231

Biochimica 0 Elsevier

et Biophysics

Scientific

Acta,

Publishing

441 (1976) 231-238 Company, Amsterdam

- Printed

in The Netherlands

BBA 56828

ABNORMAL FRACTIONS

KARL

MEMBRANE PHOSPHOLIPID CONTENT FROM THE MORRIS 7777 HEPATOMA

Y. HOSTETLER,

BRUCE

D. ZENNER

and HAROLD

IN SUBCELLULAR

P. MORRIS

Department of Medicine, University of California, San Diego, The Veterans Administration Hospital, San Diego, Calif. and Department of Biochemistry, Howard University, Washington, D.C. (U.S.A.)

(Received

March lst, 1976)

Summary 1. Mitochondrial and microsomal fractions were prepared from normal rat liver and the Morris 7777 hepatoma and characterized by the use of the marker enzymes, succinate dehydrogenase and rotenone-insensitive NADPH-cytochrome c reductase. 2. The phospholipid content per mg membrane protein of Morris 7777 hepatoma mitochondria was increased by 75% as compared with mitochondria from normal rat liver. Microsomes from this poorly-differentiated tumor were found to have a 45% decrease in the content of phospholipid. These abnormalities were independent of tumor size or age. 3. The percent phospholipid content of the subcellular fractions was determined, and revealed an increase in the percent sphingomyelin in both the microsomal and mitochondrial fractions of the tumor. Decreases in the percent phosphatidylcholine and phosphatidylethanolamine were noted in tumor microsomes as compared with normal liver. Diphosphatidylglycerol was not found in significant quantities in the microsomal fraction of this hepatoma line. 4. The content of the various phospholipid classes per mg protein in the respective mitochondrial and microsomal fractions was determined. Large increases in nearly all the major phospholipid classes were found in tumor mitochondria; tumor microsomes were characterized by an increased content of sphingomyelin but the content of nearly all other phospholipids was significantly decreased. These findings suggest the presence of disturbances in the regulation of phospholipid metabolism in subcellular organelle membranes of the Morris 7777 hepatoma. Introduction The phospholipid composition of subcellular organelles tes hepatomas has been a subject of considerable interest.

from solid and asciBergelson et al. [l]

232

have reported changes in the phospholipid composition of mito~hondria and microsomes from several hepatoma lines. Highly malignant, poorly-differentiated hepatomas (Zajdela ascites hepatoma, hepatoma 27 and hepatoma 22) exhibited some abnormal features as compared with fractions from host liver: (1) the ratio of phosphatidylethanolamine to phosphatidylcholine was similar in tumor microsomes and mitochondria; (2) the tumor microsomes contained significant amounts of diphosphatidylglycerol while normal microsomes contain virtually none; (3) increased percentages of sphingomyelin were noted in the tumor mitochondria as compared with normal mitochondria [ 11. These aberraor “chemical dedifferentiation” of subtions have been termed “equalization” cellular organelle membrane phospholipid composition by Bergelson and coworkers [l-4], It has been shown that these findings are not due to in vitro exchange of phospholipids during isolation of the subcellular fractions [ 51. The abnormalities noted above were not found in subcellular fractions from regenerating rat liver, suggesting that the changes are not due to rapid growth as such [2]. In contrast, minimal deviation hepatomas have not been found to exhibit chemical dedifferentiation of subcellular organelle membrane phospholipids. Feo et al. [6] reported that mitochondria and microsomes from Morris 5123 hepatoma resembled the corresponding fractions from normal rat liver in terms of percent phospholipid composition. Bergelson et al. [2] found that hepatoma 48, a well-differentiated, slow-growing tumor, resembled normal liver in the phospholipid composition of the respective mitochondrial and microsomal fractions. Several reviews of this subject have been published [ 3,4]. The Morris 7777 hepatoma is a poorly-differentiated, rapidly-growing rat tumor which has documented defects in t!he regulation of fatty acid synthesis 173 and cholesterol content [ 81. The phospholipid composition of this hepatoma resembles that of normal host liver except for an increase in the percent of sphingomyelin present [ 91. Increased quantities of ether-containing lipids have also been observed in the Morris 7777 hepatoma [8]. This paper presents the results of experiments in which mitochondrial and microsomal fractions were prepared from the Morris 7777 hepatoma, and their purity assessed by means of marker enzymes. The phospholipid content per mg membrane protein, the percent phospholipid composition and the amount of each phospholipid class per mg membrane protein have been determined. Methods 1. Tumors. Male rats of the Buffalo strain weighing 150-200 g, obtained from Simonson Laboratories, Gilroy, Calif., were allowed free access to water and Purina lab-chow. The Morris 7777 hepatoma line was obtained from Dr. Harold P. Morris of Howard University of Washington, D.C. lo6 viable tumor cells (as determined by exclusion of trypan blue) were injected intramuscularly into the hind limb. The tumors were removed when 1-2 cm in diameter, or as noted, and connective tissue and necrotic material were carefully removed. Normal livers were removed from non-tumor bearing male rats of the Buffalo strain after an overnight fast. 2. Preparation of subcellular fractions. The respective tissues were minced in

