histone h1 expression in human prostate cancer tissues and cell lines

Original Article

Histone H1 expression in human prostate cancer tissues andcell linespin_2755 84..92

Shinya Sato,1 Satoru Takahashi,1 Makoto Asamoto,1 Makoto Nakanishi,2 Toshiaki Wakita,3 Yuji Ogura,3

Yasushi Yatabe4 and Tomoyuki Shirai1

1Departments of Experimental Pathology and Tumor Biology and 2Cell Biology, Graduate School of MedicalSciences, Nagoya City University, 3Departments of Urology and 4Pathology and Molecular Diagnostics, Aichi CancerCenter Hospital, Nagoya, Japan

Histone H1, one of the histone superfamilies, is known todetermine chromatin structure and alter gene expression. Italso contributes to regulation of cell proliferation in breastcancer. We hypothesized a similar association in prostatecancer, and therefore examined relationships betweenhistone H1 expression and Gleason pattern, Ki-67 andandrogen receptor levels in a series of prostate cancertissues and cell lines. Histone H1 positive cancer cellsincreased with the Gleason pattern. Gleason pattern 3tumors were divided into two groups, one with high histoneH1 positivity (H1-high cases, 60–100% positivity) and theother with low histone H1 positivity (H1-low cases, 0–20%positivity). Ki-67 or androgen receptor positivity in H1-highcases was significantly higher than in H1-low cases. PC3cells demonstrated more frequent histone H1 and Ki-67positivity as compared to LNCaP cells. Silencing of histoneH1 by siRNA transfection significantly reduced cell prolif-eration in LNCaP and PC3. These findings suggest thathistone H1 expression is associated with the Gleasonpattern, cell proliferation and androgen receptor expressionin prostate cancers.

Key words: androgen receptor, Histone H1, Histone modifica-tion, hormone sensitivity, hormone therapy, prostate cancer

Prostate cancer, as with other types of malignancy, demon-strates markedly differing responses to available therapiesamong patients.1,2 The Gleason pattern, androgen sensitivity,cell kinetics and invasiveness are all prognostic parameters3–7

but they demonstrate an imperfect relationship with treatmentoutcome, necessitating a continued search for appropriatemarkers of malignant potential to assist in choice of the mosteffective therapy for each patient.

Recently, several proteins associated with chromatinremodeling (i.e. histones) have been proposed as possibleindicators of changes in malignant potential in severalcancers.8–10 Alteration in their levels can give rise to large-scale up- or down-regulation in gene expression and cellularfunctions by epigenetic mechanisms that were not wellunderstood until recently. Histone H1 is the chief proteincomponent of chromatin in histones,11–15 and it also regulatesmany gene expressions including cancer.16–19 Indeed, deple-tion of histone H1 variants was reported to cause G1 arrestand inhibit cell proliferation in breast cancer.19 Recently, itwas demonstrated that suppression of histone H1 expressionor histone H1 phosphorylation resulted in activation of andro-gen receptor (AR) in prostate cancer.20–23 Therefore, it wassuggested that histone H1 might be associated with prostatecancer growth through AR modification.24–26

In the present study, in order to clarify the significance ofhistone H1 expression in prostate cancer, we examined rela-tionships with Gleason pattern (histological architectural clas-sification of prostate cancer with prognostic feature27–29),Ki-67 expression, and AR expression in surgically resectedprostate cancer tissues as well as prostate cancer cell lines.We also performed histone H1 RNA silencing experimentsusing prostate cancer cell lines.

MATERIALS AND METHODS

Prostate cancer specimens

A total of 60 cases of surgically-resected prostate cancerwere available for this study, including 24 cases receiving

Correspondence: Shinya Sato, MD, PhD, Department of Experimen-tal Pathology and Tumor Biology, Graduate School of Medical Sci-ences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku,Nagoya 467–8601, Japan. Email: [email protected]

Received 4 June 2011. Accepted for publication 20 September2011.© 2011 The AuthorsPathology International © 2011 Japanese Society of Pathology andBlackwell Publishing Asia Pty Ltd

