CELL LINES USED IN PROSTATE CANCER RESEARCH: A COMPENDIUM OF OLD AND NEW LINES—PART 2

0022-5347/05/1732-0360/0 THE JOURNAL OF UROLOGY® Copyright © 2005 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 173, 360 –372, February 2005 Printed in U.S.A.

DOI: 10.1097/01.ju.0000149989.01263.dc

CELL LINES USED IN PROSTATE CANCER RESEARCH: A COMPENDIUM OF OLD AND NEW LINES—PART 2 R. E. SOBEL*

AND

M. D. SADAR

From the Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada

ABSTRACT

Purpose: This is part 2 of a 2-part review. Research into the molecular mechanisms underlying the various aspects of prostate cancer (PCa) requires the use of in vivo and in vitro model systems. In the last few years many new cell lines have been established by investigators from primary tissue sources and clonal derivatives of previously established lines. Therefore, the purpose of this 2-part review is to catalogue the current human cell lines developed for PCa research, as reported in the literature. Part 2 describes tissue culture cell lines derived by the insertion of transgenes, including human telomerase reverse transcriptase, SV40 T antigen and human papillomavirus genes. Part 2 also includes xenograft lines that require propagation and passage in vivo in mice. Materials and Methods: Prostate cell lines included in this review were identified by extensive searching of the literature using several strategies, including PubMed searches and book chapter reviews. Results: In total we describe the derivation, phenotype, genotype and characterization of molecular markers expressed by approximately 200 lines and sublines used in PCa research, including ones derived from primary tumors, metastases and normal prostate tissue. We paid particular attention to the expression of prostate specific antigen, androgen receptor, cytokeratins and other molecular markers used to indicate the status of PCa and the prostatic lineage of a given line. In an attempt to provide PCa researchers with a resource of information regarding new and established cell lines we have also created an online database of these PCa cell lines freely accessible via the World Wide Web at http://www.CaPCellLines.com. The web based interface allows researchers to peruse and print information regarding cell lines, add new cell lines and update or add new information regarding established cell lines. Conclusions: This compendium of cell lines currently used in PCa research combined with access to our on-line database provides researchers with a continually updated and valuable resource for investigating the molecular mechanisms of PCa. KEY WORDS: prostatic neoplasms; prostate-specific antigen; tumor cells, cultured; review (publication type); human

Understanding the molecular perturbations governing oncogenesis and subsequent tumor progression is an essential first step in the design of effective therapies. While whole animal models are often beneficial in this regard, they present significant drawbacks, including ethical concerns regarding use and treatment, housing costs and the lack of appropriate models for the cancer being studied. Prostate cancer (PCa) is no exception. Currently there is no animal model in which all aspects of human PCa can be mimicked. Older male canines are the only mammals known naturally to develop PCa but there is no way to reliably obtain such animals for research.1 To overcome these limitations cancer researchers have concentrated a significant amount of their energy and funding into developing cell culture models that mimic various cancer stages, conditions of cancer progression and tissue specific metastatic sites (eg metastasis to bone for PCa). As a result of these efforts many new cell lines and in vivo models have been developed in recent years to address problems unique to PCa. Hallmarks of the disease include multiple foci, initially slow rates of growth, increased levels of prostate specific antigen (PSA) and other molecular markers in serum, temporary shrinkage or interruption in the growth of tumors in response to androgen ablation therapy, a

transition phase during which the cancer acquires an ability to grow in the absence of hormones and metastasis to bone in late stage PCa. Initially in the PCa research field there were only 3 tissue culture cell lines used by researchers for their investigations, namely LNCaP, PC-3 and DU-145 cells, and a few clonal derivations of these lines. These cell lines are still the most popular for the majority of the published research. However, over the years a number of new cell lines and clonal derivatives of these original lines have been developed by investigators using a variety of methods, including chemical mutagenesis, genetic alterations induced by castration of host animals bearing xenograft tumors and viral transformation. These cells have also been used to address various problems encountered in understanding and treating cancer progression and metastasis. Unfortunately there is no single place for PCa researchers to access all of the information that is known about these important resources without performing tedious, time-consuming and exhaustive literature searches. For instance, there was an excellent 3-part review in 1996 to 1997 by Webber et al that summarized many cell lines in use at the time2⫺4 and a book chapter that reviewed xenograft models5 but even they do not contain complete information about established cell lines and they omitted the sublines. In the 7 years that have elapsed since the first installment of the series of Webber et al a number of significant new cell lines have been generated. In this 2-part review we have

Supported by a grant from Health Canada (MS). * Correspondence: Genome Sciences Centre, British Columbia Cancer Research Centre, 601 West 10th Ave., Vancouver, British Columbia V5Z 1L3, Canada (telephone: 604-877-6018; FAX: 604-8776011; e-mail: [email protected]). 360

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catalogued the entire array of human PCa cell lines, outlined the methods and sources used to derive them and most importantly summarized the characterizations of their expression of PSA, androgen receptor (AR), human glandular kallikrein 1 (hK2) and additional relevant molecular markers. The serum level of PSA, also known as human kallikrein 3, is the most commonly used molecular marker for screening, early detection and monitoring of the recurrence of PCa. Recently serum levels of hK2 have also been found to correlate highly with PCa, especially for organ confined disease. To accomplish our goal of presenting a complete catalogue we have also included cell lines originally delineated as PCa lines but subsequently determined to be previously established lines or lines derived from nonprostate tissues. It is important to note that in this catalogue we have not included cell lines derived from nonhuman species or primary cell lines that are commercially available. When possible the age, ethnicity, tumor stage, Gleason grade and treatment regimen in the patient from whom the source material was taken was included to characterize more fully the origins of the various lines. Unfortunately not all original reports include these data. We hope that authors will use the website database to correct these deficiencies in the literature and make these data available to the PCa research community. Undoubtedly new cell lines and model systems will be devised by PCa researchers that would normally require future revisions of this catalogue. To circumvent this problem we have established a prostate cell line database that is freely accessible to anyone via the World Wide Web. Researchers can interface with the database to 1) extract information regarding a particular cell line, 2) enter new information regarding a cell line, 3) share their problems/experiences with using a particular cell line, 4) share their problems/ experiences with using the database and 5) enter a new cell line that they have developed and are willing to provide to other researchers. The database can be accessed at http:// www.CaPCellLines.com. IN VIVO TUMOR FORMATION AND METASTASIS

An important characteristic of many cell lines is their ability to form tumors in hosts such as nude or SCID mice. Not only the ability to form tumors but also whether or not they metastasize is an important characteristic of a given cell line. Indeed, many of the sublines described below were selected for increased aggressiveness of tumor formation in vivo and/or acquisition of the ability to metastasize. Table 1 shows a summary of lines that form tumors in vivo. We make no distinction as to how the lines were derived, merely whether or not they form xenograft tumors and their respective sites of metastasis where appropriate. Lines that are maintained exclusively as xenograft tumors in mice were included only if they also showed the ability to metastasize. To save space, the derivation of these lines has been omitted from the table but can be found below in the description of the line and also in a table available for download from our cell lines website (http://www.CaPCellLines.com). CELL LINES USED IN PROSTATE CANCER RESEARCH: CELL LINES IMMORTALIZED BY EXPOSURE TO HUMAN TELOMERASE REVERSE TRANSCRIPTASE

RC-58T and RC-58T/hTERT. RC-58T cells were derived from a radical prostatectomy specimen from a 52-year-old white man. After 6 passages as monolayers, these cells senesced. Introduction of a retrovirus construct that expresses human telomerase reverse transcriptase (hTERT), the integral catalytic subunit of telomerase,6 into passage 6 RC-58T cells resulted in an immortalized line designated RC-58T/ hTERT. In contrast to RC-58T, hTERT cells exhibit a typical transformed morphology with transformed foci, adherent

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growth and lack of contact inhibition. hTERT cells express NKX3.1, cytokeratin (CK)-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) but they do not express AR or PSA. They also express prostate stem cell antigen (PSCA) and p16. Interestingly some clonal lines subsequently derived from RC-58T/hTERT express AR and PSA. However, untransduced RC-58T cells express PSA and AR as well as NKX3.1, CK-8, GAPDH and PSCA. hTERT cells are also capable of forming colonies in soft agar and large cell aggregates are capable of growth above the agar. A dose dependent inhibition of cell growth occurs in response to transforming growth factor (TGF)-␤1 or retinoic acid. Cytogenetic examination using GTG-banding revealed that hTERT cells are hypotetraploid human male (XX/XXY) with a chromosome number of 74 to 88 and a modal number (MN) of 84. Chromosomes 2, 3, 4, 10, 11, 13, 14, 15, 17, 18 and 21 have prominent marker formations and many additional chromosomal alterations (losses of Y, 2q, 3p, 4q, 10p, 11p, 14q, 17p and 18q, and gains of 16 and 20). Thus, the line is highly transformed. CELL LINES USED IN PROSTATE CANCER RESEARCH: CELL LINES IMMORTALIZED BY EXPOSURE TO SV40 T-ANTIGEN

