GTP-dependent membrane fusion during hepatocarcinogenesis and liver regeneration

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GTP-DEPENDENT

Jacques

1494-l 500

MEMBRANE FUSION DURING HEPATOCARCINOGENESIS AND LNER REGENERATION

Palement,

J. Manuel

Domlnguez,

Anne Guhette

and Line Roy

Departement d’anatomie, Faculte de medecine Universite de Montreal, C.P. 6128, Succ. A Montreal, Quebec, CANADA H3C 3J7 Received

April

2, 1991

SUMMARY: Rough microsomes were isolated from homogenates of livers of rats bearing hepatomas as well as from homogenates of livers of rats 24 and 48 h after partial hepatectomy. When incubated in the presence of GTP in a cell-free system to assay membrane fusion these membranes were observed to have a greater capacity (1.4 to 5 fold) for GTP-dependent fusion than homologous membranes from control nonproliferating liver tissue. The enhanced GTP-dependent membrane fusion may reflect 0 1991Academic Press. Inc. changes in membrane properties related to cell proliferation.

The nuclear envelope, complex

the endoplasmic

reticulum

are thought to exist as single copy organelles

During cell division these organelles membranes

(ER) network in interphase

Duplication

must be duplicated.

and redistributed

to the daughter cells.

the cytoplasmic important

organelles

in post-mitotic

constituents

reconstitution.

Included

thought to play important roles in such reconstitutive

lipids necessary to permit interaction,

GTP-binding

recognition

proteins have recently been implicated

the ER (12-16) between membranes (20,21), between Golgi membranes between secretionvesicle

membranes

1991

All

of reproductiorl

by Academic in arg

Press.

form

and membrane

amongst

the

coalescence.

in the fusion between membranes

(22,23), between endosomal and plasma membranes

1~. reserved.

constituents

of

of nuclei (17-19) between ER and Golgi membranes

$1.50

0

fusion is an

processes are proteins

membranes

1494

(24-26) and

(27-29) of non-proliferating

cells. In this investigation the question of whether rough ER membranes Copwight

membrane

cells. One might expect, therefore,

within such cells are endowed with the molecular

required to allow efficient and rapid organelle

membranes

they fuse together and form

in the daughter cells (1,4-l 1). Thus membrane

step in organelle formation

that the membranes

rights

of intracellular

The redistributed

fragments then act as templates for organelle reconstitution,

0006-291X/91

animal cells (l-3).

does not occur de nova, but rather, the maternal cytoplasmic

are fragmented

and/or

and the Golgi

from proliferating

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tissue notably livers with primary tumors and livers undergoing the presence of GTP was considered. the GTP-dependent membranes

Furthermore

fusion of such membranes

from normal non-replicating

MATERIALS

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regeneration,

using electron microscope is compared

quantitatively

can fuse in stereology with that of

tissue.

AND METHODS

induction and characterization of liver proliferation : Liver tumors were induced in male Fischer rats by administration of aflatoxin B, (Sigma Chemical Co., St-Louis, MO, USA) (30). Treatment was continued for 55 weeks. Tumors appeared after 51 weeks. Livers were used to prepare homogenates at different times before and after appearance of tumors. Portions of tissues were also fixed (31) for histopathological examination. The histological features of the tumor-bearing livers of our experimental animals were essentially similar to those described previously for rat liver tumors induced by aflatoxin B, (32). Seventy percent partial hepatectomy was carried out according to the method of Higgins and Anderson (33). Experimental and control rats were killed at intervals of 24 and 48 h after operation. In order to determine the extent of ceil proliferation occuring after various proliferative stimuli, a single injection of 13H]thymidine (New England Nuclear, Canada, sp. act. 70 Ci/mmol) was given intraperitoneally (256 p Ci) one hour before sacrifice. Pieces of liver (1-3 mm) were obtained and immersed in fixative (31). Following dehydration and embedding in paraffin sections (5 pm thick) were cut serially, put on slides and stained with haematoxylin and eosin. The sections were coated with Kodak NTB2 emulsion and processed for radioautographic analysis (34). Light microscope radioautography was carried out to determine the [3H]thymidine-labeling index of hepatocytes in tumor-bearing rats. The labeling indices for cells in tumors were compared with those of surrounding liver tissue. Results from two different experiments showed higher labeling indices for cells within tumors (4.2 to 10 fold greater t3H]thymidine incorporation, data not shown). Light microscope radioautography was carried out to determine the [3H]thymidine-labeling index of hepatocytes in liver of partially hepatectomized rats. The labeling index was very high for hepatocytes 24 and 48 h after hepatectomy (28.8 and 8.5 respectively) compared to that for hepatocytes in animals 24 and 48 h after sham surgery (1.1 and 0.9 respectively). Subcellular fractionation : To prevent interference of accumulated glycogen during fractionation of carinogen-treated livers rats were starved 48 h before they were killed (35). Partially hepatectomized animals were starved 24 h before operative procedures (36). Rough microsomes were prepared from rat liver homogenates as previously descrrbed (13). The microsomes were stripped of ribosomes by using 5 mM-sodium-immldazole buffer pH 7.4, containing 0.25 M sucrose and 5 mM sodium pyrophosphate (13). Protein concentrations were determined using the Lowry procedure (37) with bovine serum albumin as standard. Cell-free assay for membrane fusion : For the cell-free membrane fusion assay incubations were carried out in 0.25 ml of medium consisting of 40 mM Tris-HCI (pH 7.4), 7.5 mM MgCI, and 0.5 mM GTP. For quantitation 0.1 ml of microsomes (containing 200 p g of protein) were added just prior to the start of the incubation. Incubations were done at 37’ C for 120 min. For morphology microsomes were fixed and processed for electron microscopy as previously described (14). Quantitation of GTP-dependent membrane fusion was done using morphometry. Membrane lengths of embedded and sectioned rough microsomes were calculated as previously described (14, 38). The 1495

