Establishment of Two Human Prostate Cancer Cell Lines Derived … · Vol. 3. 2493-2500. Dee,nber...

Vol. 3. 2493-2500. Dee,nber 1997 Clinical Cancer Research 2493

Establishment of Two Human Prostate Cancer Cell Lines Derived

from a Single Bone Metastasis1

Nora M. Navone,2 Matilde Olive, Mustafa Ozen,

Rodney Davis, Patricia Troncoso, Shi-Ming Tu,

Dennis Johnston, Allan Pollack, Sen Pathak,

Andrew C. von Eschenbach, and

Christopher J. LogothetisThe University of Texas M. D. Anderson Cancer Center. Houston.

Texas 77030

ABSTRACTHuman prostate cancer cell lines are particularly diffi-

cult to establish, and most existing cell lines do not exhibit

features commonly seen in human prostate cancer. Most

available models either grow only in vivo as xenografts or

are androgen insensitive and fail to express prostate-specific

antigen (PSA). The lack of functionally relevant model sys-

tems of advanced prostate cancer has limited prostate can-

cer research and therapy development. Of 30 processed

samples derived from patients with prostate cancer, we

established two cell lines (MDA PCa 2a and MDA PCa 2b)

that express PSA and androgen receptor, grow in vitro, and

are androgen sensitive. Cells from these lines produced tu-

mors in nude mice when injected either s.c. or orthotopically

(intraprostatic). Both cell lines were established from a bone

metastasis of a patient whose cancer was exhibiting andro-

gen-independent growth. Although both were derived from

two samples of the same specimen, they have different ge-

netic features (as assessed by karyotype analysis) and dif-

ferent phenotypes (e.g., morphology and growth rate). It is

likely that they are distinct clones isolated by the use of

different culture procedures and reflect the genetic hetero-

geneity of the tumor. These new cell lines are the first

available derived from a bone metastasis of an androgen-

independent prostatic adenocarcinoma that grow both in

vivo and in vitro and have retained PSA expression and

androgen sensitivity. They therefore constitute important

model systems to address critical questions related to the

Received 5/1/97: revised 8/19/97: accepted 8/25/97.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

adi’ertise,nent in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported in part by grants from CaP CURE (Associ-

ation for the Cure of Cancer of the Prostate) and the John S. Dunn

Research Foundation of Houston, Texas. and by NIH Grant RRO 4999-

01.2 To whom requests for reprints should be addressed. at Department of

GU Medical Oncology. M. D. Anderson Cancer Center, I 5 15 HolcombeBoulevard, Box 13, Houston, TX 77030. Phone: (713) 792-2830: Fax:

(713) 745-1625.

androgen-independent growth of human prostate cancer

and to the complex process of bone metastasis.

INTRODUCTIONProstate cancer is the most frequently diagnosed and the

second most deadly cancer in men ( I ). Although localized

prostate cancer may be cured by radical prostatectomy or radi-

ation therapy. the prospect ofcure diminishes significantly when

prostate cancer cells escape beyond the confines of the gland (2.

3). Patients with regional or distant metastases are usually

treated with androgen deprivation therapy. Although most pa-

tients initially respond to this therapy. given sufficient time it is

believed that all eventually relapse with androgen-independent

disease. The androgen-independent phenotype of human pros-

tate cancer is defined in the clinic as the ability of prostate

cancer cells to grow despite castrate plasma concentrations of

testosterone (2-4). No current standard therapy consistently

confers a significant survival benefit to patients who develop

androgen-independent prostate cancer (2-4).

The lack of functionally relevant in vitro models has been

one of the major obstacles to advancing our understanding of the

molecular mechanisms of this disease as well as to developing

effective therapies. Most available human prostate cancer mod-

els either grow only in vito as xenografts (5-9) or (with the

exception of the LNCaP cell line: Ref. 10) lack some of the

common features of clinical prostate cancer (androgen sensitiv-

ity and expression of PSA: Refs. 5-9).

