[CANCER RESEARCH 37, 354-357, February 1977]
Lysosomal Enzyme Activities in the Regenerating Rat Liver1
Berta Fiszer-Szafarz2
and Claude
Nadal
FondationCurie-Institut
du Radium,UniversitO
de Paris-Sud,
Bátiment
111,91405Orsay,France
SUMMARY
lysosomal system in normal cells preparing for division, as
shown by the increase in activity of hyaluronidase and Ca
thepsin D at 9 hr, followed by the disappearance of 4 en
zymes studied (hyaluronidase, f3-N-acetylglucosaminidase,
acid phosphatase, and cathepsin D) from the lysosomes at
the end of G1phase, before the beginning of DNA synthesis,
and (b) that the lysosomal system slowly reorganizes to
normality after mitosis.
The activity of four lysosomal enzymes (hyaluronidase, f3N-acetylglucosaminidase, acid phosphatase, and cathepsin
D) was studied in aqueous extracts of the light mitochon
drial fraction of regenerating male rat liver. This tissue was
chosen as a model for normal cell division in vivo. In the first
wave of division, 40 to 50% of the cells divide synchro
nously. Activities were measured at 0, 9, 18 (end of G,
phase), 24 (S phase), and 30 hr (mitosis) and during regen
MATERIALS AND METHODS
eration, 4 and 11 days after partial hepatectomy. Activities
were related to fresh tissue weight, to cellular DNA, and to
Animals. Seven-week-old male Wistar rats were used.
protein content of the extracts.
The animals were kept on a standard pellet diet and water
At 9 hr, there was an important increase in hyaluronidase
and cathepsin D activities (these two enzymes act upon ad libitum. Food was not withheld prior to hepatectomy, so
macromolecules) ; @-N-acetylglucosamin idase and acid that glycogen depletion would not affect liver regeneration.
Hepatectomy. The animals were partially hepatectomized
phosphatase activities were only slightly increased. At the
under ether anesthesia. Two-thirds of the liver were re
end of the G, phase, 40 to 50% of the activity of all four
enzymes was lost, which might indicate complete loss of moved and used as controls.
In a given group of rats from the same litter, the interval
activity in cells undergoing division. This depletion per
sisted until mitosis was complete. Four days later, there was between hepatectomy and killing (the latter was always
done between 9 and 10 a.m.) was determined in order to
a slow restoration of enzyme activities; after 11 days, hyal
preserve the circadian rhythm of the animals. Sham opera
uronidase and cathepsin D exhibited about 80% of their
initial activity, whereas f3-N-acetylglucosaminidase and acid tions were also performed 9 and 24 hr after hepatectomy to
check for possible effects of the shock caused by the opera
phosphatase only regained about 50%.
These results show that the lysosomal system perhaps tion on the enzymes studied.
plays some role in cell division.
The periods elapsing between hepatectomy and killing
were chosen in accordance with kinetic data (Chart 1),
obtained as described below: 9 hr (G,), 18 hr (end of G,), 24
INTRODUCTION
hr (middle of 5), 30 hr (M), 4 days (very low thymidine incor
poration), and 11 days (regeneration accomplished).
It has been well established that hyaluronidase, an acid
Thymidine-labeling Index of Nuclei. Rats were given in
hydrolase that degrades hyaluronic acid, is present in nor
jections i.p. of 0.5 @Ci
of [6-3H]thymidine (5 Ci/mmole) 30
mai liver lysosomes (16) but is absent from these organelles
mm prior to sacrifice. Liver fragments were fixed in 4%
in hepatoma (13). Other acid hydrolases, such as acid phos
formalin and embedded in paraffin. Slides with 5-sm thick
phatase, aryl sulfatase, esterase, and ANase (9, 14, 24, 28) slices were dipped in K5 Ilford emulsion. The percentage of
have also been shown to be present in smaller amounts in
labeled nuclei was evaluated by examination of 10,000
tumors than in normal tissue. This paper sets out to exam
hepatocyte nuclei (21).
me the question of whether these changes are peculiar to
Chemicals. All reagents were purchased from Sigma
tumoral cells or common to all dividing cells, even nonma
Chemical Co., St. Louis, Mo., except hyaluronic acid, which
lignant ones. We therefore decided to study the activity of was prepared in our laboratory by the method of Mathews
various acid hydrolases in lysosomes from regenerating rat (20).
liver taken as a model for normal cell division. This model is
Preparation of Lysosomal Extracts. The method fol
particularly suitable because the 1st wave of cell divisions
lowed was essentially that described in our previous paper
following partial hepatectomy in young rats is rather well
(13), with water extraction lasting only 1 hr instead of 3. The
synchronized.
amount of distilled water used for extracting the light mito
Our results show (a) that important changes affect the chondrial pellet was calculated so that the final volume of
extract was 1 ml for 2 g of initial liver tissue.