Pathology International 2012; 62: 84–92 doi:10.1111/j.1440-1827.2011.02755.x

neoadjuvant hormonal therapy. Therapeutic grades of the 24cases were Grade 0b–Grade1. Gleason patterns of the 24cases were diagnosed using biopsy samples of the pre-treated prostate cancers. Detailed characteristics of casesare summarized in Table 1. Twenty-one cases had a singleGleason pattern (3, 4 or 5), and 39 had 2 or more patterns.They were all surgically resected at Aichi Cancer CenterHospital, Nagoya, Japan, after obtaining the approval of theInstitutional Review Board of Aichi Cancer Hospital andNagoya City University. Immediately after resection, the pros-tates were cut into 8–10 sections in the frontal plane, fixedwith a formalin and a methanol-based fixating agent (Yufix;Sakura Finetek, Tokyo, Japan) for 24–48 h and proceededfor paraffin embedding to make pathological specimens andsectioning for H&E staining and histological evaluation, aswell as immunohistochemistry. For the immunohistochemicalanalysis, glandular and interstitial cells in noncancerousareas of the prostates were defined as normal. We usedJapanese therapeutic grading criteria for assessing thera-peutic effect on prostate cancer.30

Cell lines

Human prostate cancer cell lines (LNCaP and PC3) wereobtained from the American Type Culture Collection (ATCC,

Manassas, VA, USA). A rat prostate cancer cell line, PLS-10, previously established in our laboratory31–33 was alsoemployed. All were cultured in RPMI 1640 medium contain-ing 10% FBS and 1% penicillin-streptomycin, maintained at37°C in a humidified 5% CO2 atmosphere in an incubator. Forpreparation of cell blocks from prostate cancer cell lines,cancer cells were trypsinated, accumulated by a concentra-tion cytocentrifuge technique, formalin fixed, and then paraf-fin embedded.

Immunohistochemical and immunocytochemicalstaining

Immunohistochemical and immunocytochemical stainingwas applied on paraffin-embedded sections (3 mm thick) of60 prostate cancer specimens and cell blocks from the threehuman prostate cancer cell lines. Sections were incubatedfor 60 min with antibodies specific for histone H1 (3E9,Upstate, Temecula, CA, USA; 1:4000 dilution), Ki-67 (MIB-1,Dako Cytomation Inc, Glostrup, Denmark; 1:200 dilution),and AR (N-20, Santa Cruz Biotechnology Inc, Santa Cruz,CA, USA; 1:200 dilution). Staining was achieved using two-step EnVision + System-HRP methodology, according to themanufacturer’s instructions (Dako Cytomation Inc.). Sectionswere lightly counterstained with hematoxylin to facilitate

Table 1 Clinicopathologic characteristics of 60 patients with prostate cancer

Cases without neoadjuvanthormone therapy

Cases with neoadjuvanthormone therapy

Total cases (n) 36 24Age (years) 61.3 (48–75) 63.3 (50–70)Average Gleason score 7.0 (6–9) 6.9 (6–8)

Gleason score 6 (n) 9 –Gleason score 7 (n) 20 –Gleason score 8 (n) 5 –Gleason score 9 (n) 2 –

Average Gleason pattern 3.5 (3–5) –Gleason pattern 3 (n) 34 –Gleason pattern 4 (n) 22 –Gleason pattern 5 (n) 7 –

Cases including one pattern (n) 9 –Cases including 2 or more pattern (n) 27 –Surgical margin positive case (n) 13 9Capsular invasion positive cases (n) 8 6Vascular invasion positive cases (n) 0 0Lymph node metastasis (n) 0 0Preoperative clinical stage (The Jewett system)

A1 (n) 0 0A2 (n) 0 0B0 (n) 18 3B1 (n) 12 6B2 (n) 5 11C1 (n) 1 4C2 (n) 0 0

Preoperative PSA (ng/ml) 7.18 (2.9–21.14) 1.14 (0.03–5.59)Biochemical failure (n) 5 9

Histone H1 and prostate cancer 85

© 2011 The AuthorsPathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd

orientation. Immunostained slides were evaluated by lightmicroscopy and the proportion of positive staining cells (posi-tivity) was quantified as positive cancer or normal cell nuclei/total cancer or normal cell nuclei. Approximately 1000 nucleiof cancer or normal cells per selected area were counted inall cases and the data were expressed as ratios of thenumber of positive staining cells to total counted cells.

RNA-interference

Control siRNA and histone H1 siRNA (subtype: H1o) werepurchased from Invitrogen Corp (Carlsbad, CA, USA) and2 mM aliquots were used to transfect LNCaP and PC3 cellsusing Lipofectamine 2000 (Invitrogen Corp) for 72 h accord-ing to the manufacturer’s instructions.