The purpose of this work was to develop model systems of

advanced prostate cancer. Our specific objectives were: (a to

screen human prostate cancer tissue samples derived from mul-

tiple sites for their growth in vitro and in vito: (h) to optimize

their growth conditions: and (c) to characterize the cells grown

ii? vitro. We processed 30 tissue samples derived from primary

prostate cancer of various histological grades and from prostate

cancer tissue derived from different metastatic sites. Initial

attachment and cell growth were obtained in cells from nine

specimens: of these only two were established as cell lines

(MDA PCa 2a and MDA PCa 2b). The cell lines were derived

from a bone metastasis of a patient whose cancer was clinically

progressing despite a castrate concentration of testosterone and

resulting in spinal cord compression requiring a laminectomy.

Both lines express PSA3 and AR. grow in i’ii’o and in vitro, and

are androgen sensitive. These are the first cell lines available

derived from a bone metastasis of prostate cancer that express

PSA and are androgen responsive. The lines are also the second

and third human prostate cancer cell lines to produce PSA and

are androgen sensitive. Our cell lines have now grown in vitro

3 The abbreviations used are: PSA. prostate-specific antigen: AR. an-

drogen receptor: DHT. dihydrotestosterone: EGF. epidernial growth

factor: FBS. fetal bovine serum.

Research. on August 2, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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2494 New Prostate Cancer Cell Lines

for 24 months (MDA PCa 2a is in passage 56, and MDA PCa 2b

is in passage 50) and have been used to establish tumors in nude

mice.

MATERIALS AND METHODS

Quality Control

Samples from different areas of the primary tumors ormetastatic lesions were obtained for tissue culture. Tumor sam-

pies ranged from 1.0 to 0.5 cm in the largest dimensions. A

tissue section immediately adjacent to that submitted for culture

was obtained for detailed histopathological analysis and deter-

mination of percentage of tumor in the sample.

Collection and Initial Processing of Tissue Specimens

Aseptically removed specimens were kept in cold (4#{176}C)

sterile tubes with HEPES-buffered saline ( 1 1) until processed.

The specimens were mechanically disaggregated with sterile

crossed scalpels to obtain pieces of about 1 mm3. Many of the

samples disaggregated into small clumps of tumor cells in the

washing medium as they were cut. In these cases, subsequent

enzymatic disaggregation of the tissue was not necessary. In

other cases, after cutting, the tissue was resuspended in five

volumes of digestion solution [Collagenase Sigma type 1 (40

units/mi of growth medium); Sigma Chemical Co., St. Louis,

MO] and incubated at 37#{176}C.The length of the incubation varied

from 30 mm to overnight, depending on the degree of fibrosis of

the specimen. Effective disaggregation of the tissue was moni-

tored over time by gently moving the flask.

Culture of Tissue Specimens

Every tissue specimen to be cultured was incubated with at

least three different media [HITES (12), BRFF-HPC1, and

PFMR4-1A (1 l)}. HITES medium is modified RPMI 1640

(GIBCO BRL, Gaithersburg, MD) with hydrocortisone, insulin,

transferrin, and selenium. (Estradiol was not included in our

formulation.) BRFF-HPC1 medium (Biological Research Fac-

ulty and Facility, Inc., Jamsville, MD) has a very rich formula-

tion and includes cholera toxin, DHT, insulin, EGF, and hydro-

cortisone. The formulation for PFMR4-1A (kindly provided by

Dr. Donna Peehl) includes cholera toxin, insulin, EGF, and

hydrocortisone (1 1). When enough tissue was available, we

cultured the isolated cells with and without serum. In most

cases, the presence of serum was necessary to obtain initial

attachment and cell growth. Collagen-coated dishes were also

used to encourage cell attachment and growth (1 1). Daily mon-

itoring of the cultures allowed us to alter culture conditions as

necessary.

In Vitro Cell GrowthTo determine population doubling times of the MDA PCa

2a (passage 30) and MDA PCa 2b (passage 30) cell lines, cells

were seeded onto 6-well plates at a density of 4 X 10” cells/well

in BRFF-HPC1 medium with 20% FBS and in F12K(+) me-

dium supplemented with 20% FBS. F12K(+) is F12K (Life

Technologies, Inc., Gaithersburg, MD) supplemented with 25

ng/ml cholera toxin, 10 ng/ml EGF, 40 mn phosphoetha-

nolamine, 10 pg/mI hydrocortisone, 45 m�t selenous acid, and 5

mg/mi insulin. The sources and preparation of these supple-

ments have been described previously (1 1). The cultures were

harvested at days 7 and 14, and the cells were counted using a

hemocytometer. Population doublings were calculated as the

average of three independent cell counts derived from different

wells. The anchorage-independent colony-forming assay in

semisolid medium was performed as described previously (13).

