Correlation of tumor metastasis with sterol carrier protein and plasma membrane sterol levels

Vol.

124,

October

No.

1, 1984

15,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1984

Pages

283-289

CORRELfiTION OF TUMOR METASTASIS WITH STEROL CARRIER PROTEIN AND PLGK#lCI MEMERCINE STEROL LEVELS

Friedhelm

Schroeder*,

F)nn B. Kier*,

and *Departments

of

Nary

E.

and Veterinary of Missouri MO 65212

Pharmacology

University Columbia, + Department University Minneapolis,

D. Olson T f

Carol +

Dempsey

of

F'athology

Biochemistry of Minnesota MN

55455

Received September 7, 1984 - Squalene and sterol S!GX!!KY and sterol/phospholipid molar ratios membranes were measured in cultured cell lines. SCP is abundant in all

carrier

protein whole cells

of primary

(SCP) levels and plasma and metastatic

tumor

However, cell lines. metastatic lines have significantly lower SCF levels and plasma membrane sterol/phospholipid ratios than do primary lines. The results indicate that extremely malignant, metastatic cells are unable to produce or maintain adequate levels of both SCP and plasma membrane sterols when grown in lipoprotein deficient media. This defect, Ln_ vivo --,-r probably causes excess uptake of SCF and lipid. 0 1984 Academic Press, Inc.

One generally is

process and

a

lines

derived

These

cells

from

in

cholesterol

squalene

cholesterol

of

j-n

culture compared

Gv_Q~

they

Decreases are

paralleled and sterol

or

studies

renal

to cause

by similar carrier

on by

normal

kidney

tumors

which the

changes

protein

c&lls

in

defects

malignant

in

the

(SC&') (l-21.

cell

(l-2)).

lower in

levels (3).

cholesterol content

cellular

of

level

SCF (also 0006-291X/84

283

of

culture

accumulate

cholesterol

cellular

and

(e.g.

markedly

or

metabolism

membranes

metastases

same

malignant lipid

of

focused

tumor

in

increases

new

the

exhibited

the

have

when

of

transport

human

of

regulation

formation

Our previous

regulation

cells

normal

the

permitting

proliferation.

(l-4).

in

change

transport,

However,

characteristic

accepted

these

of called $1.50

Copyright 0 1984 by Academic Press, Inc. All righrs of reproduction in any form reserved.

Vol.

124,

No.

fatty

1, 1984

acid

binding

regulator the

of studies

cultured

and

protein

lipid

(5))

ccl of

1 s,

was in

BIOPHYSICAL

is and

.here

i gnant

properties sterol

AND

metabolism

described ma1

share

BIOCHEMICAL

to

considered

to

COMMUNICATIONS

be

a

major

transport

(2,6).

The

determine

whether

other

particular

cultured

RESEARCH

highly

renal

cancer

purpose types

metastatic

cells

of of

cells,

with

regard

to

SCF

levels.

Tissue cult_ure an_d_ QA!_ Lines - LM cells were obtained from the American Type Culture Collection (CCL 1.2) and grown in suspension in a chemically defined serum free medium (7). Cells were injected into CgH/Hen (MTV-1 or athymic (nude) mice (BCILE/c, nu/nu) obtained from Charles River Laboratories, Wilmington, NY, Primary tumor and from Frederick Cancer Center, Frederick, MD. ccl 1 s and 1 ung metastasis ccl 1s were isolated and cultured in suspension in the above chemically defined serum-free medium (B-9). S-16 melanoma cell variants FlO and Fl were obtained through the generosity of Dr. E.M. Jensen, E.G. f G. Mason Research Institute, Worcester, MCI, and Dr. I.J. Fidler, Frederick Cancer Fiesearch Center, Frederick, MD. The ccl 1s were cultured monolayer with 10% heat inactivated fetal calf-serum (10,ll).

in

Membrane &plation a_ad_ Sterol/PhospholEeid ksay - Plasma membranes were prepared as described before (7,101. Plasma membranes from all cells were enriched 6-8 fold in (Na+,K+)-ATPase activity as compared to the corresponding crude homogenate. The sterol normally found in LM fibroblasts, C3H primary cells, C3H metastatic lines, Nu primary cells, and NLI metastatic lines (when the cells are cultured in the absence of serum) is desmosterol. Desmosterol/phospholipid ratios and cholesterol/phospholipid ratios (in H-lb melanoma variants) were assayed by previous methods (7). Squalene an_d_ St_gro_l cart--e_r Protein_ (SGPI f33gay - SCP was quantitated from whole cell homogenates by immunoprecipitation (6).

fhs;ul

SE

Lev_e_ls

FibroP_i2zt

quantitated

in

in were

the

of abundant

total

C3H

cultured

absence

remarkably pg/mg

Ce_lls

ratios

protein.

a_@

Discussion

pg~costernlL~hn~ehnl~@~~

Derivative

phospholipid

cell5

a!K!

ts

of

-

whole

primary

&Lies

Total

SCP

cells

and

tumor

in

suspension

serum.