Assay of Hyaluronidase (EC 3.2.1 .35). The same method
Memorial Trust, London, England.
as in Ref. 13 was used. Aliquots of 0.050 ml were taken after
2 To whom
requests
for reprints
should
be addressed.
Received June 16. 1976; accepted November 1, 1976.
0, 10, 20, 40, and 60 mm.
I This
354
research
was
supported
in
part
by
a
grant
from
the
Lady
Tata
CANCER RESEARCH VOL. 37
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Lysosoma! Enzymes in Regenerating Liver
tions in the various extracts and expressed in @kat/Iiter
[defined as @.tmoles
of reaction product liberated per sec per
liter of extract (17)].
Chemical Determinations. f3-N-Acetylglucosammne was
estimated by the method of Aeissig et a!. (26). DNA was
estimated in the initial tissue homogenate by the method of
Burton (8). Protein was estimated in the extracts by a modi
fication of the method of Lowry et a!. (12).
RESULTS
Chart1. A, evolutionof enzymeactivity/mgDNA,in percentage
of initial
activity. •,hyaluronidase; L@,/3-N-acetylglucosaminidase; 0, acid phospha
tase; A, cathepsin D. B, percentage of [3H]thymidine-Iabeled nuclei.
Assay of @-N-AcetylgIucosaminidase (EC 3.2.1 .30). For
the method used, see Ref. 13. However, the pH of the assay
was 4.0, corresponding to the optimal value determined
under our experimental conditions.
Assay of Acid Phosphatase (EC 3.1 .3.2). This enzyme
was assayed by the following procedure, which is a slight
modification of that described by Barrett (2). Four ml of 0.2
M acetate
buffer,
pH 5, were
added
to 1 ml of a solution
containing 20 to 100 p1 of the extract diluted with bovine
serum albumin aqueous solution (1 mg/mI). The mixture
was incubated at 37°for 10 mm to destroy glucose-6-phos
phatase activity. To 1.5 ml of this solution was then added
0.5 ml of an aqueous solution of 2 mM disodium p-nitro
phenylphosphate. The mixture was incubated at 37°.Ali
quots of 0.3 ml were taken after 0, 3, 6, 9, and 12 mm and
mixed with 0.3 ml of a buffer containing 1 M Tris-HCI and 0.4
M Na2HPO4,
pH 8.5, which
stops the enzymatic
reaction
and
enables measurement of the absorbance of liberated p
nitrophenol at 400 nm against a blank lacking the substrate.
Assay of Cathepsin D (EC 3.4.4.23). This enzyme was
assayed by slightly modifying the method described by Bar
rett (3). One-tenth ml of aqueous bovine hemoglobin (60
mg/mi) and 0.1 ml of lysosomal extract were added to 0.8 ml
of 0.05 M formate buffer, pH 3.5, containing 0.3 M NaCI.
(When the assayed extract was very active, it was diluted
The results concerning the enzyme activities measured
are the average of 5 experiments, corresponding to as many
groups of animals; each time point was thus the average of
10 animals. The sham operations performed at 9 and 24 hr
had no effect on the enzyme activities.
As no single parameter remained unaffected by regenera
tion phenomena, we chose 3 parameters which we assumed
to be reasonably independent: Parameter 1, fresh tissue
weight. After hepatectomy, liver weight is known to be
affected by numerous phenomena (hydration, accumula
tion of neutral fat, glycogenolysis, later followed by glyco
genesis, etc. Parameter 2, DNA. The difficulty here is the
continuous DNA synthesis during the initial days of regener
ation. Parameter 3, the protein content of the water-soluble
extract from the light mitochondrial fraction containing
most of the lysosomes. In fact, however, the lysosomes
contribute only a small percentage of the proteins in that
fraction (4).
From Table 1, which shows the ratios between the re
spective values of the 3 parameters for all the time points
studied, it is clear that the ratio of DNA to fresh tissue
weight does not vary significantly at any one point.
As to the ratio of extracted protein to fresh tissue weight,
there is an important increase 9 hr after hepatectomy.