267B1, 272E1 and 272A9. These 3 lines were the first prostate epithelial cells immortalized using SV40 transforming sequences.7 They were derived from neonatal prostatic epithelial cells, designated NP-2s, which had acquired extended culture life spans via infection with SV40 virus.8 NP-2s cells were retrieved from frozen stocks, subcultured once and transformed with pRSV-T plasmid, which contains the SV40 early region genes and the Rous Sarcoma Virus long terminal repeat,9 that had been co-precipitated with strontium phosphate.7 Individual colonies were ringed with glass cylinders and replated. The ability to form colonies on a background of senescing cells was the selection criteria. Approximate doubling times uncorrected for plating efficiency at each passage were 18 hours for 267B1, 16 hours for 272A9 and 17 hours for 272E1. All 3 original colonies shared similar morphological phenotypes and formed small aggregates that developed into tightly packed, multilayered colonies that were difficult to reduce to single cell suspension. Cells were replated to culture dishes coated with a mixture of bovine serum albumin, bovine fibronectin and type 1 collagen to promote adhesion. All 3 lines tested positive for CK-8, 18, vimentin, actin and fibronectin. Heterogeneous staining results were obtained for PSA and prostatic acid phosphatase (PAP). While the karyotypes of early passage cells were mostly diploid, by later passages a significant number of cells in each line had become tetraploid and there was a higher proportion of abnormal metaphases, monosomy, trisomy, rearrangements, dicentrics and gaps. None of the lines formed tumors in nude mice 12 months following subcutaneous or intrasplenic injection. Southern blot analysis showed that all 3 lines had only a single site genomic insertion of the T antigen. 267B1 is also mentioned in a report by Rhim of in vitro models for PCa but for some reason it is called FNC269B1.10 When 267B1 cells were infected with Ki-MSV, containing a Ki-ras oncogene, several changes were noted.11 The cells began to pile up in focal areas, form small projections, release round cells from the foci and express p21 ras protein. No such changes were observed in control treatments. In addition, a higher frequency of cells acquired cytogenetic abnormalities in chromosomes 9, 11 and 19, and a greater percent were hypotetraploid. More importantly infected cells injected subcutaneous into nude mice formed tumors at the site of inoculation within 3 to 4 weeks. Cells recultured from the tumors and re-injected into mice had a 100% take rate compared with 0% for noninfected 267B1 parental cells. Thus, the 2-fold combination of SV40 oncogenic sequences and v-Ki-ras was sufficient to transform nonneoplastic neonatal human prostate epithelium (HPE) into cells capable of producing in vivo tumors in mice.

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PROSTATE CANCER CELL LINES PART 2 TABLE 1. Lines that form xenograft tumors in mice and tissues to which they metastasize Name

BPH1(TETD)-A BPH1(TETD)-B BPH1(CAFTD)-01 to 08 BPH1(CAFTD)-04 to 06 CA7T2CL CTPE HPV-18 C-1-(Ki-ras) HPV-18 C-1-40s HPV-18 C-1-50s HPV-18 C-1-40s-129, Nu 5002-1 Tu HPV-18 C-1-40s-129-Nu 5010-5 Tu LAPC-4

Injection Site

Injection Site Tumor

Subrenal capsule Subrenal capsule Subrenal capsule Subrenal capsule Subcutaneously Subcutaneously Subcutaneously Subcutaneously Subcutaneously Subcutaneously

x x x x x x x x x x

Subcutaneously

x

Subcutaneously Prostate Intratibially

x x

Metastasis Lymph Node

Lung

Bone

Liver

Other Metastases Kidney Kidney

30 41

x

34 10

5

x x

x x 79

x x x

Subcutaneously Prostate Intraperitoneally Subcutaneously Intraperitoneally

x x x x x

Prostate Subcutaneously Intraperitoneally

12, 78

Kidney

Near bone implant LAPC-42 Subcutaneously Prostate

References

53

x

LAPC-9

P69SV40T P69SV40T-M2182 P69SV40T-M12

5

x x

x x 15

x

Diaphragm

17

x x x

x

Diaphragm

15

Prostate Subcutaneously Intraperitoneally

x x

x

Under lower abdomen Subcutaneously Near bone implant

x x

Subcutaneously Subcutaneously Subcutaneously Subcutaneously Subcutaneously Subcutaneously Subcutaneously

x x x x x x x

80 80

P69SV40T-M15 Diaphragm

17 80 80

P69SV40T-M2205 PAC-120 WISH-PC2

WPE1-NA22 WPE1-NB14 WPE1-NB11 WPE1-NB26

15

x

80

x

55

x

Benign Prostatic Hyperplasia (BPH)-1, and Sublines BPH1(TETD) and BPH1(CAFTD): BPH-1 cells were derived from a 68-year-old patient undergoing transurethral resection of the prostate (TURP) to help clear urinary obstruction consistent with BPH. The patient had not undergone any hormonal therapy and did not have malignant disease12 Tissue was processed to remove as much stromal material as possible and epithelial organoids were plated and grown in flasks for 11 days with medium changes every other day. Subconfluent cultures were immortalized by infection with the ZipneoSV virus, which carries the SV40 T antigen gene, and colonies were selected for resistance to geneticin at 800 ␮g/ml. All colonies had identical phenotypes and 1 was selected for expansion and designated BPH-1. Tissue culture phenotype is a cobblestone morphology typical of epithelial cells grown in monolayer. Staining for SV40 T antigen was positive. Cells show strong nuclear staining for p53. Positive staining was also demonstrated for CK-7, 8, 18 and 19 but not for 14. Karyotypic analysis showed the cells to be aneuploid with a number of structurally rearranged chromosomes. The MN of chromosomes is 76 (range 71 to 79) and the presence of a Y chromosome confirms the male origin. There are 6 to 8 marker chromosomes and another 5 or 6 with structural rearrangements that were designated as derivatives. Cells failed to form tumors in nude mice, although

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Muscle Muscle

small clumps of cells could be recovered from the graft sites, indicating that cells survived in the host animals. Grafting with collagen gels had no additional effects. Serum-free proliferation was inhibited by fibroblast growth factor (FGF) 2, TGF-␤1 and ␤2, and increased by epidermal growth factor (EGF), TGF-␣, FGF-1 and FGF-7. Testosterone had no effect on proliferation but 5␣-reductase and 3␣-hydroxy-steroid oxidoreductase activities were detected. Reverse transcriptase (RT)-polymerase chain reaction (PCR) and Northern analysis failed to reveal AR mRNA expression. Likewise only faint staining for PAP was observed and none was noted for PSA. RT-PCR also failed to detect mRNA for either of these proteins. A series of tumorigenic sublines of BPH-1 were derived by recombination with carcinoma-associated fibroblasts (CAFs) or by exposure to a combination of carcinogenic doses of testosterone and estradiol.12, 13 Epithelial cells recovered from these tumors were used to establish sublines that were consistently able to form tumors upon grafting under the renal capsule of nude mice. The strains were designated BPH1(TETD)-A and B for lines derived from testosterone and estradiol induced tumors, and BPH1(CAFTD)-01 to 08 for lines derived from CAF treatment. The cell morphology for all lines is generally a cobblestone or slightly elongated morphology and cells are generally less adherent than the

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parental BPH-1 cells. All lines uniformly express SV40 T antigen as well as basal cell specific markers, high molecular weight keratins and p63. The luminal cell specific marker CK-8 was also detected. Not detected were AR, estrogen receptor-␣ and progesterone receptor. In contrast to the parental line, the TD lines were able to double several times when grown in medium supplemented with charcoal stripped serum. All BPH1(CAFTD) lines except 04 were able to form colonies in soft agar, whereas BPH1(TETD) and parental cells do not form colonies. BRF-41T and 55T: These 2 lines were derived from cancerous tissue (BRF-41T) and BPH (BRF-55T) by transfection with pRSV-T.9 BRF-41T was generated from a tumor explant from a 59-year-old white man. It has a doubling time of about 62 hours and is able to produce colonies in soft agar at an efficiency of approximately 3% when low cell densities were used. Cytogenetic analysis showed that the cells are aneuploid human male (XY/XO) with chromosome counts in the hypodiploid range. BRF-55T was derived from a 60-year-old white man, has a doubling time of approximately 116 hours and produced colonies in soft agar with an efficiency of less than 0.08% when cell densities similar to those of BRF-41T were used. It is also aneuploid male (X/Y) with chromosome numbers in the hypodiploid range. RT-PCR experiments with RNA from the 2 cell lines showed the expression of H-ras, K-ras, p53, AR, PSA, CK-8 and CK-18. An interesting facet of the derivation of these cell lines involved the creation of a serum-free medium for growing prostate cell lines called BRFF-HPC1. Its formulation is outlined in the original study.9 P69SV40T and Derivatives: Nonneoplastic prostate epithelial cells were isolated from surgical specimens by collagenase digestion and immortalized by transfection with the plasmid pcc5 that contains the SV40 large T antigen gene linked to the SV40 promoter region.14, 15 The parental P69SV40T line was established and characterized. The cells exhibit an epithelial morphology, express epithelial CKs, possess an human male XY karyotype and were pseudodiploid.14 This line was injected into nude mice and produced tumors in 2 of 18 animals after a 6-month latency period.15 Cells taken from these tumors after 1 or 2 cycles in mice produced tumors in 2 of 4 and 2 of 3, respectively, with latency periods of only 12 or 25 days. EGF stimulated proliferation in the parentals and sublines, whereas an antibody to TGF-␣ inhibited proliferation under serum-free conditions. The 3 sublines M2182, M12 and M15 were subsequently generated from the parental line by serial passage through nude mice.16, 17 M2182 is characterized as tumorigenic and M12 is capable of metastases. Cytogenetic studies on the parental and 2 sublines showed that all were near diploid with several consistent abnormalities.17 Parental P69SV40T cells exhibited karyotypic changes at increasing transfer numbers. At transfer 12 the MN of chromosomes was 45 (range 39 to 100), while at transfer 60, the MN increased to 78 (range 41 to 122).17 Tumor line M2182 had an MN of 46 chromosomes (range 44 to 46) at passage 9. Tumor line M15, the most tumorigenic one, had an MN of 43 chromosomes (range 40 to 43). Thus, although the 3 lines had varying chromosomal ranges, all had an MN that tended to be near diploid, indicating that aneuploidy may not be one of the early steps in tumorigenesis. PNT1: This line was derived from the prostate of a 35-yearold cadaver donor and it was the first adult prostate cell line to be immortalized with SV40.18 The cells were transfected via liposomes with a plasmid that contained a defective SV40 replication origin (pMK16/SV40 ori-). Cells have a doubling time of approximately 40 hours, are anchorage dependent and positive for AR despite a lack of growth response to dihydrotestosterone (DHT) in medium containing growth factors and 2% horse serum. The cells express large T antigen and exhibit a morphology representative of differentiated