The

component

and

in

in these

Further-more,

the

284

and

plasma

desmosterol/ were

cell

identical Table

!A

membranes

metastatic

under data

levels

m

lines.

The

conditions I

show

tumor

cells,

level

of

that i.e.

SCP

in

and SCP I CsH

is 20-X)

a

Vol. 124, No. 1, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Table

Plasma

Membrane

Cultured

Cell

Primary

Primary

C3H C3H

Metastaeis Metastasis Metastasis

Cell

Lung

and

Metastases

C3H

DesmosterolIPhospholipid mq Whole

Cell

Plasma

+ 0.03

0.40

+ 0.02

2.2 2.3

2.07 2.20 2.03 2.10 2.17

f + f * f

0.05$$ 0.01** 0.03t$ 0.01** 0.01**

0.27 0.28 0.21 0.13 0.18

f 32 * f *

0.05 0.06 0.05 0.03 0.03

0.24 0.32 0.21 0.17 0.14

f + f f f

Metastases

2.11

f

0.03***

0.21

f 0.03

0.22

f 0.03***

and

cells cultured

chemically

and under medium

metastatic cell lines were obtained from identical conditions in serum free as described in Materials and Methods. molar ratios were determined in whole membranes of cultured cells also as described

defined desmosterol/phospholipid cells and isolated plasma in Materials and Methods. Values represent the’mean f (n=3-5). A single, double, and triple asterisk signifies p .: 0.01 and p < 0.001 by Students T test, respectively, to the C3H primary tumor cell line.

line

cell

lines

(Table

I).

is

significantly

However,

ratios

of

cell SCP

lines and

there

for

rise

to

were

also

was

and,

also

of into

nude

Furthermore,

in

or

metastatic

the

presence

cell

cells and

the

(Table

of

both

cells.

primary

on

latter

findings

external

sources

cells. into cell

CgH/Hen

apparently

subsequently

do

line

not

mice. change

to is

may

give

LM cells

nude

returned cell

mice

population, athymic

285

levels

The

primary

primary

metastatic

the

dependent

Nu primary

the

less

When

cholesterol.

immunodeficient

other

in

compared

sterol/phospholipid

serum, of

LM fibroblasts

into

mice

whole

primary

p < 0.05, as

significantly

of

than

SEM

membrane

cells.

more

C$i

than

plasma

those

by

SCP

selected

LM and

also

change

are

of

injected

is

replaced

probably,

a highly

the

approached

cells

injection

properties

(9,121).

in

sterol

membrane

Since

no

grown

metastatic

lipid

injected

is primary

were

Desmosterol indicate

ratio

either

lower

Concomitantly,

desmosterol/phospholipid I).

Q.O6$ 0.021: O.O4Y$ 0.04** 0.03**

lung

and

cell

Membrane

0.20

tumor

metastatic

of

Micea

* (3.01

C3H SCP

Levels

SCP

from

3.00

aPrimary mice

Line 1.1 1.2 2.1

all

of

Ratios and

SCP (mg/lQQ orotein)

C3H C3H Metastasis C3H Metastasis Mean

Tumors

Line

C3H

I

DesmosterolIPhospholipid

culture identical

The when (e.g. to

Vol.

124,

No.

BIOCHEMICAL

1, 1984

AND

Table Plasma

Membrane

and

Lung

Ratio

Metastases

Cell

Metastasis Metastasis Metastasis

Mean

of

Line Q 1 2

all

COMMUNICATIONS

Primary

Cultured

Micea

(Nude)