Cell Growth Induction by Androgens

The kinetics of growth induction of the MDA PCa 2a and

MDA PCa 2b cell lines by DHT was determined by the crystal

violet mitogenic assay (14, 15). Cells were seeded on 24-well

plates at a density of iO� cells/well and allowed to attach for

48 h in F12K(+) with 20% FBS. On the third day, designated as

day 0, the cultures were washed twice in PBS and cultured for

7 or 14 days in F12K(+) with 15% charcoal-stripped FBS and

10� t2 M tO 106 M DHT (Sigma). The medium was changed on

days 3, 7, and 10. Each concentration of DHT was assayed in

triplicate. The mitogenic activity of DHT was determined by the

uptake and elution of crystal violet dye by the cells in each well

(14, 15). A small aliquot of eluted dye was placed in a 96-well

plate, and the absorbance of each well at 560 nm was measured

with a Titerek Muitiscan TCC/340 (Flow Laboratories, McLean,

VA). Control experiments demonstrated that the absorbance was

directly proportional to the number of cells in each well.

Tumorigenicity of MDA PCa 2 Cells in a Nude Mouse

Assay

Six- to 8-week-old male athymic (nude) mice (BALB/c

strain; Charles River Laboratory, Wilmington, MA) were used

for the tumorigenicity assay of MDA PCa 2 cells. The animals

were housed aseptically in the Athymic Animal Facility of the

M. D. Anderson Cancer Center and manipulated under surgical

aseptic conditions in a laminar flow hood. Cells to be injected

into the mice were trypsinized, washed, counted, and resus-

pended at 4-S x 106 cells/l00 �iI of growth medium for the s.c.

injection. To perform the intraprostatic injection, 1-2 X 106

MDA PCa 2a cells were suspended in 20-30 p.1 of growth

medium.

s_c. Injection. Four mice were each injected s.c. with 5 X

106 MDA PCa 2a cells/site at each of four sites. Two sites were

injected with Matrigel ( 1 : 1 v/v; Collaborative Biomedical Prod-

uctlBecton Dickinson, Bedford, MA) and two with growth

medium. Six mice were each injected s.c. with 4 X 106 MDA

PCa 2b cells at two sites. Three mice received cell injections

with Matrigel (1 : 1 v/v) and three with growth medium.

Intraprostatic Injection. Fourteen mice were injected

with 2 X 106 MDA PCa 2a cells into the prostate (20-30 �i.l).

Mice were anesthetized with methoxyflurane, and a lower mid-

line incision was made. Tumor cell suspensions were injected

into the dorsal prostatic lobes using a 30-gauge needle with a

1-mi disposable syringe and a calibrated push button-controlled

dispensing device (Hamilton Syringe Company, Reno, NV).

The abdominal wound was closed in one layer with wound clips

(Autoclip; Clay Adams, Parsippany, NY).

Surgical Castration of Male Mice

The animals were anesthetized with methoxyflurane under

sterile conditions. A midline or bilateral scrotal incision was



Clinical Cancer Research 2495

made to expose the testicles. The testis were pulled away from

the surrounding connective tissue until the spermatic cord was

visible. The cord was ligated 1-2 cm above the junction of the

cord and the testis. The cord was subsequently cut, the testis was

removed, and the scrotum was closed with wound clips (Auto-

clip; Clay Adams) or nonabsorbable sutures. The inhalation

agent was removed before closure of the scrotum to reduce the

recovery period. Each animal was observed until it was able to

flee or defend itself. No postanesthesia analgesics were given.

The incision site was examined 24 h after the procedure.

PSA Blood Levels

Blood from the mice was obtained at regular intervals from

a small incision in the main tail vein. Serum was separated from

the blood, and PSA was measured using a dual-reactive enzy-

matic immunoassay kit with a lower limit of sensitivity of 0.4

ng/ml (Hybritech, Inc., San Diego, CA).