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luminal prostatic cells. The cells stain strongly positive using antibodies to CK-18 and 19, weakly positive for PAP and PSA, and negative for CK-14 and KL2. By passage 10 PSA expression had decreased to undetectable levels. pRNS-1–1, pRNS-1–1/ras and SCID 5045– 4: The parental pRNS-1–1 line was derived from HPE cells isolated from the normal peripheral zone of a 53-year-old man who underwent radical prostatectomy.10, 19 Immortalization was performed by transfection with pRSV-T and a plasmid containing the neomycin resistance gene. Colonies were isolated using cloning cylinders and 3 transformed lines, namely pRNS-1–1, pRSVT-1 and AS-1, were isolated. Only pRNS-1–1 became immortalized since the other 2 lines senesced after 40 to 46 population doublings. In the 1994 report of Lee et al figure 2 shows a pRNS-1–1 population doubling time of approximately 72 hours.19 Cells retain a Y chromosome and have a modal chromosome number of 49 to 52. Cytogenetic abnormalities include monosomy 5 and 16, and 3 marker chromosomes. Cells express CK-5 and 8.10, 19 PSA expression was detected in parental and early passage immortalized cells but it was absent in later passages. Growth is stimulated by EGF and insulin-like growth factor (IGF), and inhibited by TGF-␤ at physiological levels. pRNS-1–1 cells failed to grow in soft agar or produce tumors in nude mice.10, 19 Cells grown in collagen form round aggregates, which frequently disintegrate after 7 days in culture. However, when pRNS-1–1 aggregates are treated with hepatocyte growth factor/scatter factor (HGF/SF), a ligand of the tyrosine kinase receptor c-Met, they form long ducts that exhibit branching at the distal tip and are still present in culture after 20 days. These structures are reminiscent of those seen in the developing prostate. The other prostate cell lines tested, that is LNCaP, PC-3, DU-145 and 267B1, showed only minor morphological changes after exposure to HGF/SF.20 Two methods of treating pRNS-1–1 cells resulted in further transformation of this line. Introduction of the Ki-ras oncogene resulted in morphological and tumorigenicity changes.21 Within 2 to 3 weeks after infection the cells began to pile up in focal areas, form projections and release round cells from the foci. In contrast to parental cells, saturation density increased more than 2-fold, and cells formed colonies in soft agar and tumors in nude mice. Cells from one of these tumors were reestablished in vitro and designated as pRNS1–1/ras. The line grows equally well in the presence or absence of EGF, a factor that is absolutely required by the parental line. The second method involved treating the cells with differing doses of cadmium. Cells exposed twice to 10 ␮M cadmium developed an altered morphology after 10 weeks and the eighth passage. Cells began to pile up in focal areas, formed small projections and released round cells from the foci. Foci grew as islets. Unlike 0, 1 or 5 ␮M cadmium treated parentals 10 ␮M treated cells were able to grow in soft agar and form tumors at the site of inoculation when injected subcutaneously in SCID mice. Cell cultures established from these tumors were designated as the line SCID 5045– 4 and further characterized. RT-PCR testing of 267B1, pRNS-1–1 and SCID 5045– 4 cells confirmed the expression of AR, PSA, PSCA, NKX3.1, CK-8 and GAPDH in all 3. The karyotype of SCID 5045– 4 showed the line to be aneuploid male (XXYY) with a range number of 83 to 95 chromosomes. A total of 11 marker chromosomes were noted and they are the same as those found in the parental line.19 PWR-1E: Primary epithelial cell cultures derived from a nonneoplastic, adult human prostate in a 54-year-old white male were infected with Ad12-SV40 hybrid virus.22 Several immortalized clones were isolated. Single cell cloning from one of them was used to establish the cell line PWR-1E, which expresses many characteristics of normal prostatic epithelial cells. Cells have an epithelial morphology and a doubling time of approximately 35 hours. Cells express CK-8 and 18, do not express desmin or factor VIII, and show

364

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stimulated growth, induction of PSA expression and increased AR expression in response to androgens. They are positive for SV40 large-T antigen and show strong nuclear staining for p53. Cells from passages 23 and 40 failed to produce tumors in nude mice even when co-injected with Matrigel. They grow in a serum-free, defined medium and respond appropriately to EGF, basic FGF and TGF-␤. Passage 42 cells showed a human male (XY) hyperdiploid karyotype with a consistently present marker chromosome, M1, involving chromosome 8. The PWR-1E cell line is the only known Ad12-SV40 hybrid immortalized human prostatic epithelial cell line. WPMY-1: This myofibroblast cell line was derived from SV40 large-T antigen immortalized nonneoplastic stromal cells23 from the same 54-year-old white patient undergoing cryoprostatectomy as the RWPE-1 cells described.24 Cells were immortalized with the pRSVT plasmid construct using a strontium phosphate transfection procedure.25 These cells have an elongated spindle-shaped morphology intermediate between fibroblasts and smooth muscle cells. WPMY-1 cells are hyperdiploid with a range number of 56 to 68 chromosomes. Cells in the linear growth phase show a doubling time of approximately 48 hours and they are capable of forming colonies in soft agar. Immunocytochemical analysis showed that cells do not express chromogranin A, neuron-specific enolase (NSE), CK-8, factor VIII or desmin. They showed positive staining for ␣-smooth muscle actin, vimentin, fibronectin, AR, large-T antigen, p53 and pRb. They also failed to express PSA. Growth can be stimulated with EGF, basic EGF, platelet-derived growth factor-AB and BB or inhibited by TGF-␤. Cells can be maintained in serum-free medium and they form colonies in soft agar. Injection into SCID mice failed to produce tumors. Cell lines immortalized with human papillomavirus (HPV). 1510, 1512, 1519, 1532, 1535 and 1542: These lines were derived from resected benign or malignant tissue obtained from 6 patients with intermediate to high grade cancer (Gleason grades 6 to 8, tumor stages T2C to T3C) and immortalized with HPV-16 E6 and E7 transforming genes.26 A total of 16 epithelial cell lines were obtained, including 4 from normal prostate tissue, 2 from seminal vesicle and 10 from primary prostatic malignancies. Four additional fibroblast lines were also generated from prostatic stroma from the same cohort. The lines were designated 1510, 1512, 1519, 1532, 1535 and 1542 according to patient numbers. All 16 epithelial lines exhibited similar morphology regardless of the tissue of origin and all 16 tested positive for high and low molecular weight CKs, thus, confirming their epithelial origin. Only 1 line, 1519-CPTX, tested positive for PSA and PAP at passage 5 but by passage 30 the 2 markers were no longer detectable or inducible by interferon-␥, 5-aza-2⬘-deoxycytidine or DHT. Since the corresponding tumor sections stained strongly positive for PSA and PAP, the authors postulated that the loss of expression was an artifact of in vitro culture. None of the 3 lines tested were able to form tumors in nude mice. Karyotyping of the same 3 lines showed most chromosome counts in the normal male diploid range. Loss of heterozygosity analysis revealed that 4 of the 6 tumors dissected showed loss of heterozygosity at chromosome 8p12–21, a loss previously associated with PCa.27, 28 CA7T2 and CA7T2CL: The original report of Nakashiro et al named the parental line CA-7T2 with a hyphen29 but in later reports the hyphen was eliminated, and so we have also eliminated it. The parental line CA7T2 was derived from radical prostatectomy with Gleason score 3 ⫹ 3 adenocarcinoma by infection with LXSN16E6, a retrovirus vector containing the HPV-16 E6 gene.29 As soon as the cells showed outgrowth from the primary tissue specimens, they were infected. After selection with G418 the cells were injected subcutaneously into the flanks of male nude mice. Tumors were recovered and recultured. CA7T2 and CA7T2CL are clonal lines derived by limited dilution cloning of recovered

