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1 J Korean Cancer Assoc 2001;33(2): Permanent Genotypic and Phenotypic Change of Prostate Cancer Cell Line LNCaP through Cellular Interactions with Prostate or Bone Fibroblasts in vitro or in vivo Ho ng-woo Rhee, M.D., S ung Hak Kang, M.D., Tae -Kon Hwang, M.D. and Le land W.K. Chung, Ph.D. 1 Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Korea; 1 Department of Urology, University of Virginia Health Science Center, Charlottesville, USA Purpose: Cell-cell interactions determine normal pros - tate deve lopment and s ubs eque nt neoplas tic transfo r- mation. The prog res s io n of pros tate cancer fro m androge n-depe nde nt to androge n-inde pe nde nt s tates invo l- ves multiple s te ps of genetic c hanges mediated by tumor-mic roenvironment inte ractio ns. To unders tand the epigenetic facto rs that lead to prog res s ion, we s tudied if 1) androgen-dependent and non-metastatic LNCaP may inte ract with pros tate or bo ne fibro blas ts unde r mic rogravity-simulated conditions in vitro. 2) LNCaP may inte ract with prostate fibro blas ts in vivo, and acquire androge n-inde pe nde nce and metastatic pote ntial. Materials and Methods : The LNCaP s ublines we re ge n- erated as follows. 1) LNCaP cells were grown in vitro either alo ne o r with prostate o r bone fibroblas ts unde r microgravity-simulated conditions. 2) LNCaP cells were grown in vivo as c hime ric tumo rs with pros tate fibro blas ts. The LNCaP s ublines we re c haracte rized by studies of chromosomal analys is, comparative genomic hybridization and, in vivo tumo rige nic ity and metas tatic pote ntial. Results : In co mpariso n to the pare ntal LNCaP ce lls, the LNCaP s ublines unde rwe nt pe rmanent genotypic and phe notypic c hanges manifested in androge n-inde pe nde n- ce and metastatic pote ntial. Conclusion: These res ults e mphas ize the importance of cell-cell interaction as a critical determinant that could induce o r s e lect proge nies favoring e nhanced pros - tate cance r g rowth and prog res s io n. This conce pt favors the deve lopme nt of toxic ge ne the rapy targeting both prostate cancer epithelium and supporting bone stroma fo r an effective e radicatio n and prevention of pros tate cancer bone metas tas is. (J Korean Cancer Assoc 2001; 33: ) Key Words : Pros tate neoplas m, Ce ll-ce ll inte ractio n, Prog res s io n (1)., (2). 9%, (3). -, , Tel: , Fax: rhw@cmc.cuk.ac.kr , (4). NbMC-2 (basement menbrane-like synthetic matrix)matrigel - in vitro NbMC-2 (5), LNCaP (bone fibroblast) - C4-2C4-2(B) in vivo (6)., in vitroin vivo -. (microgravity-simulated condition)in vitro

Hong-Woo Rhee, et alpermanent Genotypic and Phenotypic Change of LNCaP through Cellular Interactions 169 3 - -. LNCaP, (prostate fibroblast) 3 LNCaP. In vivo LNCaP.

2 Hong-Woo Rhee, et alpermanent Genotypic and Phenotypic Change of LNCaP through Cellular Interactions LNCaP, (prostate fibroblast) 3 LNCaP. In vivo LNCaP. LNCaP in vitro in vivo - LNCaP LNCaP. 1) In vitro LNCaP Rotating-wall vessel (RWV; Synthecon, Friendswood, TX), CO2. RWV. microcarrier beads. LNCaP RWV1, 2, 3. RWV1 LNCaP 10% fetal bovine serum (FBS) T-(80% DMEM [Dulbecco's modified Eagle's medium, Gibco Laboratories, Grand Island, NY], 20% F12K [Irvine Scientific, Santa Anna, CA], 3 gm/l NaHCO3, 100 U/ml penicillin G, 100 ug/ml streptomycin, 5 ug/ml insulin, 13.6 pg/ml triiodothyronine, 5 ug/ml transferrin, 0.25 ug/ml Fig. 1. A scheme of derivation of RWV sublines from LNCaP cell line cultured alone and co-cultured either human fibroblasts of prostate or bone under the microgravity-simulated conditions and photography of RWV (rotating-wall vessel) bioreactor designed to be placed inside a humidified CO incubator.

