Prostatic binding protein: An androgen-dependent marker for prostate epithelial cells

Vol.

95, No.

2, 1980

July

31, 1980

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Pages 674-681

PROSTATIC BINDING PROTEIN:

AN ANDROGEN-

DEPENDENT MARKER FOR PROSTATE EPITHELIAL Wallace

L. McKeehan,

W. Alton

Jones

Received

June

Cell

Mary P. Rosser,

Science

Center,

Howard

Old Barn

CELLS

A. Glass

Road,

and Danna Fast

Lake Placid,

New York

12946

9, 1980 SUMMARY

Indirect immunofluorescent staining revealed that Prostatic Binding Protein, the major androgen-dependent protein in rat ventral prostate in vivo, is associated specifically with the epithelial cells in primary cell cultures derived The epithelial cells release Prostatic Binding Profrom rat ventral prostate. tein into the medium during primary culture. of Prostatic --De novo synthesis Binding Protein is demonstrable during early phases of cell culture. Prostatic Binding Protein is an excellent marker for the identification of functional prostate epithelial cells and for the study of regulation at the cellular level of the synthesis and secretion of a major androgen-dependent prostate protein. Several

laboratories

of the cytosol

prepared

of molecular

weight

the protein

and their

protein

(6-8).

exhibits Binding

40,000

nonspec Protein"

ventral

fit

at the cellular prostate

level under

it role

activity communication, in cultured

PBP is an excellent It

of a single

marker

appear

protein

of the subunits

by androgens

in vivo. -___

to be specific

of the protein

not clear,

and has been called

"Prostatic

we report

our

epithelial for

cells

identification

a good marker

of synthesis

and secretion

defined

and controlled

MATERIALS

AND METHODS

for

study

findings from

that rat

of functional of the regula-

of an androgen-dependent conditions

of tissue

culture.

from

Materials: Thirteen to 17 week old Sprague-Dawley rats were obtained Taconic Farms (Germantown, NY). Collagenase 1.8 I Type I from Worthington

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1% 1980 by ‘4cademrc Press, Inc. of reproducrion in any form reserved.

674

of The

to prostate

is

is also

the

15 to 45 per cent

consists

controlled

physiological

ed and synthesized

that

The synthesis

code for

In this

cells.

prostate

strictly

which

reported

(l-5).

steroid-binding

epithelial

from

ventral

to 50,000

the

(PBP).

prostate.

prostate

rat

mRNA's is

A though

PBP is concentrat

protein

from

and mRNA sequences

tissue

tion

have independently

it

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95, No.

2, 1980

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Biochemical Corp. (Freehold, NJ). Medium F12K was purchased from Gibco (Grand Island, NY). Donor horse serum was from Flow Laboratories (McLean, VA). Goat fluoresceinisothiocyanate-(FITC)-labeled IgG against rabbit IgG was purchased Staphylococcus aureus cells bearing from Cappel Labs. (Cochranville, PA). Protein A on their surface (Pansorbin) were purchased from Calbiochem (LaJolla, CA). [35S]methionine was from New England Nuclear (NEN) (Boston, MA). Cell Culture. Primary cell cultures were established by enzymatic dissociation of the ventral lobes of prostates from 13 to 17 week old Sprague-Dawley rats. Minced prostate glands, free of connective tissue, were incubated in 7 ml per g wet weight tissue of a solution of 675 units per ml of collagenase in Relatively homogenous aggremedium F12K and 5% horse serum for 1 hr at 37°C. gates of glandular cells were selected from the dissociation mixture by repeated settling at unit gravity at 4oC. Suspensions of cell aggregates containing 5 to 7 pg cellular DNA per ml of the culture medium indicated in the text were placed in 35 or 60 mm plastic Petri dishes. Cultures were incubated in a humidified atmosphere of 5 per cent carbon dioxide and 95 per cent air at 370C and analyzed at the times indicated in the text. Details of the cell culture methodology will be described elsewhere. Purification of PBP and Preparation of Rabbit Antiserum. PBP was isolated from rat ventral orostate bv a modification of the method of Hevns and De Moor (9). Antiserum was raised ;n rabbits according to Heyns (10).
675

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complexes with Staphylococcus aureus bearing protein A as the immunoadsorbent (13). Assay mixtures were carmut in Eppendorf tubes that contained 8.3 mM Tris-HCl (pH 7.4), 25 mM NaCl, 3.3 mM methionine, 0.0033% (w/v) NaN,, 2 mg per ml ovalbumin, 75 ~1 of a 10% solution of S. aureus, 5 ~1 of normal or immune serum and 100 ~1 of sample in a total volume of 0.30 ml. After incubation for 30 min at room temperature, the 13'S]PBP-antibody-S. aureus complexes were collected by centrifugation in a Beckman 152 Microfuge. The pellets were suspended in 0.20 ml of buffer solution, precipitated with TCA and filtered on GF/C glass fiber filters. The filters were placed directly in 10 ml of Aquasol counting fluid (NEN) and counted by liquid scintillation at an efficiency of 90 per cent. Background was determined for each sample with normal rabbit serum in the reaction mixture and this value was subtracted from the values that resulted when anti-PBP serum was used. Results were expressed as cpm I 35S]PBP produced per pg cellular DNA. Specificity of the assay for L3'SlPBP was confirmed by competition experiments using purified cold PBP.