RNA extraction and RT-PCR

Total RNAs were isolated from the prostate cancer cell linesusing an RNeasy Mini kit (Qiagen, Valencia, CA, USA) andtreated with RQ1 RNase-free DNase (Promega, Madison, WI,USA) according to the manufacturer’s instructions. Reversetranscription of RNA was carried out in a final volume of 20 mLcontaining 5¥buffer, 0.1 M DTT, Oligo (DT)20, 10 mM dNTPs,0.4 U/mL of RNase inhibitor, 1.25 U/mL of reverse tran-scriptase (Invitrogen Corp) and 1 mg of total RNA.

Quantitative reverse transcriptase-PCR

cDNAs from LNCaP and PC3 cDNAs were amplified withhistone H1o-specific primers using SYBR Green PCR MasterMix (TaKaRa, Otsu, Japan) on a LightCycler (Roche, Mann-heim, Germany). Amplification of endogenous glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) was used as aninternal control to normalize all data. Histone H1o primerswere 5′-CCACAGACCACCCCAAGTAT-3′, and 5′-GCGTGGCTACCTTCTTGATT-3′. GAPDH primers were 5′-ACAGTCAGCCGCATCTTCTT-3′ and 5′-TGGAAGATGGTGATGGGATT-3′.

Western blot analysis

LNCaP cells were homogenized in RIPA buffer (150 mMNaCl, 50 mM Tris–HCl (pH 8.0), 1% NP-40, 0.5% sodiumdeoxycholate, 0.1% SDS, 1 mM phenylmethylsulphonyl fluo-ride, 1 mM sodium orthovanadate, and protease inhibitorcocktail (Complete, Roche). Twenty mg aliquots of proteinwere resolved on SDS–PAGE and separated proteins weretransferred to nitrocellulose membranes, which were thenincubated with histone H1 (3E9, Upstate) or b-actin (AC-74,Sigma Aldrich, St.Louis, MO, USA). Immunoreactions werevisualized with the ECL-Plus detection system (GE Health-

care, Piscataway, NJ, USA) after 1 h incubation with horse-radish peroxidase–labeled anti-mouse antibodies (CellSignaling Technology, Danvers, MA, USA). ImageJ was usedfor quantitative analysis of the result of Western blot.

Cell proliferation analysis

Cell proliferation of prostate cancer cell lines was assessedusing an assay based on cleavage of the tetrazolium saltWST-1 to formazan by cellular mitochondrial dehydrogena-ses (Roche). The formazan dye formed was quantified usinga plate reader at 440 nm. Prostate cancer cells were plated in96-well microplates at 1 ¥ 104 cells/well in 100 mL of culturemedia. Cells transfected with control siRNA or histone H1siRNA were incubated for 72 h, and exposed to 10 mL ofWST-1 (Roche) per the manufacturer’s protocol.

Statistical analysis

Statistical analysis was carried out using Scheffe andBonferroni–Dunn’s multiple comparison tests, and a two-tailed Student’s t-test. A value of P < 0.05 was consideredsignificant. The results were subjected to statistical analysisby using Statview software.

RESULTS

Association between histone H1 expression andGleason pattern

Characteristics of the 60 prostate cancer cases are pre-sented in Table 1. Histone H1 positive cancer cells werescattered in Gleason pattern 3 tumors, but more frequentlyobserved in Gleason pattern 4 and 5 tumors (Fig. 1a),accounting for significantly higher percentages of cells(Fig. 1b). Almost all luminal cells, basal cells and stromalcells were positively stained for histone H1 (Fig. 1a,b) innon-cancerous areas of all cases.

Association between histone H1 expression andcell proliferation/AR expression in Gleasonpattern 3 tumors

Since there was substantial variation in the histone H1 posi-tivity among Gleason pattern 3 tumors (Fig. 1b) compared toGleason patterns 4 or 5 tumors, Gleason pattern 3 tumorswere divided into two groups, one with high histone H1 posi-tivity (H1-high cases, 60–100% of cancer cells positive, 13cases) and the other with low histone H1 positivity (H1-low

86 S. Sato et al.


cases, 0–20% of cancer cells positive, 9 cases), and differ-ences in immunohistochemical Ki-67 or AR expression wereanalyzed between the two. Ki-67 positive cancer cells werereadily observed in the H1-high cases, while relatively rare inthe H1-low cases (Fig. 2a) (P = 0.007, Fig. 2b). Androgenreceptor positive cells were more frequently observed in theH1-high cases than in the H1-low cases (Fig. 2a) (P =0.009,Fig. 2b). Ki-67 and AR positivity in other cases (20–60% ofcancer cells were positively stained for histone H1) were notsignificantly changed compared to H1-high or H1-low cases.To evaluate the relationship between histone H1 expressionand PSA in prostate cancer, the preoperative serum PSAlevels in H1-high and H1-low groups were examined. The

average serum PSA level in H1-high prostate cancer patientswas not significantly different from that in the H1-low group(P = 0.89).

