[CANCER
RESEARCH
38. 16-22. January
1978]
Location of Adult and Fetal Aldolases A, B, and C by Immunoperoxidase
Technique in LF Fast-growing Rat Hepatomas1
Antoinette Hatzfeld,2 GérardFeldmann, Jocelyne Guesnon, Christiane
Frayssinet,
and Fanny Schapira2
With the technical assistance of Marie-France Szajnert, Michèle Maurice, and Michel Wolfelsperger
Institut de Pathologie Moléculaire, INSERM U.129 et LA 85 CNRS, 24, rue du Faubourg Saint-Jacques 75014 Paris JA. H., F. S.]: Unitéde Recherches de
Physiopathologie
Hépatique, INSERM U.24, Hôpital Beau/on, 92778 Clichy-Cedex ¡G.F., J. G.]: and Institut de Recherches Scientiliques
sur le Cancer,
Bolïe Postale 8, 94800 Ville/uil, France [C. F.J
ABSTRACT
The resurgence of aldolase isozymes in cancerous
tissues is a well-known but poorly understood phenome
non. This resurgence poses the problem of whether or
not adult and fetal aldolase isozymes are produced by
the same cells.
For clarification of this question, the immunoperoxidase
technique was used to locate aldolases A, B, and C in
one type of fast-growing hepatoma, the LF hepatoma
and, by comparison, in normal adult liver. Under optical
microscopy, aldolases A and C were located in the cyto
plasm of almost all of the cancerous cells. An isozyme
antigenically identical with aldolase B was also demon
strated to be present in almost all of the cells, but the
reaction indicating the presence of this isozyme was
weaker. In normal adult liver, only aldolases A and B
were demonstrated to be present in almost all the hepatocytes. Under electron microscopy in LF hepatoma, the
three isozymes were found to be present mainly in the
cytoplasm.
These facts suggest that the three types of aldolase
are very probably present in the same cells at the same
time, and they provide indirect arguments leading us to
think that the resurgence of fetal aldolase isozymes in
cancer is not the consequence of cellular selection but is
due to a disturbance at the gene control level.
INTRODUCTION
The resurgence of fetal isozymes in cancer is a wellknown phenomenon demonstrated first for aldolase (33)
and later for several other enzymes (25, 32, 35). This
resurgence poses a problem. Do the cells that normally
synthesize the adult isozyme become capable of synthesiz
ing the fetal type during carcinogenesis? In this case genes
not expressed normally would become expressed in the
cancerous state. On the other hand, are the cells that
synthesize fetal isozymes different from the cells that syn
thesize the adult type? In this case these cells would be
fetal cells selected during carcinogenesis.
It is difficult to demonstrate whether both adult and fetal
proteins are synthesized by the same cells or by different
1 Preliminary results have been presented at the Twenty-fourth
Annual
Colloquium
'Protides of the Biological Fluids," Brugge, Belgium. 1976 (17),
and at the Conference on Onco-Developmental
Gene Expression, San Diego,
Calif., 1976 (16). This work was supported in part by Grant INSERM 7650817.
2 To whom requests for reprints should be addressed.
Received July 20. 1976; accepted
16
October
5, 1977.
cells. First, we need an adult counterpart to the fetal
enzyme. This is the case with aldolase, in contrast to afetoprotein for instance. Aldolase B is the predominant
enzyme in adult liver, and aldolases A and C are plentiful in
fetal liver (18) and in fast-growing hepatomas (34). Sec
ondly, histological methods specific for aldolase must be
used. Several of these methods have already been de
scribed (1, 22, 29). However, they demonstrate only that
aldolase is present in the cells; they do not give any
information on the nature of the isozyme. Immunoenzymatic techniques are probably superior because of their
specificity; in addition, they provide information on the
ultrastructural location of these isozymes.
In this study, we demonstrate that in 1 type of solid fastgrowing hepatoma, the LF hepatoma (5, 11), aldolases A,
B, and C are present in almost all of the cells. Using the
immunoenzymatic technique (immunoperoxidase), we were
able to infer that the 3 isozymes are probably synthesized
by-the same cell.
MATERIALS AND METHODS
Material
The LF hepatoma cells of Lafarge and Frayssinet
stemmed from a clone of fast-growing tumor cells obtained
from a 4-dimethylaminoazobenzene-induced
hepatoma (5,
11). Injection of these cells i.p. into 8-day-old Wistar-Commentry rats produced tumors within 4 days. The animals
were killed 6 to 7 days after inoculation, and their tumors
were removed at once. Normal adult liver was obtained
from adult Wistar C-Commentry rats. A fragment from each
tissue was taken for immunological studies; another was
fixed for morphological studies.
Immunological
Methods
Purification of Enzymes. Aldolase A was prepared from
rat muscle according to the method of Gracy ef al. (13).