3 170 J Korean Cancer Assoc 2001;33(2) biotin and 25 ug/ml adenine) /ml RWV. RWV2 LNCaP /ml, RWV3 LNCaP (MS cell) /ml (Fig. 1). microcarriertype I collagen-coated dextran bead (Cytodex; Amersham Pharmacia Biotech, Piscataway, NJ)5 mg/ml. RWV 2530 rpm,. RWV5% CO2, 37 o C, 2. 2) In vivo LNCaP Wu(7) chimeric LNCaP LNCaP (BALB/C strain; Charles River Laboratory, Wilmington, MA) (n=6). LNCaP ATCC (American Type Culture Collection), passage number 2023LNCaP. 8, 4. P4 ( LNCaP ) T4 ( LNCaP ) LNCaP chimeric tumor., 1 mm 5% fetal bovine serum (FBS)T-. (cell splitting). 2 P4T4 LNCaP cytokeratin. P4T4 5% FBST P4 T4 (n=6) LNCaP P4-2T4-2 (Fig. 2). 3) (conventional chromosomal analysis) LNCaPRWV Colcemid (0.02g/ml) Fig. 2. A scheme of the derivation of androgen-independent P and T sublines from LNCaP chimeric tumors maintained in intact or castrated

Hong-Woo Rhee, et alpermanent Genotypic and Phenotypic Change of LNCaP through Cellular Interactions 171 37 o C 30Hank's balanced solution (HBBS)2. 0.01% trypsin HBBS.

4 Hong-Woo Rhee, et alpermanent Genotypic and Phenotypic Change of LNCaP through Cellular Interactions o C 30Hank's balanced solution (HBBS) % trypsin HBBS. 10% FBS RPMI1640, (hypotonic solution; 0.06 M KCl) acetic acid: methanol (31, V/V)20, G-banding Giemsa (8). 4) Comparative genomic hybridization (CGH) CGHHyytinen(9). Nick translation DNAfluorescein isothiocyanate (FITC)-dUTP, DNATexas red-dutp (dutp; DuPont, Boston, MA)labelling. Labelling DNA 10glabelling Cot-1 DNA metaphasedna. 2, Vectashield antishade (Vector Laboratories, Burlingame, CA, USA) 1M 4,6- diamidine-2-phenylindole (DAPI). metaphase, 33 DNA FITC, DNA Texas Red DAPI. DNA copy labelling DNA. Background metaphase 1.0. CGH copy (chromosomal loss), 1.15 (chromosomal gain). Texas redlabelling DNA FITClabelling DNA, MCF-7. 5) 68(BALB/C strain; Charles River Laboratory).. LNCaP, 5% FBS T- RWV1, 2, 3 P4, P4-2, T4, T4-2 (1 10 6, 20 l) (dorsolateral lobe) (orthotopical injection).. 10% paraffin hematoxylin-eosin. PSA monoclonal PSA (Biogenex, Dublin, CA), fast-red AEC (Biogenex, Dublin, CA)chromogen, avidin-biotin complex. Rat anti-ig G. 1) In vitro LNCaP 2 3 (prostate organoid) (Fig. 3), organoid 5% FBS T-. RWV2, 3 RWV2, 3. cytokeratin. RWV1, 2, 3 5% FBS T-,. Fig. 3. Three-dimensional prostate organoids formed under the microgravity-simulated conditions using RWV. This 3-dimensional growth of cells or tissue cannot be done under conventional culture conditions.