Histone H1 expression in prostate cancer withneoadjuvant hormonal therapy

Immunohistochemically demonstrable histone H1 expressionwas more frequent in 12 cases receiving neoadjuvant hor-monal therapy (Gleason score before hormonal therapy: 6 or7) than in 34 cases without therapy, all being Gleason pattern3, with statistical significance (P = 0.002, Fig. 3a,b). We com-

Figure 1 Immunohistochemical analysis of histone H1 in prostate cancer and normal prostate tissue. (a) Histone H1 is positively stained innuclei of prostate cancer cells, normal luminal cells, basal cells and stromal cells. (b) Note significantly higher Histone H1 positivity in Gleasonpattern 4 or 5 tumors than in their Gleason pattern 3 counterparts (P = 0.04 and P = 0.003, respectively).



pared histone H1 positivity of prostate cancer cells in biopsysamples before hormonal therapy and surgical samplesafter hormonal therapy in the same cases (n = 4). HistoneH1 positivity in prostate cancer after hormonal therapywas higher than that in prostate cancer before hormonalthrapy, but the difference was not statistically significant(P = 0.115).

Analysis of histone H1 and Ki-67 expression andhistone H1 RNA silencing in prostate cancer cell lines

Immunohistochemically, histone H1 and Ki-67 positive cellswere more frequently observed in PC3 than in LNCaP withstatistical significance (Fig. 4a). Histone H1 protein expres-sion was decreased by histone H1 siRNA transfection inLNCaP and PC3 (Fig. 4b) by Western blot analysis. The ARexpression of LNCaP was slightly suppressed by histone H1siRNA transfection (Fig. 4c). WST-1 assay revealed that cellproliferation of histone H1 siRNA transfected cells was sig-nificantly lowered as compared to control siRNA transfectedcells (Fig. 4d,e).

DISCUSSION

The present study demonstrates for the first time that histoneH1 expression is positively correlated with the Gleasonpattern, Ki-67 positivity and AR expression in prostatecancers. Furthermore, our data provided evidence that cellproliferation increases with increasing histone H1 positivity inGleason pattern 3 cancers. Moreover, WST-1 analysis dem-onstrated that cell proliferation of human prostate cancer celllines was decreased by histone H1 suppression with a histoneH1 siRNA. Increase of histone H1 protein was previouslydemonstrated to positively correlate with cell proliferation-related CDK2 activity in HeLa cells and fibroblasts.34 Takentogether, the available data indicate that histone H1 over-expression may increase cell proliferation activity in pro-state cancer.

In the prostate cancer tissues applied in the present study,histone H1 positivity of cancer cells was about 90% in high-grade tumors (Gleason patterns 4, 5), while it was about 50%in low-grade tumors (Gleason pattern 3). Previous studiesshowed that depletion of histone H1 caused G1 arrest, cel-lular senescence and cell death in fibroblasts and breast

Figure 2 Immunohistochemicalanalysis of histone H1 andKi-67/AR in Gleason pattern 3prostate cancers. (a) Ki-67 or ARpositive cancer cells are readilyobserved in an H1-high case, butare rare in the H1-low case. (b, c)Ki-67 positivity in H1-high caseswas significantly higher than inH1-low cases (P = 0.007 andP = 0.009, respectively).

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cancer cells.17,19 Thus, histone H1 overexpression may con-tribute to avoidance of G1 arrest or cellular senescence inlate-stage prostate cancer.

The present data clearly shows that histone H1 positivity ofcancer cells in cases of high-AR positivity was significantlyhigher than that in cases of low-AR positivity. It is also notablethat histone H1 positivity of prostate cancer cells in casestreated neoajuvant hormone therapy (Therapeutic grade 0bor 1) was also significantly higher than that in cases withouthormone therapy. This result was in accordance with thehigher histone H1 positivity in PC3 cells (androgen-independent) compared with LNCaP cells (androgen-dependent). Since AR overexpression is known to be one ofthe mechanisms underlying androgen independence,35–37

these findings indicate that histone H1 overexpression maybe associated with the hormone resistant phenotype.