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profile perimeter lengths of sectioned vesicles were accumulated until a total of 737 vesicle profiles (maximum capacity for the computer program used) were measured for each experimental condition. This yielded a value for the sum of membrane lengths and this value was used to calculate the fusion index. For the fusion index the total membrane length of 737 vesicle profiles in a preparation of stripped rough microsomes incubated using non-fusion conditions was first determined. Then, the total membrane length of 737 vesicle profiles in the preparation of stripped rough microsomes incubated using fusion conditions was calculated. Since small vesicles fuse to form large vesicles under fusion conditions the sum of the membrane lengths for 737 vesicles is always higher than that for an equivalent amount of vesicles incubated using non-fusion conditions. To calculate the fusion index the total membrane length for 737 vesicles incubated using non-fusion conditions is subtracted from the total membrane length for vesicles incubated using fusion conditions and the difference is expressed as a percent value of the total membrane length for vesicles incubated using fusion conditions.

RESULTS The fusion capacity for stripped rough microsomes aflatoxin B, was compared

with that for stripped rough microsomes

rats. When stripped rough microsomes

microsomes

from livers of control

were incubated in the presence of cation only

and then examined in the electron microscope to have homogeneous

from livers of rats treated with

they were observed to be aggregated

and

vesicle sizes (Figures 1A & 2A). However when stripped rough

were incubated in the presence of both MgCl, and GTP they were observed

to be aggregated

and to have heterogeneous

vesicle sizes (Figures 18 & 28). GTP

stimulated fusion of the stripped rough microsomes vesicles (Figures 1B & 2B arrows).

and caused the formation

Stripped rough microsomes

of the large

from livers of aflatoxin

B,-treated rats consistently showed larger vesicles after incubation in the presence of GTP than those of microsomes 28).

This was confirmed

incubated

membranes

in the presence

lengths for GTP-incubated

The sum of the membrane

(see Materials and Methods). rough microsomes

analysis.

rats (compare Figures 1 B and Using electron micrographs

lengths, or perimeters

of MgCI,

and GTP.

microsomes

lengths was then used to determine

This generated a fusion index

Results are given in Table I. When compared

from livers of carcinogen-treated

statistically

rats exhibited greater (eg. 1.4 to 5.2 from livers of

rats (Table I).

To compare the fusion of membranes of membranes

of incubated

from control and treated

fold) capacity to fuse in the presence of GTP than rough microsomes control-treated

of

from livers of control rats as well as of those from livers of

rats after incubation

histograms of membrane tissues.

by morphometric

we measured the membrane

vesicles, for microsomes treated

from the livers of control-treated

from normal regenerating

from livers with hyperplastic

lesions with that

liver, rats were subjected to two thirds partial 1496

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m. Electron micrographs showing stripped rough microsomes from livers of control rats incubated 120 min. at 370 C in the presence of 7.5 mM MgCI, and in the absence (A) or presence (B) of GTP. Arrows indicate large membrane fusion products produced in the presence of GTP. Bars, 1 pm. Pla. 2. Electron micrographs showing stripped rough microsomes from livers of rats treated with aflatoxin 8, for 51 weeks. Microsomes were incubated 120 min. at 37’ C in presence of 7.5 mM MgCI, and in the absence (A) or presence (B) of GTP. Arrows indicate very large fusion products produced in the presence of GTP. Bars, 1 pm.

hepatectomy.