Chromosome Preparation and Giemsa Banding

Freshly fed (24 h earlier) and approximately 80% confluent

cell cultures were used for chromosome preparations. Prior to

harvest, cells were treated with Colcemid (final concentration,

0.04 p.g/mi) for 30 mm at 37#{176}C.After centrifugation. the cell

pellet was treated with a 0.06 M KC1 hypotonic solution for 20

mm and fixed in acetic acid-methanol (1 :3 by volume). Slides

were prepared following the routine air-drying procedure. 0p-

timaily aged slides were used for the induction of Giemsa

banding (16). At least 15 metaphase plates from each cell line

harvests were photographed using the Genetiscan (PSI, Inc.,

Houston, TX) and four metaphases from each sample were cut

and karyotyped in detail.

Protein Extraction and Western Blot

Protein extracts from cells growing in monolayer were

obtained using TRIPA buffer and following standard procedures

(17). Cell lysates were centrifuged at 100,000 X g for 45 mm at

4#{176}C.The supernatants were normalized for protein content, and

50-100 �i�g of protein per lane (depending on the molecular

weight of the protein to be detected) was fractionated on 7.5-

12% SDS polyacrylamide. Gels were blotted to a Hybond-ECL

nitrocellulose membrane (Amersham Corp., Arlington Heights,

IL), which was probed and washed following the instructions for

the enhanced chemiluminiscence Western blotting detection

system (Amersham Corp.).

Statistical Methods

The relationship between tumor size and PSA production

before and after castration was examined using the analysis of

covariance (18).

RESULTS

In 19 months, we processed 30 tissue samples, including

specimens derived from primary prostate cancer of various

histological grades. as well as prostate cancer tissue derived

from different metastatic sites. Initial attachment and cell

growth were obtained in nine specimens, but only two human

prostate cancer cell lines, MDA PCa 2a and MDA PCa 2b, have

been successfully established. These new cell lines, both ob-

tamed from one patient, express PSA and AR, grow in vito and

in vitro, and are androgen sensitive.

Clinical History

The patient was a 63-year-old black male who presented

with impending T9 cord compression at an outside hospital in

September 1993. He underwent spinal decompression and tumor

resection followed by bilateral orchiectomy. Pathological exam-

ination showed a poorly differentiated adenocarcinoma. He

came to M. D. Anderson Cancer Center in October 1993 with a

near-complete neurological recovery and good pain control.

He did well until June 1995, when he again showed evi-

dence of progression with a rising PSA in the presence of

castrated plasma levels of testosterone (14 ng/dl). He was mi-tiated on treatment consisting of suramin and doxorubicin. A

lesion at Tl2 continued to grow during treatment and was

encroaching on the spinal cord, causing significant right flank

pain. In August 1995, he underwent elective resection of a T12

paraspinal mass. At that time, his PSA was 1 14 ng/ml. Patho-

logical examination revealed metastatic adenocarcinoma. The

tumor showed strong staining for PSA. No staining for chro-

mogranin was present. The resected tumor fragments measured

in aggregate 12 X 12 X 5 cm. Two different areas of grossly

viable tumor were sampled for tissue culture studies. The con-

trol sections for both samples showed histologically similar

carcinoma.

The patient received postoperative radiation therapy but

continued to deteriorate clinically. Palliative estramustine and

etoposide were given without benefit. He developed pulmonary

lymphangitic spread of tumor in his lungs and suffered from

significant bone marrow suppression. He died of complications

from his cancer in December 1995. His PSA at the time of death

was 1158 ng/mi.

Initial Isolation of Epithelial Cells and in Vitro Growth

Two samples from different areas of the tumor resected on

August 1995 were used for culture. They were processed ac-

cording to the procedure described in “Materials and Methods”

and digested with coilagenase (Sigma type I , 40 units/mI in

growth media) at 37#{176}Cfor 3 h.

We used different culture conditions in the two tumor

samples to isolate the epithelial cells from contaminant stromal

cells and to obtain in vitro growth. The two conditions that were

successful are detailed below.

Isolation of Cells Growing in Suspension. MDA PCa 2a

cells were initially maintained in HITES with 10% FBS for 1

month. Epithelial cells did not attach under those conditions

and, therefore, were effectively separated from fibroblasts. After

1 month, the nonadherent cells were transferred to a collagen-

coated dish with BRFF-HPC1 medium and 10% FBS. After

transfer to that medium, the cells grew as monolayers.