RESULTS Indirect

immunofluorescent

that

PBP was associated

cell

cultures

of rat

of fluorescence even after lC,D).

specifically ventral

was reduced,

The remaining

not display

Therefore,

by rocket

secreted

about

slight detected

of PBP is

tent

peared

about

of the medium remained (fig.

2, arrow).

stable

Fluorography

of the

for

that first

occurred

immunoelectrophoretic

1E) 1F).

cells. cells

the cultured

(fig. cells

day of culture.

A

14 ng PBP per pg DNA was

3).

a medium change

However,

medium change

on day 7 (fig.

patterns

676

(fig.

102 ng PBP per pg cellular

another

of

(fig.

epithelial

On day 6, 35 ng PBP per pg cellular

in the medium and no increase

nuclei

culture

after

2, slot

until

(fig.

the

of prostate

Interestingly,

2, arrow),

intensity

fibroblasts

cultures

of PBP of about 2).

around

marker

DNA on the

the

8 days of culture

primary

revealed

in the medium on day 3 (fig.

of culture

during

in primary

was apparent

prostate

a specific

in medium content

at the end of day 2 (fig. reappeared

at any time

2, slot

after cells

cells,

immunoelectrophoresis

on day 2 (fig.

Although

evident

serum revealed

cells

to be concentrated

107 ng per ug cellular

increase

lA,B).

PBP was still

in the medium of primary

Analysis

anti-PBP

the epithelial

(fig.

to epithelial

the presence

rabbit

of the epithelial

fluorescence

PBP appeared

with

PBP appeared

In contrast

with

prostate

some deterioration

the cells. did

staining

of culture

the

DNA PBP con-

after

5 days

DNA reap2, slots

6,7).

medium after

2).

Vol.

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,_.-

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-

Fig. 1. Indirect immunofluorescent staining of PBP in cultured rat ventral l)r0e cells. A, C, E are microqraohs taken under phase contrast optics. B, D, E are the same fields as A, C, i under epifluorescence optics. A and C show prostate epithelial cell colonies at day 5 and B of culture, respectively. E is a field containing prostate fibroblasts on day 8 of culture. Fields were magnified 320 times and exposed for equal times. Epithelial cells treated with normal rabbit serum instead of anti-PBP serum exhibited no fluorescence. Addition of purified PBP during incubation with anti-PBP Culture serum reduced the fluorescence associated with the epithelial cells. medium was F12K containing 5 per cent horse serum and 10 ng per ml epidermal growth factor (Collaborative Research, Waltham, MA).

labeling cells the

for were

24 capable

autoradiographic

hr

with of

[35S]methionine de novo -_I_ images

synthesis associated 677

showed

that

of

PBP

(fig.

with

the

rockets

the

cultured

2b).

The of

epithelial intensity

immune

precipitate

of

Vol.

95, No.

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Analysis of PBP in the culture medium by immunoelectrophoresis. Fig. 2. Aediumwas collected from cell cultures that were prepared and labeled for 24 hr with r3'Slmeth ionine as described in Materials and Methods. Immunoelectrophoresis-was carried out as described in Materials and Methods. The numbered slots refer to cumulative days in culture at the time of collection of the medium for analysis. The rockets represent precipitates of PBP and Rocket height is proportional to the amount of PBP in the sample. antibody. Each sample represents the amount of PBP released into the culture medium by cells containing 24 pg DNA. The unnumbered slot at left in (a) contained 40 ng of purified PBP. Arrows denote a medium change. The plate in (a) was fixed and stained with Coomassie Blue dye. The duplicate plate in (b) was fixed and analyzed by fluorography as described in Materials and Methods. The culture 1.0 ~!4 dexamethasone and 1.0 uM medium was F12K containing 5% horse serum, ovine prolactin.

decreased from

the

with

increasing

background

The synthesis

[35S]PBP culture.

Protein were

complexes A as the synthesized

The level

No image

of culture.

could

be distinguished

on days 5, 6 and 7. and secretion

[35S]PBP-antibody bearing

time

of PBP was quantitated

from cells

and the

immunoadsorbent and exported

of synthesis

(fig.

culture 3).

by precipitation medium with Significant

to the medium on the

dropped

678

rapidly

of S. aureus

levels first

on day 2 and fell

of

day of to near

Vol.

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2, 1980

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01234567

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111

DAYS of CULTURE by Fig. 3. Analysis of [ 35S]PBP thatwas analyzed by electrophoresis content by reaction with anti-PBP containing Protein A as described is the mean of duplicate cultures. o, o,

PBP in the PBP in the the medium

background that

culture medium; extracts of cells was collected and

levels

between

cells

prostate

harvested analyzed.

cells

the

same

content

of

changes

and deserves

same culture medium for its [35S~PBP with S. aureus Each data point

cultures

from

[35SlPBP

on day 1 and 7 of culture.

and medium may reflect

epithelial

from

The cellular

by day 7.

in the medium except

immunoprecipitation. The in fig. 2 was analyzed serum and then precipitation in Elaterials and Methods.