Desmosterol/Fhospholipid Cell

Whole

Nu Nu Nu

of

Athymic

from

Line

Nu Primary

RESEARCH

II

Desmosterol/Phospholipid

Tumors

Cell

BIOPHYSICAL

Metastases

Line

Fl asma

Membranes

Q.21*Q.Q3

Q.5Q_+Q. 05

0. lQ_+Q.Q6 0.20~0.02 Q.22fQ.04

0.21fQ.Ql$$L Q.29fQ.041: Q.27rLQ.Q5$

Q.2Q_+Q.Q1

Q.25*0.02$$1

aFrimary athymic

tumor cells and lung metastatic cell lines were obtained from (nude) mice and cultured under identical conditions in a serum-free .chemically defined medium as described in Materials 2nd A single Values represent the mean 2 SEM (n=3-10). M!ztheds. and triple asterisk signify p .< 0.05 and p < 0.001, respectively, by as compared to the Nu primary cell line. Students T test,

the

original

Ltl

composition

and

mice

C3H

membrane

(Table

derived

I)

from

cell

fibroblast

lung

metastases

of ratios

cell

observation

is

cul.t~ed II)

there

is

cell

ratios SE

plasma

of

under membrane

when

compared

comparison

to

whole

difference

in

the

to

115-X)

cells

total

pg/mg

B-16+10 of

identical

the

less

cell

for

melanoma metastatic

cholesterol/phopholipid

membrane

and

I and

I),

is

SCP

cell

line

H-l&-F1

SGF are

.the

in

abundant

levels

in

strikingly line

Similarly, of

whole

II.

melanoma

III).

r

Melmem

Again,

ratio 286

However

_of 13~16

of

2.4-fold

and

ratios.

Tables

(Table

conditions

ratios I)

plasma

protein).

the

important

cell

(Table

lines lower

ratios

(Table

cf.

with

significantly

have

2.O-fold

lines;

LPI

findings

sterol/phopholipid

are

to

to

potentially

in

Similar

lipid

metastatic

~he.l_..~~rerelL~~~~e~e~.~e~.~ B_ati_o -

those

mice

Another

cell

the

nude

cells

metastatic

than

of

membrane

metastatic

cells

highly

plasma

apparent

Cells

melanoma

cultured

no

II).

to

Similar

tumor

higher

and

v_araan&

lower

that

primary

(Table

the

(Table

l,ine

regard

(9).

membranes

desmosterol/phospholipid primary

with

properties

plasma

I

line

highly

the

Vol. 124, No. 1, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table SW Cell

and

III

Cholesterol/Fhospholipid

Line

Ratios

Phenotype

In

E-16

SCF

Melanoma

Variantsa

Cholesterol/Fhospholipid

(mg/lO9

mg)

Whole

Cell

Fl asma

Membrane

P-l&.-F1

Low

Metastatic

1.93

*

0.03

0.40

f

0.93

0.66

*

0.04

R-16-FlO

High Metastatic

1.47

f

9.03fl

0.21

f

0.02**

0.24

f

0.

aB-16 HG2!2dS.

asterisk

melanoma Values

variants represent signifies p <

were 0.01

in Materials and a double as compared

cultured as described the mean -+ SEM (n=3) by Students T test,

lo**

_and to

the

P-l&F1

line.

met&static

cell

metar-;tatic

line.

metastatic

cell

phospholipid

line

(Table

These

conditions,

the

presence

of

indicate

that

the

calf

on external

dependent

AS seen

with

in

there

LM primary

P-16-F1.O II

and

protein

similar

studies

called

abundant

degree

to

the

the

cells,

than

the

media

are

and

membrane R-l&F1

the

whole

cell

ratio

in

these

values

(Table the

highly

more

cells.

cells

III).

lines;

They

even

plasma

cell

low cells

metastatic

LM

for

also

culture

cells

metastatic

metastatic

is

ccl 1 media.

less

LM

sterol/

LM cell

of

to

cell

metastatic

melanoma

melanoma lipid

less

the

LM

cells

cell

in

with

Al

is 50,

metar;tatic cf.

Tables

I,

III.

In these

level

in

of

tumor

no dii:ferences ccl 15,

serum

serum

ratio

comparison

are

of

sources

cholesterol/phospholipid higher

of

less

the

whole

melanoma

ratio

metastatic

highly

the

may reflect

absence

of

findings

II),

metastatic cell

that

to

I and

differences

fetal

than

contrast

highly whole

the

i.e.,

in

(Tables

of

to

III).

r

lines

ratio

lower

markedly

However

when compared

the

is

in

of of

we

concentrated

SCP (2,6). highly

SCP in

The results

malignant these

malignancy

on

the

!jhoW that

LN and melanoma

cultured

cells

or metastatic

is

ability. 287

inversely

ubiquitous

abundant,

SW is

cells.

also

However, related

Furthermore,

to the

the the

Vol.