cells. The cells are maintained in keratinocyte serum-free medium with 50 ␮g/ml bovine pituitary extract and 5 ng/ml EGF.29 Whether the CA7T2L and CA7T2CL lines are different clones is not clearly defined in the published literature, and so the 2 lines may be the same clone. The cells produce poorly differentiated carcinoma in nude mice, and are negative for the expression of PSA and AR,30 and positive for CK-AE1/3, thus, confirming their epithelial origin. Unfortunately the use of this pancytokeratin reagent does not differentiate between basal or luminal lineages because it recognizes all human cytokeratins. Tumor growth for CA7T2 cells was enhanced by co-inoculation with prostate, bone or skin derived stromal cells.29 Tumors derived from cells injected with or without stromal cells were microscopically indistinguishable. CA7T2 cells express c-met/HGF/SF receptor mRNA, as determined by RT-PCR.29 In the normal prostate gland c-met/HGF/SF is only expressed in basal epithelia.31 HPV-18 A2 and HPV-18 C1: HPE cells taken from a 54year-old white man undergoing cystectomy for bladder carcinoma were transfected with pHPV-18, a plasmid that contains the entire HPV-18 genome.32 Two clones, designated as A-2 and C-1, were selected and further characterized. Cells from the 2 lines grew in the typical polygonal arrangement of epithelial cells but they were less polygonal than parental HPE cells. Western immunoblot analysis determined that expression of at least the E7 gene product was required for transformation. Interestingly p53, originally detected in parental cells, is not detectable in either clone, consistent with E7 destabilizing p53. This destabilization has been proposed to account for the loss of p53 tumor suppressor activity.33 C-1 and A-2 cell lines are aneuploid and have 5 and 9 marker chromosomes, respectively. Each line retains at least 1 Y chromosome. The 2 lines stain positive for CK-8 and 18, and early passages stained strongly positive for PSA with later passages staining only weakly. Neither line was capable of forming colonies in soft agar or producing tumors in nude mice. When the C-1 line was further altered by introduction of the Ki-ras oncogene by infection with Kirsten murine sarcoma virus, cell morphology was altered dramatically. Cells began piling up in focal areas. p21 was expressed at a low level and tumors developed in nude mice 3 to 4 weeks after injection at the inoculation sites. Tumors consisted of large, poorly differentiated carcinomas with poorly differentiated epithelial cells, hyperchromatic and irregularly shaped nuclei with rapid cell division and a high mitotic index. Treatment of the C-1 line with N-methyl-N-nitrosourea (MNU) also yielded additional lines.10, 34 Two clonal sublines were subsequently isolated from HPV-18 C-1 cells that differed in chromosomal changes and were designated 40s and 50s. The 40s population is XO, and shows losses of chromosomes Y, 3, 7, 13 and 18, and gains of 5, 20 and minute-M4. Other abnormalities were also noted. The 50s cells are XYq⫹ and Yq⫹, and there is single copy loss of chromosomes 12, 18 and 21 due to marker formation. They also gained a copy of 5, 20 and minute-M4 as well as of 4 and 14, and 2 copies of 15. The 40s cells were treated with MNU 3 times with 50 or 100 ␮g/ml. After several passages cells treated with 50 ␮g/ml showed anchorage independent growth and formed colonies in soft agar. When inoculated into nude or SCID mice, tumors developed from 5 of 5 inoculations in 3 months while 100 ␮g/ml treated cells were significantly less tumorigenic and only developed tumors in 1 of 4 inoculations into nude mice after 4 months. Parental C-1 cells were unable to form tumors even after 6 months in nude or SCID mice. Karyotype analysis showed the further loss of 1 copy of chromosome 22 in MNU treated cells. Duplication of 11(q14⬎q24) and translocation of part of the 9q to 11q terminus were observed. No loss of chromosome 3 was observed in NMU treated cell lines. Two additional sublines were independently isolated from tumors generated from the MNU exposed cell line and they

PROSTATE CANCER CELL LINES PART 2

were designated as 129 Nu 5002–1 Tu and 129 Nu 5010 –5 Tu. They showed further loss of the p arms of chromosomes 8 and 10, and gain of the q arm of chromosome 8. P4E6: TURP biopsies of well differentiated PCa were used to establish primary PCa epithelial cultures.35 Cells with an extended life span were obtained by recombinant retrovirus PLXSN16E6 infection to introduce the HPV E6 gene and designated PxE6, where x represents the patient number. The genetic identity of the target cells was assessed by allelotyping using microsatellites located on chromosome 8p, and microdissection of tissues and primary cell cultures. Compared with cells immortalized with SV40 virus, E6 expressing epithelial cells were stable and remained diploid, while SV40 immortalized cultures were more unstable and tended to become tetraploid. All PxE6 cells eventually senesced but 1 immortalized epithelial culture, P4E6, emerged from 1 culture. P4E6 remains close to diploid with a karyotype similar to that of early prostate cancer cells and it retains PSA and AR expression. Also expressed are CK-8 and low levels of vimentin. It lacks expression of E-cadherin and chromogranin A. When layered onto collagen rafts, the cells form distinct epithelial clusters and lack the differentiation features exhibited by normal epithelial cells, which form proto-glands. Although not discussed specifically, all PxE6 cells showed doubling times of 60 to 120 hours, and so P4E6 probably also falls into this range. PZ-HPV-7 and CA-HPV-10: These lines were derived from normal peripheral zone epithelium and cancerous tissues of human prostatectomy specimens and, hence, the designations as PZ and CA, respectively.36 Wedges of tissue were dissected from radical prostatectomy specimens from Gleason grade 4/4 adenocarcinoma, minced and digested with collagenase. Cells were then grown in medium that selected for pure epithelial cells. Cells were transfected with pBR322HPV-18 using liposomes. The 2 lines are morphologically distinct from each other and from parental cells with PZHPV-7 appearing as a highly regular, large cobblestone shape with relatively thick membranes. CA-HPV-10 also appeared cobblestone-like but the cells were smaller and the membranes appeared thinner at confluence. Few elongated cells were seen in either line, while parental cancer and normal HPE cells have a high percent of elongated cells. Karyotype analysis showed that PZ-HPV-7 has 35 to 105 chromosomes with an MN of 46 at passage 38. By passage 99 the MN had shifted to a near tetraploid 106 (range 103 to 108). Most chromosomes had 4 or 5 copies. CA-HPV-10 was aneuploid at passage 26 (range 40 to 132) with an MN of 73. Double minutes were observed in 10% of the spreads. By passage 89 the range was 69 to 75 with an MN of 72 with double minutes in 43% of the spreads. In the original description the 2 lines stained positive for CK-5 and 8 and HPV-E6, and were negative for PSA when tested by immunocytochemistry and Northern blot analysis.36, 37 Subsequent testing using the more sensitive method RT-PCR revealed the presence of transcripts for PSA and prostate specific membrane antigen (PSMA) in each line. Testing for AR by immunocytochemistry was negative.38 The authors claimed that neither line forms tumors when injected into nude mice, and yet subcutaneous injection of the 2 lines gave rise to occasional lumps, which were nonprogressive and slowly regressed. These lumps were composed of keratin forming squamous epithelia arranged in a nodular pattern. Why they referred to the growths as nonprogressive lumps instead of benign tumors was not detailed and the fact that they regressed with time is in our opinion insufficient evidence to deny the existence of tumor formation. However, what this designation points out is the need to identify a clear set of parameters that researchers can test for to determine what is or is not a xenograft tumor and whether a borderline tumor is or is not benign. RWPE-1 and 2, Clonal Derivatives WPE1-NA22, NB14,