Table 2 gives the values of the respective activities of
hyaluronidase, /3-N-acetylglucosaminidase, acid phospha
tase, and cathepsin D in relation to DNA. At 9 hr after
hepatectomy there is a rise in the levels of activities of all 4
enzymes in relation to the 3 parameters, as can be calcu
lated from Tables 1 and 2. If the increases are related to the
initial activities, they appear lower when related to protein
content than with the other 2 parameters; this might be due
to an increase in nonlysosomal proteins belonging to parti
des coprecipitating with the Iysosomes, as shown by the
increase in that parameter in Table 2. After 9 hr, the evolu
twice with distilled water before the assay.) The mixture was
incubated at 37°.Aliquots of 0.1 ml were taken after 0, 2, 4,
8, and 12 mm and mixed with 0.5 ml of 3% trichloroacetic
acid. The precipitated protein was pelleted, and the liber
ated acid-soluble peptides in the supernatant were esti
mated by the method of Lowry et al. (19), adjusted to suit
the present conditions as follows. One-tenth ml of superna
tent was mixed with 0.6 ml of Aeagent E, in which Reagent
A was 0.125 N NaOH instead of 0.1 N (the extra concentra
tion being exactly the amount needed to neutralize the
trichloroacetic acid), and with 0.06 ml of the Folin-Ciocal
teu reagent. Readings were made at 750 nm with tyrosine as
standard.
All enzyme activities were evaluated as activity concentra
Table 1
Relationship between DNA, fresh tissue weight,
regenerationTime
content of Iysosomal extract during rat liver
after
hepatectomy
(mg/g)0
hr
DNA/fresh liver
(mg/g)
(13)―
1.90 ±0.06―
and protein
Protein/fresh liver
(13) 2.33 ±0.12
9hr
(9)
1.99±0.04
(9)2.91
18 hr
24 hr
30 hr
4 days
0.14a
11 days
(4)
(4)
(1)
(4)
(4)
1.91 ±0.11
1.82 ±0.08
2.02
2.08 ±0.09
1.72 ±0.06
(4) 2.26 ±0.22
(4) 2.32 ±0.16
(3) 1.93 ±0.11
(1) 2.38
(4) 2.33 ±
Numbers
in parentheses,
number
of
lysosomal
±0.09
extracts
stud
ied.
b Mean
± S.E.
FEBRUARY 1977
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355
B. Fiszer-Szafarz and C. Nadal
Table 2
Lysosoma!enzymeactivities in regenerating rat liver
D0
cosaminidaseAcid
EnzymeHyaluronidase/3-N-Acetylglu
0.067 ±0.004―(7) 2.07 ±0.25(12)
0.049
hr(14)―
phosphataseCathepsin
5.03 ±0.43(1
2) 0.58 ±
hr(9)
0.121
0.110 ±0.015(9)
2.37 ±0.20(9)
5.32 ±0.51(9)
0.87 ±
0.0824
8 hr(4)
0.032 ±0.004(4)
1.47 ±0.23(3)
2.28 ±0.20(3)
0.37±
hr(2)
0.0430
0.035 ±0.008(3)
1.15 ±0.20(4)
1.92±0.17(4)
0.30 ±
0.032 ±0.003(3)
0.41 ±0.12(3)
1.08±0.10(3)
0.22 ±
2.58 ±0.37(3)
0.47 ±0.09
0.034days(4)
hr(3)
0.043±0.004(4)0.77±0.08(4)2.12±0.15(4)0.40±0.041
b Mean
1.14 ±0.07(4)
0.059 ±0.007(4)
1 days(4)
a Numbers
in
± S.E.,
parentheses,
expressed
number
in
nkat/mg
of
experiments.
DNA.
tion of all the activities in relation to any of the 3 parameters
is the same. This shows that our results do not depend on
the variation of a single parameter but reflect a real phe
nomenon.
If, as in Chart 1, we superimpose the curves for the 4
enzyme activities in relation to DNA and compare them with
the curve indicating thymidine incorporation into DNA, we
can see how lysosomal activity varies during the cell cycle
and liver regeneration.
Enzyme activities start increasing between 0 and 9 hr but
afterward begin to decrease; by 18 hr, at the end of the G,
phase,thedecreaseisbetween40 and 50%. Ifthisdrop is
linked to the replicative cell cycle, our results can be ac
counted for by the complete lysosome depletion occurring
in those cells entering the cycle, since the 1st wave of
division in 7-week-old rats involves about 40% of the hepa
tocytes. Values were lower at 24 hr and still lower at 30 hr,
when the synchronous 1st wave ended and asynchronous
multiplication began.
On the 4th day, when mitosis reverted to a low level, our
results show that the level of lysosomal enzyme activity was
still low. By the 1ith day activity had considerably increased
but was still not quite normal.
Quite apart from the above phenomena we observed that
the enzymatic activities measured in lysosomal young rat
liver extracts were much more variable than the corre
sponding activities measured in adult extracts. Conse
quently, the same variability was observed in regenerating
liver, since the animals used were necessarily young. How
ever, this problem was minimized by using, as far as possi
ble, animals from the same litter for a given kinetic experi
ment.