233

iced 0.25 M sucrose containing 5 mM Tris - HCl (pH 7.4) and 2 mM EDTA, and washed several times. A 10% (w/v) homogenate was prepared in 0.25 M sucrose/5 mM Tris (pH 7.4)/2 mM EDTA, with three strokes of a motor-driven Potter-Elvejhem homogenizer. The respective homogenates were subjected to differential centrifugation essentially according to the method of Sarzala et al. [lo] with the following modification: the step gradients of Sarzala et al. were replaced with linear sucrose gradients, prepared with a Beckman linear density gradient-former (Beckman Instruments, Palo Alto, CA). Mitochondria which had been washed three times in 0.25 M sucrose were applied in 4-ml aliquots to the top of a 29-ml continuous, linear sucrose gradient (20-55s sucrose) and a 5-ml cushion of 60% sucrose in 38.5 ml cellulose nitrate tubes. The gradients were centrifuged in a Beckman L265B ultracentrifuge in the Beckman SW-27 rotor at 82 000 X g (25 000 rev./min) for 30 min. The bands representing the purified mitochondria were isolated with a Beckman fraction recovery device. Tumor mitochondrial preparations gave a significantly wider band than the mitochondrial preparations from normal liver. No differences were noted in the sedimentation properties of the respective microsomal fractions. 3. Marker enzymes. Succinate dehydrogenase, a mitochondrial marker enzyme, was determined by the method of Green et al. [ll]. Rotenone-insensitive NADPH-cytochrome c reductase (microsomes) was measured by the method of Sottacasa et al. [ 121. 4. Analytical procedures. Protein was measured by the method of Lowry et al. [ 131. Two-dimensional thin-layer chromatography of the total lipid extracts was done by a minor modification of the method of Rouser et al. [ 141. A slurry was prepared with 90 g of silica H, 5 g magnesium acetate and 240 ml glass-distilled water, and then spread in 0.5-mm layers on 20 X 20 cm glass plates. The plates were allowed to air-dry and were subsequently activated for 1 h at 120°C before use. 300-600 nmol of phospholipid phosphorus were applied to the origin and the chromatogram was developed in the first dimension with chloroform/methanol/concentrated ammonia/water (60 : 30 : 2 : 1.5 by vol.). The plates were dried for 30 min in a chamber flushed with dry nitrogen and were developed in the second dimension with chloroform/acetone/methanol/glacial acetic acid/water (3 : 4 : 1 : 1 : 0.5, by vol.). Lipid spots were visualized with iodine vapors and scraped into tubes for phosphorus analysis by the method of Rouser et al. [14]. Recoveries of lipid phosphorus were 95% or more. 5. Chemicals. Sucrose, ultrapure, was obtained from Schwartz-Mann, Orangeburg, N.Y., cytochrome c from Boehringer, San Francisco, Calif., NADPH from Sigma, St. Louis, MO. Other chemicals were of analytic reagent grade obtained from the usual commercial sources. All solvents were redistilled before use. Results and Discussion The purity of the mitochondrial and microsomal fractions from normal rat liver and the Morris 7777 hepatoma was assessed by measurement of succinate dehydrogenase and rotenone-insensitive NADPH-cytochrome c reductase and the results are shown in Table I. Rotenone-insensitive NADPH-cytochrome c

234

TABLE

I

MARKER

ENZYME

LEVELS

IN

are given

in nmol/mg

NORMAL

LIVER

AND

MORRIS

1771

HEPATOMA

SUBCELLULAR

FRACTIONS The

values

tests

of

significance

Subcellular

were

per min;

performed

P values

using

are for Morris

Succinate

fraction

t test

Student’s

of

dehydrogenase

7777

hepatoma

the

difference

vs. normal between

liver. unpaired

Statistical groups.

Rotenone-insensitive NADPH-cytochrome

c

reductase Liver

mitochondria

Morris Liver

7171

(4)

mitochondria

microsomes

Morris

7171

18.6

? 6.7

13.0

+ 5.2

1.34

f 0.7

0.79

f. 0.6

(8)

(4)

microsomes

(8)

(N.S.)

0.73

i. 0.7

0.71

+ 0.5

41.3 (N.S.)

(N.S.)

f 8.2

7.4

I 2.9

(P