The Gleason pattern is one of the major indicators ofmalignant potential of prostate cancers,38,39 often applied as aprognostic factor.27–29 Since histone H1 positivity was found toincrease with increasing Gleason pattern in prostate cancer

tissues, histone H1 expression might also serve as a prognos-tic marker of prostate cancer. However, because all casesused in the present study were of relatively early stage (Patho-logic stage pT2a or pT2b, pN0) and all the patients remainedalive, no analysis of prognostic potential was possible. Wealso examined the relationship between PSA failure andhistone H1 expression immunohistochemically, but histoneH1 positivity in prostate cancer cells was not significantlydifferent between cases of PSA failure and cases of non-failure (P = 0.3825, data not shown). One reason for this is thelow number of recurrent cases in this study (n = 5). Further-more, a high Gleason pattern was related to the risk of PSArecurrence in prostate cancer,40,41 but three of the five patientswith recurrent PSA showed Gleason pattern 3. Furtherresearch is needed to clarify the association between clinicalcharacteristics and histone H1 expression using moreadvanced cases.

In this study, the AR expression in the H1-high group wassignificantly higher than that in the H1-low group, but theserum PSA level was not. The PSA expression was regulated

Figure 3 Immunohistochemical analysisof histone H1 in prostate cancer with orwithout neoadjuvant hormonal therapy.(a) Many histone H1 positive cancer cellsare observed in cases with neoadjuvanthormonal therapy, but there is only a scat-tered presence in cases without therapy.(b) Histone H1 positivity in cases with neo-adjuvant hormonal therapy is significan-tly higher than in cases without therapy(P = 0.002).



by AR, but AR activation was not induced only by AR ampli-fication. In fact, AR amplification was observed in hormone-refractory prostate cancers, but many of these cancers havealternative mechanisms of AR activation or mechanisms thatcan independently stimulate AR-responsive genes.42 More-over, some reports have shown that the correlation betweenPSA expression and AR expression is changed by the andro-gen sensitivity of AR.43 Therefore, we think that the differencebetween serum PSA level and AR expression in relation withhistone H1 expression was not an abnormal result.

The AR and Ki-67 positivity of prostate cancer cells inpatients receiving neoadjuvant hormonal therapy was higherthan that in patients who did not receive therapy (data notshown). It has been reported that prostate cancer that per-sists after hormonal therapy exhibits amplified AR expression

and that AR expression and cell proliferation are correlated inprostate cancer.44–46 Thus, the result that the AR and Ki-67positivity of prostate cancer cells in patients receiving neo-adjuvant hormonal therapy was higher than that in patientswho did not receive therapy was reasonable.

Although the histone positivity of prostate cancer cells inpatients receiving neoadjuvant hormonal therapy was signifi-cantly higher than that in patients not receiving therapy, thehistone H1 positivity of prostate cancer cells was not signifi-cantly different between biopsy samples before hormonaltherapy and surgical samples after hormonal therapy. In thisstudy, we compared the histone H1 expression before andafter hormonal therapy in only four cases because the biopsyfor almost all the patients included in this study had beenperformed in other hospitals. We were thus unable to

Figure 4 Histone H1 siRNA transfection in androgen-dependent and independent prostate cancer cell lines. (a) Histone H1 positive cells arereadily apparent in the PC3 case, but are only scattered in LNCaP. (b) Western blot analysis of histone H1 protein expression in LNCaP andPC3 cells. conR, control siRNA (mock) transfection; si, Histone H1 siRNA transfection. (c) Western blot analysis of androgen receptor (AR)protein expression in LNCaP cells. Con, control; conR, control siRNA (mock) transfection; si, Histone H1 siRNA transfection. (d, e) WST-1assay in LNCaP or PC3. Cell viability of histone H1 siRNA transfected LNCaP and PC3 cells was significantly decreased as compared tocontrol siRNA transfected cells in a dose dependent manner. (LNCaP; P < 0.0001 and P = 0.0016, PC3; P = 0.006 and =0.03, respectively).

90 S. Sato et al.


examine more cases for comparing histone H1 expressionbefore and after hormonal therapy in the same cases. In thefuture, we will obtain more sets of biopsy samples beforehormonal therapy and surgical samples after hormonaltherapy for examining histone H1 expression.