Use of Low Percentage Serum in the Growth Medium

to Favor Epithelial Growth over Fibroblasts. MDA PCa 2b

cells were grown as a monolayer in the presence of fibroblasts

in BRFF-HPC1 supplemented with 2% FBS for 2 months until

the fibroblasts died, at which time the FBS was increased to

10%.

The MDA PCa 2 cell lines have sustained continuous in

vitro growth as monolayer cultures since their initial isolation



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Fig. 1 Phase-contrast micrographs of human prostate cancer cell lines.A, MDA PCa 2a passage 21 ; B, MDA PCa 2b passage 15. Note thatMDA PCa 2a cells are larger and, unlike MDA PCa2b, form tightintercellular junctions.

(24 mo). MDA PCa 2a is in passage 56, and MDA PCa 2b is in

passage 50. MDA PCa 2a and MDA PCa 2b are propagated in

either BRFF-HPC1 with 20% FBS or F12K(+) with 20% FBS.

Several vials of each cell line (5 X 106 cells/vial) have been

frozen at every third passage. Recovery of frozen cells after 6

months was good; 60-70% of cells were viable and grew. The

cells are free of Mycoplasma as assessed by PCR amplification

of tissue culture supernatant using genus-specific PCR primers

(19).

MDA PCa 2 cells were originally derived from different

areas of the same tumor specimen and have some elements of

morphology in common; they grow in clumps, form layers, and

never reach confluence. However, as shown in Fig. 1 , there are

some morphological differences between the two cell lines.

MDA PCa 2a cells are bigger, and unlike MDA PCa 2b, form

tight intercellular junctions as seen under light microscopy.

The population doubling times also differed in the two cell

lines. For MDA PCA 2a growing in BRFF-HPC1 with 20%

FBS, the population doubling time was 82 h at 7 and at 14 days.

When grown in F12K(+) with 20% FBS, the doubling time was

87-93 h at 7 and at 14 days. For MDA PCa 2b cells growing in

BRFF-HPC1 w/20% FBS, the doubling time was 42-53 h at 7

and at 14 days; growing in F12K(+) w/20% FBS, it was 6 1-73

h at 7 and at 14 days. The difference in growth rates and the

morphological differences described above suggest that the two

cells lines originated from different clones within the tumor

isolated by the different culture methods.

Clonal Growth Assay in Soft Agarose

Both MDA PCa 2a and MDA PCa 2b formed colonies in

soft agarose. Their colony-forming efficiency was 2-4%, which

is within the expected range for human cancer cell lines.

Karyotype Analysis of MDA PCa 2a and MDA PCa 2b

Giemsa-banded karyotype analysis was performed on

these two cell lines before and after growth in nude mice.

Before injection in nude mice, MDA PCa 2a at passages 6

and 18 contained between 49 and 94 chromosomes, with a

mode at 63. There were five consistent clonal marker chro-

mosomes present in this cell line (Ml-M5), tentatively iden-

rifled as: Ml, del(3q); M2, t(5q;?;l5q); M3, del(8p); M4,

t(5q;7q;14q); and MS. del(17p12). A typical Y chromosome

was present in most metaphases, and some cells had more

than one copy of the Y chromosome. A karyotype showing

marker chromosomes and other anomalies is shown in Fig.

2a. MDA PCa 2b at passage 10 contained between 44 and 92

chromosomes, with a mode at 47. This cell line had seven

clonal marker chromosomes, including the five that were

found in MDA PCa 2a (Fig. 2b). The two new marker

chromosomes were tentatively identified as ml, del(2p) and

m2, t(4;?). A typical Y chromosome was absent in some cells

but not in others.

To characterize the cells that produced tumors in nude

mice, tumor samples from mice were minced, processed with

coliagenase, and grown in vitro for 3-6 weeks prior to harvest-

ing for karyotype analysis. This analysis showed human cells

with the same characteristic markers found in the original cell

lines.