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NB11 and NB26, and CTPE: The nontumorigenic RWPE-1 line was derived from epithelial cells taken from the peripheral zone of a nonneoplastic adult human prostate in a 54year-old white man. The cells were immortalized with HPV18. A single cell clone from limiting dilution was selected to generate the RWPE-1 line and then a clonal derivative of this line was superinfected with v-Ki-ras to generate the RWPE-2 line.24, 32 RWPE-1 cells were incapable of tumor formation in nude mice, while RWPE-2 cells formed tumors in 4 of 5. The 2 lines stain positive for CKs 8 and 18, and negative for desmin and factor VIII, and they respond to the androgen R1881 with increased growth, PSA production and increased AR levels. Doubling times are 48 hours for RWPE-2 and 58 hours for RWPE-1. Karyotypic analyses showed that the 2 lines are aneuploid human with counts in the hyperdiploid range. Marker chromosomes common to the 2 lines were noted. RWPE-1 cells were also exposed to the carcinogen MNU and transformed cells were injected into the flanks of nude mice. Cells from first-generation tumors were recovered, grown in culture and re-injected into nude mice to form second-generation tumors. These tumors were recovered and grown in culture in soft agar. Individual colonies were excised and expanded to form the lines designated as WPE1NA22, WPE1-NB14, WPE1-NB11 and WPE1-NB26. The NA or NB descriptor indicates original MNU treatment of 50 or 100 ␮g/ml, respectively.39 Table 2 summarizes the characteristics of these lines. All lines were stimulated in growth to some degree by EGF and inhibited by TGF-␤. RWPE-1 cells are not anchorage independent, while all MNU lines generated showed some ability to form colonies in soft agar (table 2). Cell morphology showed increasing cell elongation from typical epithelial NA22 cells to elongated NB26 cells. Karyotypic analysis of RWPE-1 cells showed 2 cell populations, that is 1 with an MN of 45 chromosomes (X, ⫺Y) and 1 with an MN of 51 (XY).24 All 4 MNU lines lost copies of Y and 22, and gained copies of 5 and 20. All lines revealed a loss of material from the long arms of 7 and 18, and all showed duplicated material from the long arms of 9 and 11. There were a variety of other abnormalities shared between different combinations of the lines with the general observation that the more tumorigenic a line, the more genetic rearrangements it displayed. Evidence exists that treatment of cells with cadmium leads to malignant transformation.40 RWPE-1 cells maintained in 10 ␮M CdCl2 showed minor morphological changes after 8 weeks.41 The treated cells were designated as CTPE and subsequently cultured in cadmium-free medium. Cells formed mounds prior to attaining confluence and, contrary to parentals, they were able to form tumors in nude mice. In addition, 80% of the tumors showed local invasion and 1 lung metastasis was noted. Cells stained positive for PSA. CELL LINES USED IN PROSTATE CANCER RESEARCH: XENOGRAFT CELL LINES

Since tissue culture PCa cell lines are notoriously difficult to establish from primary cancer specimens, another strategy that researchers have exploited is to passage cells as xenografts in immunocompromised hosts. The cells often flourish in the host, which allows the amplification of small amounts of starting material as well as enrichment for homogeneous cell populations from the original heterogeneous tumor sample. Xenografted tissue often mimics 1 or more clinical features of PCa, such as metastases and progression to androgen independence in castrated hosts. Thus, xenografts are another excellent source of material in which to examine the genetics and molecular mechanisms of PCa. The main drawbacks of forming long-term xenograft lines are the technical expertise required for passaging the lines, and the additional care and costs associated with host animal maintenance.

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PROSTATE CANCER CELL LINES PART 2 TABLE 2. General characteristics of RWPE-1 and its MNU derived clonal derivatives

Cyk-8 Cyk-18 Synthetic androgen treatment response: AR PSA Doubling time (hrs) Colony forming efficiency % % Invasion Tumor characteristics in vivo: Tumorigenicity at 7 wks (cm3 tumor size)* Invasion Adapted from Webber et al.39 * Cells were injected with Matrigel.

RWPE-1

WPE1-NA22

WPE1-NB14

WPE1-NB11

WPE1-NB26

⫹ ⫹

⫹ ⫹

⫹ ⫹

⫹ ⫹

⫹ ⫹

⫹ ⫹ 120 0 1

⫹, Heterogeneous ⫹ 96 0.06 9

⫹, Heterogenous ⫹ 45 1.85 30

⫹ ⫹ 40 2.04 73

⫹ ⫹ 40 2.58 95

None ⫺

0.2 None

0.3 None

0.3 Slight

0.7 High

Another critical problem is our current inability to archive reliably xenograft lines in liquid nitrogen for subsequent retrieval (for example, UCRU-PR-2). A central facility to maintain important xenograft lines independent of researcher grant funding would go a long way to ensuring that these valuable lines do not perish due to researchers changing projects, retiring or lacking funds/facilities to maintain a given line(s). 9479. This line was derived from a metastatic bone lesion from the iliac crest of a 70-year-old white man who had previously undergone bilateral orchiectomy and extensive drug treatment, including vincristine, cytoxan, methotrexate, prednisone, 5-fluorouracil and acyclovir.42 Tissue was submitted for in vitro tissue culture and also implanted into nude mice. Long-term growth in tissue culture failed to generate an immortal line, although the tissue implanted into nude mice grew successfully with a tumor take rate of 81%. Tumors in mice appear to form 2 distinct populations. One group has a rapid doubling time of 2 weeks, while the other has a lag phase time of 8 weeks, after which tumors enter a rapid growth phase, doubling approximately every week. The 2 types grow equally well in male or female mice, supporting the hormone independent status of the patient. Interestingly some mice bearing tumors are hypophosphatemic. No metastases have been observed. Histological analyses of tumor sections show that the cells are cuboidal to polygonal in shape and oriented back to back without intervening stroma or a well-defined basal lamina. Intermediate junctions and poorly formed desmosomes joined the cells. Cells were often clustered in small irregular acini, which were lined to microvilli. Most cells had uniform round to elongated nuclei and rarely lipid droplets were noted. Pseudo-inclusions of cytoplasm were seen in some nuclei. CWR21, CWR22, CWR31 and CWR91. The CWR21, 22, 31 and 91 lines are serially transplantable xenograft lines established from TURPs or radical prostatectomies. Tissue was minced, mixed with Matrigel and injected subcutaneously into nude mice supplemented with testosterone.43 All patients from whom CWR21, CWR22 and CWR31 xenografts were taken had stage D carcinoma with osseous metastases, and Gleason grades 8, 9 and 9, respectively. CWR91 was from a patient with stage C disease and Gleason grade 7. Although initially transplanted as pieces of minced tissue, CWR22 is now passaged by dissociation of tissue into cell suspensions and subsequent injection of the suspension into testosterone supplemented nude mice.44 The xenograft regresses substantially after castration of the host and in some cases it completely regresses.45 PSA levels decrease dramatically following castration (up to 3,000-fold) and begin to increase after 2 to 7 months with tumor growth recurring 3 to 10 months later.45, 46 The relapsed tumor is then designated CWR22R. High levels of AR are expressed at castration and after recurrence. The expression of PSA and hK2 normally depends on androgen and shows similar profiles in recurrent tumors.

The same pattern was noted for other AR regulated genes, such as ␣-enolase, IGF binding protein-5, Nkx3.1, ARA-70, Cdk1 and Cdk2, thus, supporting the notion that AR is transcriptionally active in recurrent tumors even in the absence of testicular androgen.46 Interestingly IGF binding protein-5 mRNA expression was localized to stromal cells but absent from epithelial cells, whereas the protein was bound to adjacent epithelial cells.47, 48 The tumor expresses members of the EGFR family, including erbB1, erbB2/neu, erbB3 and the neu differentiation factor, a ligand for these receptors. The cells express CK-8 and 18, and contain an A273H mutation in p5349 and H874Y mutation in AR.50 Interestingly although CWR22R is able to grow and form tumors in female mice, whereas CWR22 cannot, CWR22R tumors tend to grow more slowly than CWR22.45 The reasons for this discrepancy are not known. CWR22R also responds less vigorously to testosterone when growing in culture in soft agar than does CWR22.45 Cytogenetic analysis of CWR22 showed loss of chromosome 2, additional copies of chromosomes 7, 8 and 12, an additional isochromosome for 1q, a derived chromosome 4 with an extra piece on the q arm and an unidentified marker chromosome.44 LAPC-1 to 9 and 42. Each of these lines was derived independently from patients with locally advanced or metastatic (stage C, D1 or D2) PCa. Biopsy tissue was minced into 2 to 3 mm3 sections and immediately implanted subcutaneously into SCID mice in the presence of Matrigel.51 LAPC explants 2 and 6 failed to proliferate, and LAPC-1 and 5, which failed to pass a PCR test for human DNA composition, are not discussed in this review. LAPC-7 and 8 showed human DNA, PSA expression (LAPC-8) and preferential growth in male vs castrated male mice in early passages but by passage 5 the cells were mostly CD45 expressing human hematopoietic cells. LAPC-3 was derived from TURP in a patient with stage D disease, LAPC-4 was obtained from lymph node metastasis in a patient with stage D disease and LAPC-9 was taken from femoral metastasis in a patient who had disease progression while receiving hormone ablation therapy.51, 52 LAPC-3 and 4 retained the most consistent molecular and histological features of PCa. Cytogenetic analysis showed a range of 79 to 92 and an MN of 89 chromosomes for LAPC-4, and a range of 68 to 81 and an MN of 69 for LAPC-3, suggesting hypotetraploid and near triploid karyotypes, respectively. The 2 xenografts exhibit chromosomal abnormalities, such as loss of Y (LAPC-4) and 16 (LAPC-3 and 4), and each contains a deletion at chromosome 12p12, an abnormality not previously associated with PCa. Importantly the 2 lines can be subjected to repeat frozen storage and recovery. Of all of the LAPC xenografts only LAPC-4 was successfully established as a cell line in tissue culture. LAPC-3, 4, 5 and 8 expressed PSA at levels from 1% to 100% of the levels found in LNCaP cells.51 The LAPC-4 and LAPC-9 lines express wild-type AR and secrete high levels of PSA. The 2 lines grow as tumors in male SCID mice, respond to androgen ablation and eventually progress to androgen independence. Androgen