Rats were killed at 24 and 48 h after surgery and rough microsomes

purified from liver homogenates.

stripped rough microsomes

GTP-dependent

membrane

fusion was assessed

using

from livers of rats obtained 24 and 48h after hepatectomy

well as using similar membranes

as

from animal livers 24 and 48 h after sham surgery.

quantitative

analysis of fusion indicated that stripped rough microsomes

undergoing

rapid proliferation

from quiescent

were

were able to fuse better in the presence

A

from livers of rats of GTP than those

livers of control rats (Table II).

DISCUSSION Membranes primary tumors

of rough microsomes and livers undergoing

fusion when incubated membrane

in the presence

fusion was first described

liver (12). It was suggested

from proliferating regeneration,

underwent

of GTP and MgCI,.The using stripped

that this nucleotide 1497

tissue, notably

efficient and significant

involvement

rough microsomes

may be important

livers with

of GTP in RER from normal rat

for the maintenance

of

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I

MORPHOMETRIC COMPARISON OF GTP-DEPENDENT FUSION OF STRIPPED ROUGH MICROSOMES FROM LIVERS OF CONTROLAND AFLATOXIN-TREATED RATS Time after initiation of treatment (weeks)

Total membrane b-N

Mean membrane length &ml

Standard deviation

Fusion index W)

25

SRM control SRM treated

209 308

.284 .418*

.270 .537

7.3 29.8

30

SRM control SRM treated

255 275

346 .373**

.442 .679

15.3 21.5

34

SRM control SRM treated

238 373

.323 .505*

.306 .760

9.2 42.1

51

SRM control SRM treated

218 326

.296 .44.2*

.334 .720

7.3 38.0

59

SRM control SRM treated

282 385

,383 .522*

.365 .736

28.7 47.8

60

SRM control SRM treated

283 397

.383 .539*

.423 .860

28.9 49.3

Morphometry l

Type of microsome

and the fusion index are defined in the Materials

and Methods,

P vs control, P < 0.001. P vs control, 0.4 > P > 0.2.

l *

TABLE

II

MORPHOMETRIC COMPARISON OF GTP-DEPENDENT FUSION OF STRIPPED ROUGH MICROSOMES FROM LIVERS OF SHAM OPERATEDAND PARTIALLY HEPATECTOMIZED RATS Hours after Type of surgery microsome

Total membrane

Mean membrane length Qm)

24

SRM control SRM hepatectomy

286 433

388 .588*

48

SRM control SRM hepatectomy

308 416

.417 .565*

Morphometry Methods. * P vs control,

and calculation

of the fusion

Standard deviation

Fusion index (W

.451 .807

29.4 48.1

.534

34.2

1.044

index are described

P < 0.001. 1498

51.4

in the Materials

and

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the organization

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of the RER in hepatocytes

involved in membrane GTP

may be an important

membrane

traffic in non-dividing

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(12). GTP has since been shown to be

traffic between divers organelles

Therefore

RESEARCH

from interphase

factor permitting

cells, as well as, organelle

This would be consistent with the suggestion

membrane reconstitution

cells (20-29). assembly

and

in mitotic cells.

of Warren (39) that the fusion mechanism

that operates during mitosis may be similar to the one that operates during interphase and allows intracellular The enhanced

transport. capacity for fusion of RER membranes

animals as well as that of RER membranes changes

in the content

dependent peroxidation

membrane

of GTP-binding fusion and/or

from carcinogen-treated

from regenerating proteins

required

to other changes

known to occur in proliferative cell populations

liver may be related to for the initiation

of GTP-

such as changes

in lipid

(40). Studies are underway

to examine some of these possibilities.

ACKNOWLEDGMENTS

We thank Jean tiveille for photographic work and Chantal Joseph for typing the manuscript. The work was supported by grants from the Medical Research Council of Canada, the Fonds de la Recherche en Sante du Quebec, the Cancer Research Society Inc. (MT-7325, 850039 and MD37960 to JP), and by the Fonds pour la Formation de Chercheurs et I’Aide a la Recherche (studenship, J.M.D.).

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