These

in secretory

which

paralleled differences

activity

of the

investigation.

DISCUSSION Up to 45 per

cent

androgen-dependent tein

binds

protein

steroids

prostatic

carcinoma

and

the

binds

Forsgren

agent,

estramustine

that

is

tory

based

plasma

prostate

Prostatic

polyamines

to the

(15)

immunologically on steroid

plays

role

(14)

(4).

and seminal in

human prostate

tissue

PBP. binding

679

Preliminary support

the in vitro

fluid

prostate

binding (3).

properties

suggest

that

function.

contains results

their

The pro-

and the anti-

These

rodent

to rat

(PBP).

DNA and chromatin

tissue

of a single

inhibits

of spermatozoa

have shown that

and carcinogen

Protein

A subunit

to isolated

a central

similar

consists

and carcinogens (5).

membrane

cytosol Binding

(3)

of PBP in prostate

probably

et al.

called

complexes

abundance

the protein

ventral

(1,4),

of androgen-receptor PBP also

of rat

results.

a protein in our labora

Vol.

95, No.

2, 1980

In this cultured with

BIOCHEMICAL

report,

prostate

epithelial

epithelial

cellular

we showed

cells

stores

that

was demonstrable

for

appear

in the

were

in primary

culture

of extracellular

corticoids,

synthesized

the majority

is

probably

to inadequate

the level

cell

of PBP synthesis conditions

polypeptide

culture

growth

which

was declining

conditions.

include

in intra-

to and

of PBP synthesis Preliminary

results

can be modulated

androgen, matrix

of PBP

PBP continued

the decline

culture

factors,

over

de novo synthesis -__

and secretion

by

of PBP associated

Unlabelled

we attribute

and secreted

carried

in the animal,

Presently,

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synthesized

medium even when --de novo synthesis

undetectable.

variety

Although culture

RESEARCH

stored,

up to one week in culture.

eventually

that

BIOPHYSICAL

PBP is

cells.

in primary

culture

suggest

that

AND

prolactin,

by a gluco-

and nutrient

concentra-

tions. These

results

of regulation

at the

androgen-dependent identification

demonstrate cellular

prostate of functional

and may be of value

that level protein.

of the

synthesis

PBP is also

prostate

in the early

PBP is an excellent

epithelial

detection

marker

for

and secretion a useful

cells

of prostate

marker

--in vitro cell

the study of a major for

the

and -__ in vivo

hyperplasia

or

malignancy. ACKNOWLEDGEMENTS We are grateful to Dr. W. Heyns, Catholic University of Leuven, Leuven, Belgium for a enerous gift of reference rabbit anti-PBP serum. The study was supported by N? I Contract No. CP6576, NC1 Grant No. CA26110 and the W. Alton Jones Foundation.

REFERENCES :: 1: 5. 6. 7. 8.

103, 1090-1095. Heyns, W., Van Damme, B., and De Moor, P. (1978) Endocrinology B., MOUS, J., Rombauts, W., and De Moor, P. (1979) J. Heyns, W., Peeters, Steroid Biochem. 11, 209-213. Chen, C., Hiipakka, R.A., and Liao, S. (1979) J. Steroid Biochem. 11, 401-405. F.S. (1979) J. Bio . Chem. 254, Lea, O.A., Petrusz, P., and French, 196-6202. B ., and Forsgren, B., Bjb'rk, P., Carlstrijm, K., Gustafsson, J.- i ., Pousette, Hb'gberg, B. Proc. Natl. Acad. Sci. USA 76, 3149-3153. B., Rombauts, W., and Heyns, W. (1977) Biochem. Biophys. Mous, J., Peeters, Res. Commun. 79, 1111-1116. (1978) Biochem. J. 170, Parker, M.G., Scrace, G.T., and Mainwaring, M.I.P. 115-121. Parker, M.G., and Scrace, G.T. (1978) Eur. J. Biochem. 85, 399-406.

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Heyns, W., and De Moor, P. (1977) Eur. J. Biochem. 78, 221-230. Heyns, W. (1977) FEBS Letters 81, 43-47. Laurell, C.-B. (1972) J. Clin. Lab. Invest. 2, 21-27 (Suppl.). Kapuscinski, J., and Skozylas, B. (1977) Anal. Biochem. 83, 252-257. Goding, J.N. (1978) J. Immunol. 20, 241-253. FlcKeehan, W.L., and Fast, D. (1980) Cancer Res., submitted. orsgren, B., BjGrk, P., Carlstrom, K., Ekman, P., Gustafsson, J.- 8 ., i . , and Hogberg, B. (1979) Cancer Treatment Rep. 63, 1186 (Abstr.).

681

Pousette,