PROSTATE CANCER CELL LINES PART 2

withdrawal has slightly differing effects on the 2 lines. Withdrawal causes tumor regression and a decrease in PSA expression in LAPC-4, while in LAPC-9, the PSA decrease occurs without affecting tumor size. LAPC-9 tumors are capable of survival in castrated mice but tumors remain dormant. However, even after a dormancy period of 110 days tumors responded to subcutaneous administration of androgen by a 30-fold increase in PSA levels 14 days after administration.52 In contrast, LAPC-3 tumors express low levels of AR and PSA, are androgen independent (AI) and have not been tested for their ability to metastasize.5, 51 LAPC-4 cells also express CKs 5, 8 and 18, have 2 mutations in p53 protein (P72R and R175H) and have a mutation in the tumor protein 53 (TP53) gene, which produces a A175H mutation in p53.49 It is a bit of a mystery how the same protein could exhibit 2 substitutions at the same site, namely residue 175. Either the line is not clonal or the 2 TP53 alleles contain the respective mutations. LAPC-4 and 9 can be implanted subcutaneously, orthotopically or intratibially.5 Subcutaneous tumors show less tendency to metastasize, while orthotopically established tumors metastasize to the regional lymph nodes and lung. Intratibial injection yields osteoblastic tumors typical of those seen in human metastases. Bony tumors can be harvested and re-injected, and they subsequently develop an enhanced ability to grow in bone. Injection of LAPC-4 cells near a human bone core previously implanted into the hind limb of a SCID mouse resulted in the creation of a more aggressive line, termed LAPC-42 (read as LAPC-4 squared).53 Specifically adult cancellous bone was harvested from the femoral heads of male patients and implanted intramuscularly in the hind limb muscles of 6 to 8-week-old SCID mice. Ten weeks later LAPC-4 cells were suspended in Matrigel and injected into the hind limb muscles 5 mm from the bone implant. Tumors that formed around the bone implant were harvested, and a single cell suspension was prepared comprising bone selected cells and re-injected into SCID mice. These cells were termed LAPC4.2 This line forms larger tumors more rapidly, develops higher serum PSA levels and, sooner than parental cells, progresses to androgen independence and metastasizes to bone after orthotopic injection. In contrast to the parental cells and in support of their AI status, LAPC-42 cells are able to form tumors in female mice. Like the parental cells, they express PSA, PSCA, PSMA, STEAP (6-transmembrane epithelial antigen of the prostate), hK2, CK-8 and CK-18. While LAPC-4 parental cells only express bone sialoprotein, LAPC-42 cells expresses sialoprotein, osteocalcin and osteopontin bone specific cytokines. Orthotopic injection of parental cells did not metastasize to the implanted bones, whereas LAPC-42 cells did in 1 of 3 animals. MDA Pca-31, 40, 43 and 44. These xenograft lines were recently introduced in a review article on PCa model systems but little additional information is currently available.54 MDA Pca-31 and 40 were derived from liver metastases, Pca-43 was derived from an adrenal metastasis and Pca-44 came from a skin metastasis. PAC-120, and HID-16, 19, 25, 28, 33 and 34 variant lines. Material for the PAC-120 xenograft line was obtained from TURP in a 51-year-old male with multiple lung metastases and a Gleason score of 9 (5 ⫹ 4). Resected tissue was implanted directly into nude mice under the wall of the lower abdomen near the prostate.55 The implanted resection consisted of Gleason pattern 5 tissue. Glandular differentiation was rare and the mitotic index was high. The phenotype of the xenograft tumor resembled that of the original resected tumor. Tissue in the clinical tumor samples and the xenograft exhibited positive immunostaining for CK-KL1, CD57 antigen, NSE, epithelial membrane antigen and PAP. Samples were negative for vimentin, chromogranin A and synaptophysin. PSA was also absent except in Gleason 2 foci of the original tumor. PSA transcripts were detected in Northern

367

blots using total RNA, in Western blots using PAC-120 protein extract and a polyclonal antibody, and by RT-PCR in the serum of nude mice bearing PAC-120 tumors of median size (2,200 m3). Karyotype analysis yielded a near triploid number with a range of 55 to 69 chromosomes, of which approximately half were rearranged. Most rearrangements were in duplicate and they were confirmed using chromosome painting. This indicates a tumor that underwent tetraploidization and should be considered hypotetraploid. The authors postulated a certain scheme for karyotype evolution, that is at early stages many chromosomes were lost or rearranged, leading to a hypodiploid karyotype. Endoreplication occurred, generating duplicate derivative chromosomes such as der(3)t(3;17) and del(9). Rearrangements continued to occur after endoreduplication, leading to a single copy of new derivatives such as del(8)(q11q22) or complex single copy chromosomes such as der(1)t(1;3;8;17), which was probably derived from der(1)hsr(1;17). Micrometastases were identified by RT-PCR in the lungs but not in the brain or liver of mice bearing PAC-120 xenografts. PSA in mice with xenografts was decreased following antiandrogen therapy, indicating that tumors were initially hormone sensitive. Xenografts established in male mice were not transplantable to females or castrated males. Surgical castration of mice already bearing tumors led to almost complete regression but tumors eventually recurred locally 160 to 220 days after castration. HID variant lines were derived from PAC-120 xenografts in mice that were castrated when the local tumor reached 250 to 500 mm3. After several months tumors started to regrow, from which 6 variant lines were established, namely HID-25, 28, 33, 16, 19 and 34. Recurrence latencies for the variants ranged from 8 to 15 months. Latency periods for HID-28 tumors transplanted into intact or castrated males were 45 and 180 days, and doubling times were 18 and 35 days, respectively. Phenotypically the variants ranged from similar to the parental xenograft (HID-33) to large sheets of pleiomorphic tumor cells without glandular differentiation (HID-19) to tumors with a focal neuroendocrine-like pattern (HID-34). HID-28 and 5 presented mixed forms composed of well limited mucoid areas contiguous with areas similar to parental PAC-120. As measured by RT-PCR, PSA expression in the variants was variable. HID-25, 16 and 34 were positive, whereas HID-19 and 33 were negative. R198. This interesting line was derived from papillary (transitional cell) carcinoma of the human bladder in an 81-year-old white man with symptoms of urinary tract obstruction.56 At 15 years earlier the patient had undergone transurethral prostatectomy. Suprapubic prostatectomy was performed and during the operation a papillary mass adjacent to the right ureter was also removed. The prostatic mass was determined to be BPH. It showed no signs of prostatic carcinoma but it contained neoplastic epithelial tissue clinging to the surface of the resected fragments. The clinging neoplastic epithelial tissue was minced and injected subcutaneously into 6-week-old male BALB/c nude mice. Karyotype studies revealed the range number of chromosomes to be 64 to 90. Marker chromosomes were present, including 3 to 6 large acrocentrics, of which 1 contained 2 large, intensely staining bands adjacent to the centromere and 1 similar band in a terminal position. A medium-sized ring was also observed in some metaphases. In nude mice the tumor line exhibits properties attributable to prostatic and transitional epithelium. In tumor bearing animals given androgens the neoplasm has a rapid growth rate, possesses low levels of measurable androgen receptors, produces tartrate inhibitable acid phosphatase and forms well encapsulated cystic tumors composed of transitional, glandular and squamous cells. Early passages failed to grow in intact male mice without androgen supplements but in later passages some transplants were able to grow in untreated males, in castrates and