Figure 4 shows that the percentage of histone H1 positivitydiffered between LNCaP and PC3 cells in immunohis-tochemical analysis, but histone H1 protein expression inLNCaP and PC3 cells did not differ on Western blots. Wecould not understand the reason for this because the histoneH1 antibody used in the immunohistochemical and Westernblot analyses was the same, but the sensitivity for histone H1protein might differ between cell block samples and proteinsuspended in RIPA buffer. Further investigations are requiredin this regard.

Histone H1 depletion affects the transcription status ofmany genes associated with prostate cancer cell prolifera-tion, such as IGF-2 and Bcl6.16 Histone H1 also affects CDKphosphorylation.34 Therefore, histone H1 may regulate cellproliferation by changing the expression of these genes.

Although we could compare the differences between thehistone H1-high group and the histone H1-low group in thecase of prostate cancer patients with a low Gleason pattern,we could not compare the differences between the histoneH1-high group and histone H1-low group in the case of pros-tate cancer patients with a high Gleason pattern because thehistone H1 positivity for these patients was so high that wecould not discern a low H1 positivity group.

Our results revealed that normal luminal cells, basal cellsand stromal cells in noncancerous areas of all cases stronglyexpressed histone H1 immunohistochemically. Actually,histone H1 is one of the most abundant chromatin-bindingproteins, and histone H1 stabilizes the compact and higher-order chromatin structure and inhibits DNA-dependentactivities such as transcription and replication in normal dif-ferentiated cells.47 In this study, histone H1 positivity of pros-tate cancer cells in H1-high cases were not significantlydifferent from that of normal luminal cells in the same cases,but Ki-67 positivity of cancer cells in H1-high cases wassignificantly higher than in the respective normal luminalcells. Therefore, the function of histone H1 in normal luminalcells may be different from that in malignant transformedprostate cells. Actually, histone H1 plays several roles inother histone modifications. For example, histone H1 affectsthe acetylation or phosphorylation of histone H2A and H2B,which regulate androgen receptor expression in prostatecancer cells.16 Histone H1 also affects the function ofsenescence-associated proteins, such as p21.17 Moreover,histone H1 function is altered by the status of cancer cells.48

In conclusion, histone H1 expression is closely associatedwith malignant potential of prostate cancer and may be auseful marker of cell proliferation. Histone H1 might also be atherapeutic target of prostate cancer, but further studies are

needed to assess mechanisms of histone H1-associated cellproliferation and AR expression.

ACKNOWLEDGMENT

The authors thank Koji Kato for assistance in processingspecimens. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education,Science, Sports and Culture of Japan, a Grant-in-Aid forCancer Research from the Ministry of Health, Labour andWelfare of Japan, and a grant from the Society for Promotionof Pathology of Nagoya, Japan.

REFERENCES

1 Fairley L, Baker M, Whiteway J, Cross W, Forman D. Trends innon-metastatic prostate cancer management in the Northernand Yorkshire region of England, 2000-2006. Br J Cancer 2009;101: 1839–45.

2 Fitzgerald TJ, Wang T, Goel HL et al. Prostate carcinoma andradiation therapy: Therapeutic treatment resistance and strate-gies for targeted therapeutic intervention. Expert Rev Antican-cer Ther 2008; 8: 967–74.

3 Jani AB, Hellman S. Early prostate cancer: Clinical decision-making. Lancet 2003; 361: 1045–53.

4 Merglen A, Schmidlin F, Fioretta G et al. Short- and long-termmortality with localized prostate cancer. Arch Intern Med 2007;167: 1944–50.

5 Gillatt D. Antiandrogen treatments in locally advanced prostatecancer: Are they all the same? J Cancer Res Clin Oncol 2006;132 (Suppl. 1): S17–26.

6 Ahlgren G, Pedersen K, Lundberg S, Aus G, Hugosson J,Abrahamsson PA. Tumor cell proliferation in prostate cancerafter 3 months of neoadjuvant LHRH analogue treatment is aprognostic marker of recurrence after radical prostatectomy.Urology 1999; 54: 329–34.

7 Bolla M, van Poppel H, Collette L et al. Postoperative radio-therapy after radical prostatectomy: A randomised controlledtrial (EORTC trial 22911). Lancet 2005; 366: 572–8.