124,

No.

cellular

1, 1984

level

BIOCHEMICAL

of

SCP

correlates

membrane/phospholipid ability.

levels

is

cancer

cells

many

in

tissues

associated others

are

growing is

a

cells

are

able

will

that

low

levels

human

tumors

higher

than

that

receptors up

highly

the

to presence

that,

1. Gonzalez, Invest. Ckol.. 2. Dempsey, 63-86.

it

of of

SCF

modified

5erum.

This cells

by

rapidly

and

lymphotoxin

SGP

cell

blood

1ik::ely ability

Cell

lipid

apparent,

and

is

its

lipids.

and

various contain

it

lipoprotein

division

adequate

SCP

becomes in

will

loses

and

than

with

cell

SCF’

that

grown

sterol

Therefore, a

defect

membrane

tumors

(l-2).

BO

malignant

experience most

of

highly

and

malignancy,

are

unregulated

_vi;lo?

levels

become

are

rapidly

j-5

_v%&~g,

deficient

F(.V., Current

and Topics

288

when

media

proliferation,

!lipoprotein)

to surface

gn_

vjv_os

supply.

This investigation was supported primarily and in part by NIH grants GM-31651, HL-25JlL3 was also supported by American Cancer grant the recipient of a Special Emphasis Career (RR-CKW13).

R., Clayman, 1 I l-3. M.E. (19841

is

reports

in

(15)

Y past

j-5

normal

Acknowledqements: NIH grant CA-24;53? and HL-33176. It SC-37G. A . B . K . is Development Award

occur

most

of

However

levels

of

where

cholesterol/phospholipid

culture,

levels

cells.

malignant leads

in

lower

maintain

Also,

(16).

development

from

decreased

renal

Related

lymphocytes

that,

normal

may

taken

appears

serum,

lipid

stern1

(1-Z).

(13).

and

and

human

cells

from

increasing SW

in

adrenal SW

leukemic

suggests

or

a

populations

normal

rat

fraction

and

of

cell

during

produce

(141,

plasma with

findings

SW

COMMUNICATIONS

between

previous

of

,the

decreases

correlation

accumulate

lipoprotein

exhibit or

to

RESEARCH

with

also

and

the

it

primary

in

fibroblasts

our

with

L Thus,

and

and

property

resistant

close

with

hepatomas

ratio

which

This

accord.

BIOPHYSICAL

directly

ratio,

metastatic

AND

Dempsey, in

M.E. Cellular

by

(1981) Regulation

24,

Vol.

124,

No.

1, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Clavman, R., Gonzalez, K., Elliott. A.Y.. and Dempsey, M.E. (1980) Biochem. Hiophys. Res. Commun. 92, 13X5-1.361. 4. Clayman, R.V., Gonzalez3 R., Elliott, cI.Y., Gleason, D.E.? and Dempsey, M. E. (1983) J. Urol. 129, 621-624. 5. Gordon, J-I., Fllpers, D.H. I Ockner-, R.E., and Strauss, cI.W. (1983) J. Eciol. Chem. 258, 3356-3363. 6. McGuire, D.M., Olson, C.D., Towle, H.C.I and Dempsey, M.E. (1984) J. Viol. Chem. 259, 5968-5371. 7. Schroeder, F., Perlmutter, J.F., Glaser, il., and Vagelos, r.R. (1976! J. Eiol. Chem. 251, 5015-5026. Feller, D.J., Schroeder, F., and Eylund, D.E(. (1983) Pharmacol. 32, 2217-2223. Hi ochem. andSchroeder, F. (19132) Transplantation 33, 9. Kier, A. B. 274-279. 10 . (19134) Cancer Research 44, Schroeder r F. and Gardener, J.M. 3262-3269 . (1984) Eiochem. Biaphys. r in Press. 11. Schroeder I F. 12. (1978) in “The Nude Mouse in Friedman, V. H. and Shin, S. Experimental and Clinical Research”, J. Fogh and E. Giovanella, X33-384, Academic Press, New York. eds., pp. 13. Dempsey, M.E. I O1sonl C.D., Cunningham, B.C. I and Headon, D.R. (1984) Circulation, in press. Mishl::in, S., Morris, H.P., Murthy, F.V, and Halperin, M.L. 14. (1777) J. Eciol, Chem. 252, 3626-3628. 15. Gottfriend, E.L. (1977) J. Lipid Res. 8, 321-327. Rosenau, W. (17811) in “Cancer Campaign; Metastatic Tumor lb. Growth, Volume IV”, E. Grundman, ed. 3 pp. 117-122, Gustan Fischer Verlag F’ress, New York. 3.

a.

289