368

PROSTATE CANCER CELL LINES PART 2

in females. Tumors in castrated, untreated mice appeared 7 to 8 weeks after inoculation and generally grew more rapidly than those in intact or androgen treated hosts. The administration of estrogens or transplantation of the tumor into female mice causes tumor regression. Compared with tumors inoculated into castrated males an even smaller percent of the transplants placed into females or estrogenized animals resulted in tumors that can grow under these conditions. Interestingly these tumors are infiltrating, scirrhous carcinomas that closely resemble squamous cell carcinomas of the bladder. They grow slowly and do not possess AR or secretory material. They are composed of a homogeneous population of squamous cells that are locally invasive. Molecular marker analysis revealed that PAP levels were significant in R198 tumors and more importantly they were influenced by sex steroids. There was no detectable PAP in tumors grown in females. Low levels of cytosolic AR were found in male mice with and without DHT treatment. No detectable levels of AR were found in tumors grown in females or castrated males. The paradox of a bladder tumor with some prostatic characteristics may be explained by the fact that the tumor was derived from the trigone region of the bladder, which embryologically is formed by an admixture of tissue from the wolffian duct and the urogenital sinus.57, 58 Some trigone derived neoplasms have characteristics of the bladder and the prostate.59 Thus, the authors hypothesized that R198 is a tumor that phenotypically expresses the embryological origins of the bladder and the prostate. As such, it may represent a useful model for investigating sex steroid responsive cells of the urogenital epithelium. RP22090. This line was established from TURP in a patient with stage D2 adenocarcinoma (Gleason grade 3 ⫹ 4).60 To date the line can only be maintained in nude mice. The original tumor had aneuploid and diploid cells but by passage 26 in the mice only aneuploid cells were detected by flow cytometry. Xenografts express PSA in mouse serum. PSA levels decrease after bilateral orchiectomy in the mice but growth continues. Karyotyping determined the cells to be hypertriploid with an MN of 68 chromosomes. The only study that references these cells failed to characterize the cells for AR, PAP or any CK markers, concentrating instead on the use of comparative genomic hybridization between it and LuCaP 23.1. Other than the comparative genomic hybridization results that it was obtained from an adenocarcinoma and secretes PSA no additional study has been reported to verify this as a unique PCa cell line. UCRU-PR-2. This xenograft was established from TURP for primary small cell carcinoma of the prostate in a 75-yearold male.61 This line was the first prostate small cell carcinoma line developed. The donor tumor and xenograft share the common morphological and ultrastructural features of small cell, undifferentiated carcinoma, including irregular nuclei, scant cytoplasm and neurosecretory-type granules, and also elaborate epithelial membrane antigen, carcinoembryonic antigen and NSE. Cells express somatostatin but they do not express calcitonin. The line expresses a diploid DNA complement. Mean tumor doubling time was 12.5 to 14.7 days depending on the implantation site. Androgen and estrogen receptors are not expressed, although PAP is present in serum from tumor bearing mice at low levels. The authors postulated a possible common stem cell origin for adenocarcinoma and small cell, undifferentiated carcinoma of the prostate. Further studies of a cell line derived from this tumor may clarify the issue. As of passage 9, it still maintained the features of small cell, undifferentiated carcinoma but also showed epithelial as well as neuroendocrine characteristics.62 Tumors were NSE positive and PSA negative, and they synthesized and secreted the peptide hormones Adrenocorticotropic hormone, ␤-endorphin and somatostatin in vivo. Single cell suspen-

sions generated from tumors and placed in short-term tissue culture also expressed these 2 hormones with an initial surge of production between days 3 and 24, then decreasing but still detectable by day 58. This suggests that the gene for proopiomelanocortin, the precursor of these 2 hormones, is expressed and molecule processing occurs. As a result of these findings, the authors suggested that the line may yield insights into the histogenesis of the subtypes of prostate cancer and also aid studies of the regulation of ectopic hormone production. The effect of local environmental factors on tumor growth and behavior were investigated in nude mice by implantation under the spleen, liver or kidney, subcutaneous injection into the scapular region, intramuscular injection into the thigh, intravenous injection into the tail vein or intraperitoneal injection.63 Tumors grew subcutaneously, intramuscularly, intraperitoneally or after implantation in the kidney. No growth was observed intravenously, or after implantation in the liver or spleen. All tumors that grew were histologically identical and they were small cell carcinomas. Only fragments implanted within muscle and under the kidney capsule were locally invasive. Tumors that grew subcutaneously or intraperitoneally showed no invasion. No induced metastases were observed in the lung after intravenous injection. Tumors expressed epithelial membrane antigen and NSE but failed to express PAP, PSA or keratin. The implantation sites did not allow the outgrowth of subpopulations, as detected by the parameters used, namely light and electron microscopy, tumor marker expression, hormone production levels and DNA flow cytometry. Electron microscopy of tumors showed ultrastructural features of poorly differentiated, small cell neuroendocrine carcinomas. Unfortunately only 1 vial of frozen cells of this line still exists and its caretakers are attempting to resurrect the line. Due to the slow growth rate of these cells (approximately 6 months to establish a tumor) it may be some time before we know if the line is available for study or has died off (P. J. Russell, personal communication). WISH-PC2. This xenograft was established from TURP in a 67-year-old white male diagnosed with T3N1M1 adenocarcinoma with a Gleason score of 8 (3 ⫹ 5).64 Pathological examination of the resected material revealed poorly differentiated carcinoma infiltrating the smooth muscle with typical NE differentiation. Minced tissue fragments from the surgical samples were implanted subcutaneously into SCID mice and began growth after a latency period of 4 months. Subsequent passages showed the growth rate to be rapid. When 3 ⫻ 106 cells were implanted or injected subcutaneously with Matrigel, tumor doubling occurred every 11 and 13.5 days in the presence and absence of androgen, respectively. Without Matrigel the times were 15 and 18 days, respectively. Although WISH-PC2 is classed as a novel neuroendocrine xenograft of small cell carcinoma, it was derived from mixed tissue and, therefore, the authors tested for the presence of some residual adenocarcinoma. Since all tests for PSA and AR were negative, enhanced growth in the presence of androgen is probably not due to the presence of adenocarcinoma tissue. It grows within the prostate, liver and bone, inducing osteolytic lesions with foci of osteoblastic activity. Orthotopic injection into the dorsal lobes results in metastases to bones ⬎ lymph nodes ⬎ liver. Intrahepatic injections also resulted in metastases. WISH-PC2 secretes chromogranin A to the mouse and expresses prostate plasma carcinoma tumor antigen 1 and members of the Erb-B receptor family. It does not express PSA, PSCA, PSMA or AR and, although it is androgen responsive, it grows independently of androgen. In further support of its prostatic epithelial cell origin it also expressed CK-8 and 18, prostate carcinoma tumor antigen-1, a surface marker for PCa but not BPH or normal prostate tissue,65 and STEAP, a cell surface marker

PROSTATE CANCER CELL LINES PART 2

commonly expressed in cancers but the normal expression of which is limited to the prostate and bladder.66 LuCaP lines. A group headed by Dr. Robert Vessella, University of Washington, Seattle, Washington has developed a number of xenograft lines designated by the prefix LuCaP and followed by a number. They are described. LuCaP 23 and Sublines: Independent xenograft lines were derived from 2 separate lymph node metastases and a liver metastasis from a 63-year-old white man with stage D1 adenocarcinoma, Gleason grade 3 ⫹ 5, and designated as LuCaP 23.1, 23.8 and 23.12, respectively.67 Cells do not grow in culture and they must be maintained in hosts by serial transplantation. Tumor doubling times are approximately 11, 15 and 21 days for 23.1, 23.8 and 23.12, respectively. No tumors are generated in female mice and tumor take is lower in castrated mice. All 3 lines produce PSA and PAP in about a 1:10 ratio of serum concentrations. In all 3 lines PSA levels decrease to a nadir following castration and then 3 possible outcomes were noted, namely a prolonged lapse of greater than 50 days with little or no recovery of PSA levels, termed responders, recovery of high PSA levels between 20 and 50 days, termed partial responders, and recovery of high PSA levels less than 20 days following castration, termed minimal responders. These responses were independent of the source of the xenograft. Xenograft morphology shows a glandular pattern that resembled Gleason grade 3– 4 adenocarcinoma with abnormal nuclear morphology, scattered neuroendocrine cells and little stroma. Electron microscopy revealed desmosomes, plasma membrane interdigitations between epithelioid cells surrounding the pseudoglandular lumen and pseudo-inclusions of cytoplasm into the nuclei with some nuclei exhibiting prominent nucleoli. Karyotype analysis revealed chromosome numbers ranging from 62 to 112 and an MN of 78. Due to poor morphology a definitive karyotype could not be prepared. LuCaP 35 and 35V: Tissue for establishing the LuCaP 35 was obtained from the left inguinal lymph node of a 66-yearold white man 3 years after presentation with a diagnosis of T4 (stage C) cancer. Eight bits of tissue from the lymph node containing metastatic AI PCa were implanted into athymic mice. Initial growth occurred after 5 weeks and passage 1 occurred after 9 weeks. A xenograft, designated as LuCaP 35, was maintained by serial passage in Balb/c nude mice.68 The take rate in intact vs castrated males is 87% and 33%, respectively. Tumor volume doubling time during initial growth phase is approximately 10 days in intact males and approximately 8 days in castrated males. The castration of mice with LuCaP 35 tumors leads to an immediate decrease in PSA and by day 7 the levels achieve a nadir of less than 0.1 ng/ml. Tumor volume also decreases. PSA begins to increase after approximately 50 days and by 100 days tumors begin to grow in an AI manner. Thus, LuCaP 35 is an androgen sensitive, PSA producing xenograft. Generally the levels of PSA increase slowly and correlate with tumor volume. The cells are polygonal and epithelial in appearance, express wild-type AR and exhibit deletions in chromosome 8p but not in chromosome 10. Attempts to culture LuCaP 35 have been unsuccessful. An AI variant of LuCaP 35, designated as LuCaP 35V, was established by taking tumor bits from a LuCaP 35 tumor that had completely regressed following castration and implanting them into castrated male CB.17 Fox Chase SCID mice. The LuCaP 35V take rate was only assayed in castrated males and it was 85%. Doubling time was approximately 16 days. Unfortunately no staining for molecular markers was performed and no karyotyping other than allelotyping of a particular locus on chromosome 8p, which exhibited loss of heterozygosity, was assayed. Hopefully, future reports will rectify these deficiencies. LuCaP 49: This neuroendocrine/small cell prostate carcinoma xenograft was taken from an omentum metastasis in a