5 172 J Korean Cancer Assoc 2001;33(2) 2) In vivo LNCaP 5% FBS T- 2 P4, P4-2T4, T4-2 LNCaP cytokeratin. P4, P4-2, Table 1. Conventional chromosomal analysis of LNCaP and RWV sublines Cell type Marker chromosome Note LNCaP M1 to M6 RWV1 M1 to M6 same as LNCaP RWV2 M1 to M6 different, however 2 new marker chromosomes LNCaP lineage (m & m1) RWV3 M1 to M6, m & m1 same as RWV2 T4, T4-2 5% FBST-,. 3) LNCaP 80 M1 M6 (marker chromosome), telometric association. RWV1 LNCaP. RWV2 80 M1 M6 LNCaP. 2 (m, m1) 3 9. Telometric association hyperploid metaphase. RWV3 RWV2 (Table 1). 4) Comparative genomic hybridization (CGH) LNCaP RWV2 Table 2. Comparative genomic hybridization of LNCaP, RWV and P & T sublines Cell type Chromosomal loss Chromosomal gain LNCaP 2, 4q23-qter, 6pter-q16, 13q, 19p, 20p 3q21q24, 3q27-qter, 17 RWV1 2, 4p15-qter, 6pter-q16, 13q, 20p 3q21q24, 3q27-qter, 17, 19p RWV2 1p34, 2, 4q22-qter, 6pter-q16, 13q13-qter 3q RWV3 2, 4p15-qter, 6pter-q16, 13q, 18, 20p 3q21q24, 3q27-qter, 17, 19p P4 2, 4q23-qter, 6 pter-q16, 13q, 20p 16 P4-2 2, 4p15-qter, 6 pter-q16, 7p, 13q, 20p 16p, 19p T4 2, 4q23-qter, 6 pter-q16, 13q 16p, 17, 19p T4-2 2, 4p15-qter, 6, 13q, 20p none Table 3. Tumorigenicity and metastatic potentials in castrated hosts administered LNCaP, RWV and P & T sublines orthotopically Cell type Tumorigenicity Metastasis (%)* Primary (%)* Lymph node Lung Liver Bone LNCaP 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) RWV1 7/8 (87.5) 7/8 (87.5) 1/8 (12.5) 0/8 (0) 5/8 (62.5) RWV2 7/8 (87.5) 7/8 (87.5) 1/8 (12.5) 1/8 (12.5) 7/8 (87.5) RWV3 8/8 (100) 7/8 (87.5) 2/8 (25) 2/8 (25) 5/8 (62.5) P4 9/ 10 (90) 9/ 10 (90) 2/ 10 (20) 1/ 10 (10) 6/ 10 (60) P4-2 10/ 10 (100) 10/ 10 (100) 3/ 10 (30) 9/ 10 (90) T4 10/ 10 (100) 10/ 10 (100) 7/ 10 (70) T4-2 8/9 (88.8) 8/9 (88.8) 3/9 (33.3) 6/9 (66.6)

6 Hong-Woo Rhee, et alpermanent Genotypic and Phenotypic Change of LNCaP through Cellular Interactions 173 Fig. 4. Histomorphologic and PSA immunohistochemical characterization of both the primary and metastatic prostate tumors of RWV sublines. A. Primary tumor (H & E, 200). B. Primary tumor (PSA staining, 200). C. Metastatic tumor, bone (H & E, 200). D. Metastatic tumor, bone (PSA staining, 400). E. Metastatic tumor, lymph node (H & E, 200). F. Metastatic tumor, lung (H & E, 200). G. Metastatic tumor, liver (H & E, 200). Arrows indicate tumor areas and arrow heads indicate the ( ) PSA staining areas (reddish-brown color).

174 J Korean Cancer Assoc 2001;33(2) Fig. 5. Histomorphologic and PSA immunohistochemical characterization of both the primary and metastatic prostate tumors of P & T sublines. A. Primary tumor (H & E, 200).

7 174 J Korean Cancer Assoc 2001;33(2) Fig. 5. Histomorphologic and PSA immunohistochemical characterization of both the primary and metastatic prostate tumors of P & T sublines. A. Primary tumor (H & E, 200). B. Primary tumor (PSA staining, 200). C. Metastatic tumor, lymph node (H & E, 400). D. Metastatic tumor, lymph node (PSA staining, 200). E. Metastatic tumor, bone (H & E, 100). F. Metastatic tumor, lung (H & E, 100). G. metastatic tumor, liver (H & E, 200). Arrows indicate tumor areas and arrow heads indicate the ( ) PSA staining areas (reddish-brown color).

8 Hong-Woo Rhee, et alpermanent Genotypic and Phenotypic Change of LNCaP through Cellular Interactions 175, RWV1, 3 RWV. P4T4 LNCaP, P4-2T4-2 P4, P4-2, T4, T4-2 (Table 2). 5) 620 LNCaP, RWV1, 2, 3 P4, P4-2, T4, T4-2,, (Table 3)..,. LNCaPRWV P4, P4-2, T4, T4-2. PSA LNCaP (Fig. 4, 5). (10).,, - (11). -, - (4). - (rat fetal urogenital sinus)in vivoin vitro (12,13). in vivo - - in vitro. in vitro NASA 3. Prewette(14) in vitro,,, cells/ml. 3 (shearing force)3. 3 (cell assembly) (multicelluar response) (15). 3 RWV LNCaP,. RWV LNCaP LNCaP, CGH LNCaPRWV, RWV1, 2, 3 LNCaP. LNCaP 19pRWV1, 3 19p (16), 19p, 1p34 1p36(17) RWV2 1p34, RWV318 (18). RWV.

