig. 7)Citation . Thus, demethylation or inhibition of HDACs could partially restore expression, but both together further increased expression of AR. This observation is similar to that described by Cameron et al. (26) and suggests a framework for combination therapies in certain forms of cancer.



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  		Fig. 7. Effect of treatment of the follicular lymphoma cell line RL with demethylating and HDAC inhibiting agents on expression of AR mRNA. The RL cells alone show no detectable expression, but treatment with the demethylating agent 5-Aza or the HDAC inhibitor TSA resulted in some expression. The combined use of 5-Aza and TSA produces maximal mRNA.

 
Thus, our findings suggest that methylation of the AR promoter DNA impacts on but is not entirely responsible for gene silencing. Most likely, DNA methylation and histone acetylation are jointly important in gene silencing through formation of repressive complexes that involve not only methylation as an inciting event but also the subsequent attraction of methyl-binding proteins, recruitment of HDAC molecules, and concomitant chromatin remodeling. Although the impact of AR DNA methylation has not previously been reported, the role(s) of histone acetylation in AR expression has been studied (27, 28, 29) . It is clear that AR can bind histone acetyltranferases such as p/CAF, as well as coactivating factors, and in so doing, acetylates histones in the AR, as well as additional histones within the nucleosome, and this results in gene transcription. However, this is cell cycle dependent as cells at the G1-S phase show decreased AR expression and dissociation of p/CAF secondary to cyclin D1 binding, resulting in histone deacetylation (27) . Reacetylation using TSA then allows partial but not complete AR expression. In this study, we reaffirm the partial reactivation of AR after TSA treatment of a follicular lymphoma cell line but additionally show that treatment with the demethylating agent 5-Aza also only partially reactivates AR. However, a synergistic recovery occurs in the presence of both 5-Aza and TSA. This observation strongly suggests that both DNA methylation and HDAC are involved in AR silencing in the follicular lymphomas and likely some DLBCL cases.

DISCUSSION

This study provides important new information regarding AR in NHL. However, it should also be noted that a brief report on 26 cases of FLI/FLII and 14 cases of MCL were previously examined, and it was noted that CDKN1C (20) was hypermethylated in MCL but not FLI/FLII, but the AR gene promoter was methylated in FLI/FLII but not MCL. Gene expression for AR was inversely correlated with methylation (20) . First, B-cell NHL is associated with promoter DNA methylation of AR in a differentiation stage-selective manner; those cases arising in the germinal center (FLI/FLII/DLBCL) are preferentially methylated. The pregerminal center and postgerminal center tumors do not show the same methylation pattern. Second, these methylation patterns hold across both genders, although a normal pattern of X chromosomal inactivation via methylation is present in women. Third, although demethylating agents can potentially reverse the methylation, full re-expression of AR requires both demethylation and reacetylation. Thus, examination of AR methylation in NHL has provided provocative data and new mechanistic insights, but the overall effect of AR silencing in NHL is not clear because it potentially affects numerous other genes.

Although speculative, the reason(s) for this may relate to the unique activities that take place in the germinal center reaction. The state of methylation has been correlated with immunoglobulin gene regulation; both the µ heavy chain and {kappa} light chain gene are hypermethylated in cells in which they are not recombined (30) . Concomitant with V(D)J rearrangement, these loci become unmethylated and transcriptionally active. Regulated demethylation is thought to control the specificity of the V(D)J recombinase (31) . Furthermore, it has been shown that Pax5, a gene expressed in normal B cells until plasmacytic differentiation, is under control of DNA methylation (32) .

How AR might function with respect to germinal center NHL is also speculative. One of the hallmarks of FLI/FLII is surface expression of CD10 or NEP, an enzyme thought to identify lymphoid cells with specific cycling and apoptotic abilities (33) . An interesting relationship between AR and the NEP gene has been reported previously (34) . In prostate cell lines, steroid regulation of NEP involves at least two elements, including a typical ARE that binds receptors for androgen, progesterone, and glucocorticoids, and a unique androgen-responsive region that only binds AR. Thus, it is tempting to speculate on the possible impact of AR methylation silencing and NEP expression. Although NEP expression in lymphoid cells relates to specific cycling and apoptotic abilities, the t(14,18) translocation that is characteristic of FLI/FLII leads to high levels of bcl-2 protein and may protect cells from apoptosis (35) . Both bcl-2 and c-myc interact in progression of follicular lymphomas (36) and c-myc increases transcription of a large number of genes in B cells that are necessary for acceleration of G1 progression. Also, c-myc is an AR-regulated gene in some tumors, with a reciprocal relationship between AR mRNA levels and c-myc overexpression (37 , 38) .

The loss of AR expression in prostate cancer may be an important event in tumor progression, moving from a hormone-dependent to a hormone-independent condition that appears to be dependent on the acquisition of autocrine pathways (39) . It is possible that NHL with silenced AR might be more susceptible to the activities of other autocrine mechanisms such as IL-6 signaling. Cross-talk between the IL-6 and AR signal transduction pathways has been reported in which IL-6 induced several ARE-driven reporters that are dependent on AR and functioned via the mitogen-activated protein kinase and signal transducers and activators of transcription 3 pathways (40) . IL-6 also regulates the human DNA methyltranferase-1 gene promoter and resultant enzyme activity, which requires transcriptional activation by the Fli-1 transcription factor via signal transducers and activators of transcription 3 (41 , 42) . A convincing role for IL-6 in plasmacytic differentiation of B-cells and autocrine tumor growth has been demonstrated, but an even more convincing link involves a murine model of extraosseous plasmacytoma (43) . Upon stimulation with pristane, all of the transgenic mice carrying a widely expressed IL-6 transgene developed lymphoplasmacytic tumors. Unexpectedly, ~30% of these developed follicular lymphomas or DLBCL, thus demonstrating a connection between IL-6 dysregulation of an inflammatory response and development of germinal center lymphomas. Thus, it is conceivable that interactions between IL-6, AR methylation and silencing, and potentially IL-6-driven override of the silenced AR may be involved in development of FLI/FLII and DLBCL. However, at this time, any definitive mechanism(s) is speculative, but the data presented create many opportunities for additional exploration of the role(s) of AR in NHL.

FOOTNOTES

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

1 To whom requests for reprints should be addressed, at Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri School of Medicine, 115 Business Loop 70 West, Columbia, MO 65203. Phone: (573) 882-1283; E-mail: caldwellc{at}health.missouri.edu Back

2 The abbreviations used are: NHL, non-Hodgkin’s lymphoma; MCL, mantle cell lymphoma; FLI, grade I follicular lymphoma; FLII, grade II follicular lymphoma; ARE, androgen response element; AR, androgen receptor; DLBCL, diffuse large B-cell lymphoma; LPL, lymphoplasmacytic lymphoma; MM, multiple myeloma; B-CLL/SLL, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma; MSP, methylation-specific PCR; TSA, trichostatin A; 5-Aza, 5-azacytidine; HDAC, histone deacetylase; NEP, neutral endopeptidase 24.11; IL, interleukin. Back

Received 1/ 3/03; revised 5/16/03; accepted 5/16/03.

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