Regulation of MDA PCa 2 Cell Growth by Androgens in

Vitro

To determine if the MDA PCa 2 cells were androgen

sensitive, we evaluated the in vitro mitogenic effects of DHT

in charcoal-stripped FBS. As shown in Fig. 3, peak responses

were seen with 1 X lO_8 M DHT after 14 days in MDA PCA

2a (P < 0.001) and after 7 days in MDA PCa 2b cells (P <

0.001), when compared with controls grown in hormone-free

medium.

Expression of AR and PSA in MDA PCa 2 Cells

Expression of AR and PSA was examined by Western blot

analysis of MDA PCa 2 cell lysates. The human prostate cancer

cell line LNCaP was used as a positive control for AR and PSA

expression (5, 10, 20, 21); the androgen-insensitive cell lines

PC3 and DU14S served as negative controls (5, 20, 21). As

shown in Fig. 4, both AR and PSA are expressed in each of the

MDA PCa 2 cell lines. These results are in agreement with the

androgen-sensitive phenotype of MDA PCa 2 cells described in

the previous section.



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Fig. 2 Giemsa-banded karyotypes of human prostate cancer cell lines.

A, MDA PCa 2a at passage 6: B, MDA PCa 2b at passage 10. M.

markers (Ml to M5) common to both cell lines. ni. unique markers ml

and m2, present only in MDA PCa 2b. Both types of marker chromo-

somes from an additional metaphase spread are shown at the bottom of

each karyotype. Note the presence of two copies of chromosome Y in

MDA PCa 2a and an apparent absence of a typical chromosome Y inthis cell ofthe MDA PCa 2b. Unidentified markers (UM) present in both

cell lines are also shown.


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Tumorigenicity of MDA PCa 2 Cells in an Athymic Mouse

Assay

MDA PCA 2a

The tumorigenicity of MDA PCa 2a was assessed by two

methods: s.c. injection and intraprostatic injection in athymic

mice.

s.c. injection. Seven weeks after injection, six of the

eight sites injected with tumor cells and Matrigel had a 47-170-

mm3 tumor. Nine weeks after injection, three of eight sites

injected with medium (no Matrigel) had tumors larger than 40

mm3. No metastases were detected by macroscopic examination

of mice organs at necropsy. Tumor volume was calculated as

length X width X height X 0.5236 (the formula for an ellip-

soid). PSA blood levels of the tumor-bearing mice were deter-

mined at regular intervals and were found to increase over time

after s.c. injection of MDA PCa 2a cells. At week 8, PSA blood

levels ranged from 40 to 150 ng/ml.

Intraprostatic injection. Ten weeks after injection, 10

of 14 mice injected showed increased values of PSA in their

blood (24-170 ng/mi). Eleven weeks after injection, 7 of 14

mice that received injections had palpable tumors. Seventeen

weeks after injection, the tumors were 200-800 mm3.

MDA PCa 2b

The tumorigenicity of MDA PCa 2b cells was assessed

through s.c. injection. Six weeks after injection, all six sites

injected with Matrigel had tumors ranging in size from 40 to

80 mm3; three of six sites injected with growth medium had

small tumors. The mice injected with Matrigel had serum

PSA values ranging from 370 to 667 ng/ml, whereas the mice

injected without Matrigel had values ranging from 53.4 to

129.5 ng/ml. Eight weeks after injection, all 12 sites injected

had tumors. Intraprostatic injections were not done with

MDA PCa 2b cells, due to the high rate of tumor induction

achieved through s.c. injections. In vivo, MDA PCa 2b grew

faster than MDA PCa 2a.

The ability of MDA PCa 2 cells to induce tumors that

secrete PSA as a function of tumor size in athymic mice is

compelling evidence that these cell lines originated from human

prostatic cancer. In addition, these results suggest that this

system is a useful model to study human prostate cancer. Ex-

pression of PSA is an invaluable marker of prostate cancer

progression (2-4), because tumor growth can be monitored by

PSA blood levels, and loss of PSA expression occurs only in

very late stages during the progression of the disease to a more

aggressive phenotype.