9 176 J Korean Cancer Assoc 2001;33(2) CGH G-band, (polyploidy) G-band (19). CGH2 (9) CGH. RWV,,. LNCaPRWV, PSA LNCaP RWV. 3 -, RWV (multistep carcinogenesis),. Thalmann (6)in vivo LNCaP MS chimeric tumor LNCaP C4-2. C4-2 LNCaP. LNCaP.. --, clone, LNCaP in vivo -- LNCaP...., (tumor-restricted and promoter-mediated toxic gene delivery system). Gardner(20) thymidine kinase osteocalcin promoter bystander effect.. LNCaP , Nowell PC. The clonal evolution of tumor cell populations. Science 1976;194: Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61: Carter BS, Beaty TH, Steinberg GD, Childs B, Walsh PC. Mendelian inheritance of familial prostate cancer. Proc Natl

10 Hong-Woo Rhee, et alpermanent Genotypic and Phenotypic Change of LNCaP through Cellular Interactions 177 Acad Sci 1992;89: Chung LWK. The role of stromal-epithelial interaction in normal and malignant growth. Cancer Surv 1995;23: Freeman MR, Bagli DJ, Lamb CC, Guthrie PD, Uchida T, Slavin RE, Chung LWK. Culture of prostatic cell line in basement membrane gels results in an enhancement of malignantproperties and constitutive alterations in gene expression. J Cell Physiol 1994;158: Thalmann GN, Anezinis PE, Chang SM, Zhau HE, Kim EE, Hopwood VL, Pathak S, von Eschenbach AC, Chung LWK. Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer. Cancer Res 1994;54: Wu HC, Hsieh JT, Gleave ME, Brown NM, Pathak S, Chung LWK. Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: role of bone stromal cells. Int J Cancer 1994;57: Pathak S. Chromosome banding techniques. J Reprod Med 1976;17: Hyytinen ER, Thalmann GN, Zhau HE, Karhu R, Kallioniemi OP, Chung LWK, Visakorpi T. Genetic changes associated with the acquisition of androgen-independent growth, tumorigenicity and metastatic potential in the prostate cancer model. Br J Cancer 1997;75: Neubauer BL, Best KL, Hoover DM, Slisz ML, van Frank RM, Goode RL. Mesenchymal-epithelial interactions as factors influencing male accessory sex organ growth in the rat. Federation Proc 1986;45: Camps JL, Chang SM, Hsu TC, Freeman MR, Hong SJ, Zhau HE, von Eschenbach AC, Chung LWK. Fibroblast-mediated acceleration of human epithelial tumor growth in vivo. Proc Natl Acad Sci 1990;87: Gleave ME, Hsieh JT, Gao C, Chung LWK, von Eschenbach AC. Acceleration of human prostate cancer growth in vivo by prostate and bone fibroblasts. Cancer Res 1991;51: Zhau HE, Hong SJ, Chung LWK. A fetal rat urogenital sinus mesenchymal cell line (rugm): accelerated growth and conferral of androgen-induced growth responsiveness upon a human bladder cancer epithelial cell line in vivo. Int J Cancer 1994;56: Prewett TL, Goodwin TJ, Spaulding GF. Three-dimensional modeling of T-24 bladder carcinoma cell line: a new simulated microgravity culture vessel. J Tissue Cult Methods 1993;15: Jessup JM, Goodwin TJ, Spaulding GF. Prospects for use of microgravity-based bioreactors to study three-dimensional hosttumor interactions in human neoplasia. J Cell Biochem 1993; 51: Gao AC, Lou W, Ichikawa T, Denmeade SR, Barrett JC, Issacs JT. Suppression of the tumorigenicity of prostatic cancer cells by gene(s) located on human chromosome 19p Prostate 1999;38: Gibbs M, Stanford JL, McIndoe RA, Jarvik GP, Kolb S, Goode EL, Chakrabarti L, Schuster EF, Buckley VA, Miller EL, Brandzel S, Li S, Hood L, Ostrander EA. Evidence for a rare prostate cancer-susceptibility locus at chromosome 1p36. Am J Human Genetics 1999;64: Padalecki SS, Troyer DA, Hansen MF, Saric T, Schneider BG, O'Connel P, Leach RJ. Identification of two distinct regions of allelic imbalance on chromosome 18q in metastatic prostate cancer. Int J Cancer 2000;85: Ha MJ, Park SJ, Kang HW, Kim HC, Kim HJ. Chromosomal alterations in gastric cancer cell lines detected by comparative genomic hybridization. J Korean Cancer Assoc 1999;31: Gardner TA, Ko SC, Kao C, Shirakawa T, Cheon J, Gotoh A, Chung LWK. Exploiting stromal-epithelial interaction for model development and new strategies of gene therapy for prostate cancer and osteosarcoma metastases. Gene Ther Mol Biol 1998;2:41-58.

Can032.hwp

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歯Can022.PDF