8 Mimori K, Ogawa K, Okamoto M, Sudo T, Inoue H, Mori M.Clinical significance of enhancer of zeste homolog 2 expressionin colorectal cancer cases. Eur J Surg Oncol 2005; 31: 376–80.

9 Collett K, Eide GE, Arnes J et al. Expression of enhancer ofzeste homologue 2 is significantly associated with increasedtumor cell proliferation and is a marker of aggressive breastcancer. Clin Cancer Res 2006; 12: 1168–74.

10 Baylin SB, Ohm JE. Epigenetic gene silencing in cancer—amechanism for early oncogenic pathway addiction? Nat RevCancer 2006; 6: 107–16.

11 Woodcock CL, Skoultchi AI, Fan Y. Role of linker histone inchromatin structure and function: H1 stoichiometry and nucleo-some repeat length. Chromosome Res 2006; 14: 17–25.

12 Grewal SI, Moazed D. Heterochromatin and epigenetic controlof gene expression. Science 2003; 301: 798–802.

13 Henikoff S. Nucleosome destabilization in the epigenetic regu-lation of gene expression. Nat Rev Genet 2008; 9: 15–26.

14 Pereira SL, Reeve JN. Histones and nucleosomes in Archaeaand Eukarya: A comparative analysis. Extremophiles 1998; 2:141–8.



15 Bednar J, Horowitz RA, Grigoryev SA et al. Nucleosomes, linkerDNA, and linker histone form a unique structural motif thatdirects the higher-order folding and compaction of chromatin.Proc Natl Acad Sci U S A 1998; 95: 14173–8.

16 Fan Y, Nikitina T, Zhao J et al. Histone H1 depletion inmammals alters global chromatin structure but causes specificchanges in gene regulation. Cell 2005; 123: 1199–212.

17 Funayama R, Saito M, Tanobe H, Ishikawa F. Loss of linkerhistone H1 in cellular senescence. J Cell Biol 2006; 175: 869–80.

18 Kostova NN, Srebreva LN, Milev AD et al. Immunohistochemi-cal demonstration of histone H1(0) in human breast carcinoma.Histochem Cell Biol 2005; 124: 435–43.

19 Sancho M, Diani E, Beato M, Jordan A. Depletion of humanhistone H1 variants uncovers specific roles in gene expressionand cell growth. PLoS Genet 2008; 4: e1000227.

20 Jia L, Shen HC, Wantroba M et al. Locus-wide chromatinremodeling and enhanced androgen receptor-mediated tran-scription in recurrent prostate tumor cells. Mol Cell Biol 2006;26: 7331–41.

21 Metzger E, Yin N, Wissmann M et al. Phosphorylation of histoneH3 at threonine 11 establishes a novel chromatin mark fortranscriptional regulation. Nat Cell Biol 2008; 10: 53–60.

22 Ai J, Wang Y, Dar JA et al. HDAC6 regulates androgen receptorhypersensitivity and nuclear localization via modulating Hsp90acetylation in castration-resistant prostate cancer. Mol Endo-crinol 2009; 23: 1963–72.

23 Zhu P, Zhou W, Wang J et al. A histone H2A deubiquitinasecomplex coordinating histone acetylation and H1 dissociation intranscriptional regulation. Mol Cell 2007; 27: 609–21.

24 Attard G, Swennenhuis JF, Olmos D et al. Characterization ofERG, AR and PTEN gene status in circulating tumor cells frompatients with castration-resistant prostate cancer. Cancer Res2009; 69: 2912–8.

25 Quero L, Giocanti N, Hennequin C, Favaudon V. Antagonisticinteraction between bicalutamide (Casodex) and radiation inandrogen-positive prostate cancer LNCaP cells. Prostate 2010;70: 401–11.

26 Mendiratta P, Mostaghel E, Guinney J et al. Genomic strategyfor targeting therapy in castration-resistant prostate cancer. JClin Oncol 2009; 27: 2022–9.

27 Murphy GP, Busch C, Abrahamsson PA et al. Histopathology oflocalized prostate cancer. Consensus Conference on Diagnosisand Prognostic Parameters in Localized Prostate Cancer.Stockholm, Sweden, May 12-13, 1993. Scand J Urol NephrolSuppl 1994; 162: 7–42. discussion 115-27.

28 Pan CC, Potter SR, Partin AW, Epstein JI. The prognosticsignificance of tertiary Gleason patterns of higher grade inradical prostatectomy specimens: A proposal to modify theGleason grading system. Am J Surg Pathol 2000; 24: 563–9.