DISCUSSION
Little is known about the life cycle of lysosomes. The
present work indicates that at least 2 distinct processes
affec@tthem during the cell cycle: (a) their function before
mitosis and (b) the reorganization of the lysosomal system
in a new cell.
Normal cell division, as exemplified by regenerating rat
356
liver, is accompanied by important changes in lysosomal
enzyme activity. The 1st wave of division, which is fairly well
synchronized, enables these changes to be related to the
various phases of the cell cycle.
During the 9 hr following hepatectomy, Becker and Lane
(5) observed the presence of a large number of autophago
somes (lysosomes in the process of digesting the cytoplasm
of the cell in which they are located). The number of auto
phagosomes gradually diminished, afterward, and within 24
hr they had disappeared altogether. Verity et al. (29) found a
decline of acid phosphohydrolase, f3-glucuronidase, fJ-glu
cosidase, and f3-galactosidase 24 hr after hepatectomy in
female mice. Allison and Mallucci (1) also observed that
reorganization of all constituents and loss of lysosomes
preceded mitosis in HeLa cells. Finally, Quintart and
Baudhuin (25) noticed a drop in lysosomal activity in cul
tured hepatoma cells during mitosis (see Ref. 7 for review of
posthepatectomy reorganization). This situation is reminis
cent of that encountered in starvation (11) or after sublethal
hepatotoxic treatment (18), since in both cases the cell
degrades certain constituents by autophagy in order to
build up others.
The present study concerns developments occurring 9 hr
after partial hepatectomy and thereafter.
Events at 9 hr are characterized by variations in lysosomal
enzyme activity. All activities increased: those of hyaluroni
dase and cathepsin D, which act upon macromolecules, by
about 50%, whereas others such as f3-N-acetylglucosamini
dase and acid phosphatase increased by about 10% (/3-Nacetylglucosammnidase is known to act on small molecules,
which is probably also the case for acid phosphatase). The
end of the G, phase (18 hr) is characterized by a general
drop in lysosomal activity, which seems to correspond to
complete enzyme depletion in cells entering a division cy
cle. This conclusion stems from the parallel between the
percentage of reduction in enzyme activity and the percent
age of dividing cells. No notable change in the activity of the
4 enzymes studied was observed during subsequent stages
of the division cycle.
In regenerating liver the dividing cells probably reorga
nize their structure, one aspect of which is an increase in
the activity of the lysosomal enzymes responsible for organ
CANCER RESEARCH VOL. 37
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Lysosomal Enzymes in Regenerating Liver
elle degradation and of constituents not involved in divi
sion. These enzymes are then gradually used up and are not
renewed. In fact, they might originate in the Golgi apparatus
(6, 22, 23), part of which has been observed to disappear
before division (10, 27). This phenomenon would then be
the direct cause of the loss of lysosomes before mitosis, as
observed microscopically by the authors mentioned above
and, biochemically, by ourselves.
The 2nd stage of lysosome development takes place in
the daughter cell after division. This stage is characterized,
among other events, by the reconstitution of the Golgi
apparatus (10), which is a slow process. Such reconstitu
tion is also reflected in the increased lysosomal activity
measured between the 4th and 11th days afterhepatec
tomy. Horvat and Acs (15) observed an increase in lysoso
mal enzyme activities in cultured cells reaching the stage of
contact inhibition.
The observation that mitosis is accompanied by complete
loss of the 4 lysosomal enzymes studied is similar to the
observation we reported in a previous work on actively
growing hepatoma (13). In this malignant tissue, we also
observed complete loss of lysosomal hyaluronidase and a
considerable drop in /3-N-acetylglucosaminidase, which
can probably be explained by the depletion of cells in the
process of division. The similarity in the behavior of both
normal and tumoral growing tissue is interesting as regards
the role of lysosomes in cell division.
6. Boutry, J. M., and Novikoff, A. B. Cytochemical Studies on Goigi Appara
tus, GERL, and Lysosomes in Neurons of Dorsal Root Ganglia in Mice.
Proc. NatI. Acad. Sci. U. S., 72: 508-512, 1975.
7. Bucher, N. L. R., and Malt, A. A. Regeneration of Liver and Kidney, pp.
23-54. Boston: Little Brown and Co, 1971.
8. Burton,K.A Studyof theConditionsandMechanism
of Diphenylamine
9.
10.
11.
12.
13.
14.
ity and Neoplastic
15.
16.
17.
18.
19.
20.
21.
ACKNOWLEDGMENTS
We would like to express our appreciation to C. Pinon for her skillful
technical assistance.
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FEBRUARY1977
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357
Lysosomal Enzyme Activities in the Regenerating Rat Liver
Berta Fiszer-Szafarz and Claude Nadal
Cancer Res 1977;37:354-357.
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