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71-year-old male diagnosed 4 years earlier with clinical stage B-II prostate carcinoma. Tissue fragments (20 to 40 mg) were propagated as serial subcutaneous implants in SCID mice for greater than 4 years.69 This line is particularly valuable since virtually no animal models of a neuroendocrine/small cell variant of prostate carcinoma were previously available for experimental studies. All tumor passages exhibit neuroendocrine/small cell carcinoma phenotype insensitivity to androgen deprivation, expression of neuroendocrine proteins and lack of expression of PSA or AR. The tumors have a doubling time of 6.5 days. Although such a rapid doubling rate is unique for PCa xenografts, small cell carcinoma is an aggressive form of the disease, which may account for the rapid growth rate. Morphologically the cell populations are composed of uniformly round to slightly spindle-shaped cells with scant cytoplasm and nuclei with a fine pattern of heterochromatin. Cytoplasm stained positive for low molecular weight keratins and negative for high molecular weight keratins. Cells were PSA negative and chromogranin positive. Only 1% of tumor cells stained faintly positive for AR. Cells expressed nuclear MIB1/Ki67 and a majority of the cells expressed synaptophysin in the peripheral cytoplasm. Adrenocorticotropic hormone, calcitonin, gastrin, glucagons, insulin, pancreatic polypeptide and somatostatin stained negative. Karyotype analysis revealed loss of heterozygosity for the short arm of chromosome 8 with a near diploid complement of chromosomes ranging from 32 to 49 and an MN of 38. This xenograft should prove useful for investigation of the molecular mechanisms underlying this uncommon and aggressive form of prostate carcinoma. It can also be used for comparisons to the more common forms to determine similarities and differences among the different types of PCa. LuCaP 58, 69, 70 and 73: These lines are only briefly mentioned in a report of Porkka et al of the pHYDE gene.70 Their development is attributed to Dr. Robert Vessella, at whose laboratory at University of Washington, Seattle, Washington all previously described LuCaP lines were generated. All LuCaP lines were established from hormone refractory prostate carcinomas and propagated in intact male mice except LuCaP 49 and LuCaP 58. Deletion of the pHyde locus was detected in LuCaP 69 and 70 but it was expressed in all lines. No further information is known about these lines. CELL LINES USED IN PROSTATE CANCER RESEARCH: ROTTERDAM XENOGRAFT PC- MODELS

This panel of xenograft models, as designated by the prefix PC followed by a number, were developed during many years by a group in Rotterdam and they were reviewed in a recent article on xenograft models of PCa by that group.71 In that report they are referred to as The Rotterdam PC- models. For the sake of convenience and as a tribute to their long history we have retained that classification and, as for the LuCaP lines above, we treat them in a separate section, although to our knowledge neither of these 2 groups differ significantly from other xenograft lines. PC-82. This was the first human PCa xenograft line ever developed. Transplantation of tissue removed from a cribiform human PCa by total peritoneal prostatectomy in a 58year-old white man resulted in a serially transplantable tumor line in nude mice, that is PC 82.72 The tumor did not change its histological appearance in 21⁄2 years of growth and passages, and it remained moderately differentiated adenocarcinoma. Doubling time for tumor diameter is about 4 weeks, whereas doubling time for tumor volume is about 18 days. The cells secrete and contain large amounts of PAP. Tumors show a slow rate of growth and, since they regress after castration and estrogen treatment, they are androgen dependent.73 Tumors also fail to grow in female mice. At passage 9 the chromosome number was diploid. No further

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karyotyping was performed. No metastases were observed. Endogenous AR translocates to the nucleus in response to testosterone.74 These cells lack the enzyme 5␣-reductase.73 PC-295, 310, 329, 346, 324, 339 and 374. These lines were derived from individual patients by placing small pieces of solid tumor tissue specimens (approximately 30 mm3) subcutaneously into the 2 shoulders of intact males or male and female NMRI athymic nude mice supplemented with testosterone.75 Except for 310 and 346 no specific patient details are provided for individual lines other than the fact that specimens were obtained from radical prostatectomy, TURP, lymph node dissection and metastatic lesions in hormone refractory patients. PC-310 was obtained from radical prostatectomy in a previously untreated patient76 and PC-346 was from obtained an untreated primary tumor.75 The tumors were subsequently passaged in intact or testosterone supplemented nude males. The assessment of androgen dependence was determined by passage in intact females. After the lines were established in NMRI nude mice they could also be serially passaged in BALB/c nude mice. Volume doubling time for tumors 295, 310 and 329 is greater than 12 days with a lag phase of more than 3 months. Line 346 has a doubling time of less than 10 days and a lag phase of 1 to 2 months. Lines 324 and 374 double in volume approximately every 10 days with a lag phase of 1 to 2 months. Line 339 has the slowest growth rate and it doubles in volume approximately every 20 days with a lag phase of 1 to 2 months. Four xenografts (295, 310, 329 and 346) are androgen dependent and fail to grow in female mice. Tumors from these 4 lines show varying degrees of regression (partial to complete) upon androgen withdrawal but they do not progress to an AI state. All 4 lines express PSA. Unfortunately the 329 line was recently lost.71 Interestingly in the PC-295 and PC-310 lines androgen ablation leads to neuroendocrine differentiation, a common phenotype seen in AI PCa.71, 76 The remaining 3 lines (324, 339 and 374) are AI and grow equally well in male or female mice, although the take rate and growth for 374 are slower in females. PC-324 and 339 do not express PSA or AR and, thus, they may represent poorly differentiated PCa. All lines except 324 and 339 express PSA mRNA.75 Like PC-346, PC-374 is androgen responsive but not androgen dependent. Tumors continue to grow after androgen ablation, albeit at a slower rate.71 Except for line 374 all tumors consist mainly of diploid cells. PC-295 appears to have a small population of tetraploid cells, while 374 is entirely tetraploid.75 Several lines were subsequently developed from the PC346 and PC-346B lines (346I and 346BI), which were derived from different TURP specimens from the same patient.71 Previous sections provide a description of the PC-346C line. PC-EW. This line was derived through heterotransplantation of tumor tissue from a lymph node metastasis.77 Like PC-82, which was developed by the same group,72 PC-EW is androgen dependent and similar to the original tumor in terms of histological pattern, the amount of PAP secreted and the absence of a hormonally independent subline. Unfortunately in later years this line was found to be contaminated with mouse hepatitis virus and was it removed from the Rotterdam panel of available tumor models.71 DISCUSSION

In part 2 of this 2-part review we describe the derivation and characterization of tissue culture cell lines generated by the insertion of mammalian and viral transgenes. We also describe xenograft lines that require maintenance in living mouse hosts for growth and serial passage. The use of transgenes to establish transformed immortalized tissue culture cell lines has enabled researchers to develop lines from noncancerous prostatic cells, including phe-

notypically normal or BPH zones. They provide valuable resources to look at the cytogenetic and molecular changes that occur as cells progress from normal to cancerous phenotypes. Examples include using various methods of co-culture (BPH-1 sublines) and mutagenesis (RWPE-1 and its MNU derivatives) to create tumorigenic sublines from parental cells that were nontumorigenic in mouse hosts or create invasive cells from noninvasive parentals. As can be seen by the diversity of phenotypes described, the PCa research community is fortunate to have a wide variety of xenograft models to use for studying different aspects of PCa. Of particular interest are the androgen dependent vs independent (eg PC-295, 310 and 326 vs PC-324, 339 and 374) lines and those from rarer forms of the disease, such as small cell neuroendocrine carcinoma (eg UCRU-PR-2 and WISH-PC2). In contrast to the growth and maintenance of tissue culture lines, xenograft lines are labor and cost intensive to maintain and propagate. While some xenograft lines have been archived in frozen storage, the storage methods are not reliable for all lines and, thus, the field of PCa research stands to lose a number of these important lines in the event of laboratory misfortunes, investigator retirement or other unpredictable future events. The UCRU-PR-2 xenograft exemplifies this kind of problem. A possible solution to these problems would be the formation of a publicly financed, independent facility dedicated to the maintenance, preservation and dispersal of xenograft lines to the research community. After a line has become characterized and established according to the guidelines of such a facility researchers could then deposit a representative sample of their line(s), which would then be propagated and made available to the research community for a nominal fee. CONCLUSIONS

In this 2-part review we have attempted to provide an up-to-date catalogue of the approximately 200 human cell lines that have been and are currently being used to investigate the molecular mechanisms underlying PCa oncogenesis, progression to androgen independence and metastasis to tissue specific sites such as bone. To our knowledge except for a few commercially available primary tissue lines this review is complete. We apologize in advance for any oversight on our part in the event that any cell line has been missed. For new lines recently submitted to the literature of which we had no foreknowledge we invite their insertion into our online database. In the future researchers will develop additional new lines and further characterize the ones described in this review. Thus, we have created a freely accessible online database, http://www.CaPCellLines.com, where investigators can supplement and access this information in a timely and efficient manner. We hope that this provides a useful tool and resource for prostate cancer researchers in the years to come. Steven Quayle assisted with database construction. REFERENCES

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