Effect of Castration of Mice Bearing Tumors Produced by

MDA PCa 2 Cells

The effect of castration on the in vivo growth of MDA PCa

2 cells was assessed by monitoring tumor size and PSA blood

levels after castration of mice bearing tumors produced by s.c.

injection of the cells. Male athymic mice were injected with 4 X

106 cells with medium (no Matrigel). Mice were castrated when

their tumors were 40 mm3 or larger. This occurred at 8-1/2

weeks in 3 of 10 mice injected with MDA PCa 2a cells and at

6 weeks in S of 9 mice injected with MDA PCa 2b cells. As

shown in Figs. 5 and 6, the tumors stopped growing or de-

creased in size, and PSA blood levels dropped dramatically after

castration of the mice injected with either cell line. These results

demonstrate that the MDA PCa 2 cell lines are androgen re-

sponsive.

PSA Blood Levels as a Function of Tumor Size

Four regression lines (MDA PCa 2a before and after cas-

tration, and MDA PCa 2b before and after castration) using

tumor weight to predict PSA were calculated. Because tumor

weight is a measure of cellular mass and PSA is normally



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DHT(M) DHT(M)

AR � llOkD

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PSA .� 33kD

Fig. 4 AR and PSA expression in human prostate cancer cell lines.Western blot analysis was performed with polyclonal antibody to human

AR (Novocastra) and with antibody to human PSA (Dako, Inc.,

Carpenteria, CA). �3-Actin (Mr 43,000) was used as a loading control.

DU145, negative control for AR and PSA; LNCaP, positive control for

AR and PSA; PC3, negative control for AR and PSA.


MDA PCa 2a I 14 days MDA PCa 2b I 7 days

Fig. 3 Growth of MDA PCa 2cells in vitro in the presence

of different concentrations of

DHT. Each data point is the av-erage of three independent cx-

periments: bars, SD. O.D., ab-

sorbance at 560 nm.

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analyzed as a logarithm, both weight and PSA were log (base

10) transformed for the analysis. The correlation of all re-

gression lines was above 0.89, and for every unit of log

increment in tumor weight, there was 2/5 of log increments in

PSA produced by MDA PCa 2a and 3/4 of log increments in

PSA produced by MDA PCa 2b. The amount of PSA pro-

duced by tumor weight remained the same in MDA PCa 2b

after castration but the relationship was lost in MDA PCa 2a

after castration. Based on these calculations, we estimated

that MDA PCa 2a produces 0.43 (± 0.15) ng/mi of PSA per

g of tumor, and MDA PCa 2b produces 0.67 (± 0.08) ng/ml

of PSA per g of tumor.

DISCUSSION

We have established two new human prostate cancer cell

lines, MDA PCa 2a and MDA PCa 2b. Evidence that these cell

lines originated from human prostatic cancer includes the pres-

ervation of PSA expression and androgen sensitivity.

MDA PCa 2 cells constitute an important model system for

the study of the clinically defined androgen-independent growth

of human prostate cancer. These cell lines were derived from a

patient with prostate cancer who relapsed two years after sur-

gical castration. At the time of relapse, the tumor from which the

cells were derived was actively growing, producing symptoms

that necessitated surgery. This objective evidence of disease

progression in the presence of castrate plasma levels of testos-

terone (14 ng/dl) is the clinical definition of androgen-indepen-

dent growth used at the University of Texas M. D. Anderson

Cancer Center and indicates the presence of an androgen-inde-

pendent tumor (4, 22).

Recurrent prostate cancer is a spectrum of diseases that

differ in clinical presentation and prognosis (2-4). A recent

report proposes a classification of prostate cancers as hormone-

naive, androgen-independent/hormone-sensitive. or hormone-

independent based on their hormone sensitivity (4). Androgen-

independent/hormone-sensitive cells are categorized as

androgen-independent because they grow in the presence of

castrate plasma levels of testosterone, but their growth rate

responds to other hormonal manipulations (e.g. , adrenal andro-

gen blockade and antiandrogens). The importance of this dis-

tinction is that it is generally recognized that a subpopulation of

patients who have relapses after one hormone treatment may

still respond to a second hormone treatment (4). The MDA PCa

2 cells, which are derived from an androgen-independent tumor

but are sensitive to androgens in vitro and in vito, are presumed

to represent the androgen-independent/hormone-sensitive tumor

type.