29 Humphrey PA. Gleason grading and prognostic factors in car-cinoma of the prostate. Mod Pathol 2004; 17: 292–306.

30 Kitagawa Y, Koshida K, Mizokami A et al. Pathological effects ofneoadjuvant hormonal therapy help predict progression of pros-tate cancer after radical prostatectomy. Int J Urol 2003; 10:377–82.

31 Shirai T. Significance of chemoprevention for prostate cancerdevelopment: Experimental in vivo approaches to chemopre-vention. Pathol Int 2008; 58: 1–16.

32 Kawai N, Ito A, Nakahara Y et al. Anticancer effect of hyper-thermia on prostate cancer mediated by magnetite cationicliposomes and immune-response induction in transplanted syn-geneic rats. Prostate 2005; 64: 373–81.

33 Nakanishi H, Takeuchi S, Kato K et al. Establishment and char-acterization of three androgen-independent, metastatic carci-noma cell lines from 3,2′-dimethyl-4-aminobiphenyl-inducedprostatic tumors in F344 rats. Jpn J Cancer Res 1996; 87:1218–26.

34 Contreras A, Hale TK, Stenoien DL, Rosen JM, Mancini MA,Herrera RE. The dynamic mobility of histone H1 is regulated bycyclin/CDK phosphorylation. Mol Cell Biol 2003; 23: 8626–36.

35 Linja MJ, Savinainen KJ, Saramaki OR, Tammela TL, VessellaRL, Visakorpi T. Amplification and overexpression of androgenreceptor gene in hormone-refractory prostate cancer. CancerRes 2001; 61: 3550–55.

36 Donovan MJ, Hamann S, Clayton M et al. Systems pathologyapproach for the prediction of prostate cancer progression afterradical prostatectomy. J Clin Oncol 2008; 26: 3923–9.

37 Attar RM, Takimoto CH, Gottardis MM. Castration-resistantprostate cancer: Locking up the molecular escape routes. ClinCancer Res 2009; 15: 3251–5.

38 Gleason DF. Classification of prostatic carcinomas. CancerChemother Rep 1966; 50: 125–8.

39 Epstein JI, Allsbrook WC Jr, Amin MB, Egevad LL. The 2005international society of urological pathology (ISUP) consensusconference on gleason grading of prostatic carcinoma. Am JSurg Pathol 2005; 29: 1228–42.

40 Hofer MD, Kuefer R, Huang W et al. Prognostic factors in lymphnode-positive prostate cancer. Urology 2006; 67: 1016–21.

41 Harnden P, Shelley MD, Coles B, Staffurth J, Mason MD.Should the Gleason grading system for prostate cancerbe modified to account for high-grade tertiary components? Asystematic review and meta-analysis. Lancet Oncol 2007; 8:411–9.

42 Edwards J, Krishna NS, Grigor KM, Bartlett JM. Androgenreceptor gene amplification and protein expression in hormonerefractory prostate cancer. Br J Cancer 2003; 89: 552–6.

43 Kawata H, Ishikura N, Watanabe M, Nishimoto A, Tsunenari T,Aoki Y. Prolonged treatment with bicalutamide induces andro-gen receptor overexpression and androgen hypersensitivity.Prostate 2010; 70: 745–54.

44 Tso CL, McBride WH, Sun J et al. Androgen deprivation inducesselective outgrowth of aggressive hormone-refractory prostatecancer clones expressing distinct cellular and molecular prop-erties not present in parental androgen-dependent cancer cells.Cancer J 2000; 6: 220–33.

45 Vis AN, Schroder FH. Key targets of hormonal treatment ofprostate cancer. Part 1: The androgen receptor and ste-roidogenic pathways. BJU Int 2009; 104: 438–48.

46 Waltering KK, Helenius MA, Sahu B et al. Increased expressionof androgen receptor sensitizes prostate cancer cells to lowlevels of androgens. Cancer Res 2009; 69: 8141–9.

47 Misteli T. Beyond the sequence: Cellular organization ofgenome function. Cell 2007; 128: 787–800.

48 Ninios YP, Sekeri-Pataryas KE, Sourlingas TG. Histone H1subtype preferences of DFF40 and possible nuclear localizationof DFF40/45 in normal and trichostatin A-treated NB4 leukemiccells. Apoptosis 2010; 15: 128–38.

92 S. Sato et al.


histone h1 expression in human prostate cancer tissues and cell lines

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