To our knowledge, the LNCaP cell line is the only other

existing human prostate cancer cell line that is androgen sensi-

tive. LNCaP was derived from a metastatic site (supraclavicular

lymph node) of a patient with an androgen-independent pros-

tatic adenocarcinoma (10). In contrast to MDA PCa 2a and 2b,



A VCastration B150�

Castration

Fig. 5 Tumor size (A) and PSA blood levels

(B) produced by s.c. injection of MDA PCa 2a

cells into athymic mice. At 8-1/2 weeks after

cell injection, 3 of 10 mice that received injec-

tions had tumors larger than 40 mm3. Each

symbol represents a different mouse. The ar-

row indicates the time when the mice were

castrated. Note that after castration of the mice,

the tumors decrease in size or stop growing,

and the PSA blood levels drop dramatically.

A Castration

Sc N � 0’� © � r� � ‘4 (0 �

- ,- ,- ,-

Weeks after cell injection Weeks after cell injection

300�

250�

200�

150�

100�

50�

CastrationB

E

a

.�

00

(�1 ‘l� �C 00 ©

Weeks after cell injection

Fig. 6 Tumor size (A) and PSA blood levels (B)produced by s.c. injection of MDA PCa 2b cellsinto athymic mice. At 6 weeks after injection. five

of nine mice that received injections had tumors

larger than 40 mm3. Each symbol represents a

different mouse. The arrow indicates the time

when the mice were castrated. Note that aftercastration of the mice, the tumors decrease in sizeor stop growing, and the PSA blood levels dropdramatically.

Weeks after cell injection


EE

0

Ea

EE

1�

0

Ea

the LNCaP cell line is only weakly tumorigenic when injected

s.c. into male nude mice, but the tumorigenicity can be increased

by coinoculation with Matrigel or fibrobiasts (23). The complex

role of androgens in prostate cancer progression has been high-

lighted in a recent publication (24). In that report, an androgen-

repressed human prostate cancer cell line derived from a patient

with androgen-independent prostate cancer was characterized.

Those findings imply that a subpopuiation of cells is inhibited

by androgens. The authors suggest that the androgen-repressed

phenotype may represent late events in prostate cancer progres-

sion (24).

A clinical hallmark of prostate cancer progression to the

androgen-independent state is the development of osseous me-

tastases: up to 84% of patients have bone metastases at autopsy.

This is the only malignancy to consistently produce osteobiastic

lesions (2, 23, 25). The marked clinical affinity that prostate

cancer has for bone is responsible for much of the clinical

morbidity that is a result of this cancer. Our cell lines may serve

as a useful model of the complex process of prostate cancer

metastasis and progression within bone. The availability of this

new model system is essential for the study of these interactions.

The marker chromosomes characteristic of these prostate

cancer cell lines contain regions that have been identified by

allelotyping studies and comparative genomic hybridization

analysis as sites of common allelic gains or losses in clinical

prostate cancer specimens, i.e., 8p (26-28), 7q (27, 29-32), Sq

(27, 28), 3q (27), 2q (27), and l7p (22, 27). Ofparticular interest

is the presence of an interstitial or intercalary deletion of Yip 12,

with preservation of Y/pl 1 and Ylpl3. This may suggest the

presence of an additional tumor suppressor gene in 17p along

with p53, which is located at Y7pl3.l (22). It is also of interest

that Cher et a!. (27) found an increased frequency of ailelic

gains in the 4q25-28 region among African-Americans, because

the MDA PCa 2 cell lines which were derived from an African-

American patient have three to four copies of chromosome 4,

reflecting amplification of the 4q25-28 region. A marker chro-

mosome involving chromosome 4 was also present in the MDA

PCa 2b cell line.

The MDA PCa 2 cell lines, although obtained from differ-

ent samples of the same specimen, have unique genetic features

(as assessed by karyotype analysis) and phenotypes (different

morphology and different growth rate). It is likely that these two

cell lines are distinct clones that reflect the genetic heterogeneity

of the tumor and will provide important model systems to

cellular interaction that may participate in prostate cancer pro-

gression within bone.

ACKNOWLEDGMENTSWe gratefully acknowledge the technical assistance of Dr. P. A.

Steck in the colony-forming assay and C. R. Sikes for technical assist-

ance in the surgical procedures in mice.




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1997;3:2493-2500. Clin Cancer Res N M Navone, M Olive, M Ozen, et al. from a single bone metastasis.Establishment of two human prostate cancer cell lines derived

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