The extracted supernatant was brought to 45% saturation
with saturated ammonium sulfate. This solution was centrifuged at 37,000 x g for 1 hr, and the precipitate was
dissolved in 10 mM Tris-HCI buffer, pH 7.5, with 1 mM
EDTA-10 mM /3-mercaptoethanol. The solution was then
applied to a cellulose phosphate Whatman column equili
brated with the same buffer. Elution was performed with a
linear NaCI gradient (0 to 350 mM) in 10 mM Tris-HCI
buffer, pH 7.5-1 mM EDTA-2.5 mM fructose 1,6-diphosphate.
The purity of the protein was checked by acrylamide gel
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Location of Aldo/ases A, B, and C by Immunoperoxidase
electrophoresis. Only 1 NhL-terminal amino acid was found
(15). The specific activity of the purified enzyme, measured
by the colorimetrie method of Sibley and Lehninger (34),
was 32 lU/mg with fructose 1,6-diphosphate as substrate
and 0.7 lU/mg with fructose 1-phosphate as substrate. The
activity ratio of fructose 1,6-phosphate to fructose 1-phos
phate characteristic of each isozyme (30) was 50.
Aldolase B was prepared from rat liver. It was purified in
the same manner as was aldolase A with the following
differences. The 45 to 65% ammonium sulfate fraction was
applied to a Whatman CM 52 column. The elution was
performed with 2.5 mM fructose 1,6-diphosphate in 10 mM
Tris-HCI buffer, pH 7.5-1 mM EDTA. The protein was com
pletely purified by isoelectrofocusing in a LKB 8101 column
filled with 1% ampholines (pH 9 to 11). The purified protein
showed only 1 band on polyacrylamide gel electrophoresis
with or without sodium dodecyl sulfate. The specific activity
with fructose 1,6-diphosphate was 2.5 lU/mg; with fructose
1-phosphate, it was 2.5 ID/mg; thus the activity ratio of
both substrates was 1.0.
Aldolase C was prepared from rat brain (15). It was
purified as for aldolase A with the following differences.
The 45 to 65% ammonium sulfate fraction was applied to a
Whatman DEAE 52 cellulose column. The elution was
performed with 0.5 mM fructose 1,6-diphosphate-150 mM
NaCI in 10 mM Tris-HCI buffer-1 mM EDTA, pH 7.5. The
enzyme was further purified by electrophoresis on a potato
starch block and then by isoelectrofocusing in a LKB
column 8101 filled with 1% ampholines (pH 4 to 6). The
purity of the protein was checked by electrophoresis and
by determination of the NH.-terminal amino acid. The spe
cific activity with fructose 1,6-diphosphate as substrate
was 6.0 IU/mg; with fructose 1-phosphate, it was 0.8 ID/
mg. The aldolase C activity ratio was 7.5 (15).
Preparation of Antisera. Antisera to aldolases A, B, and
C were prepared in rabbit by immunization with pure
crystalline enzymes. The first injection of purified aldolase
[1 mg in 1 ml of phosphate-NaCI buffer with an equal
volume of complete Freund's adjuvant (Difco Laboratories,
Detroit, Mich.)] was made into the hind footpads. Ten days
later, i.m. and s.c. injections were made, followed 10 days
after by an i.m. injection. After the last injection, antisera
were tested by immunodiffusion and by immunoelectrophoresis against the supernatant from tissue extracts of liver,
brain, muscle, and normal rat serum (39).
After a washing, the immunodiffusion and immunoelectrophoresis plates were stained by the method of Penhoet
ef al. (30).
Monospecific antisera were obtained by absorption with
the use of normal rat tissue extracts (7). Anti-aldolase A
and C antisera were absorbed by normal rat serum and
liver; anti-aldolase B antiserum was absorbed by normal
rat serum and brain.
Morphological Methods
We used a method similar to that used to demonstrate
albumin in human liver cells (9). Rat tumor and normal
adult liver fragments were fixed for 10 hr at 4°in a solution
of 2.75% paraformaldehyde with 0.2% picric acid buffered
with 100 mM phosphate buffer, pH 7.4 (37). The conserva
JANUARY
Method
tion of the immunological structure of fixed aldolase was
demonstrated in vitro by adding this fixative to purified
aldolase under the same conditions as those used in tissue
fixation. When, after dialysis, the fixed preparation was
tested by immunodiffusion against antialdolase antibodies,
no difference in immunological reactivity between it and
unfixed aldolase was detectable.
After fixation, tissue fragments were washed 2 times
in phosphate buffer with 250 mM sucrose for 48 hr at
4°.One hr before cutting, the fragments were immersed in
a 10% glycerol solution with 100 mM phosphate buffer, pH
7.4. Sections (8 /urn thick) were cut with a cryostat and
divided into 4 groups, 3 groups being incubated for 1 hr at
room temperature with a different aldolase antiserum (1/25
dilution) and the fourth group acting as control. The sec
tions were then washed 3 times, 20 min each, and incubated
with sheep anti-rabbit y-globulin antibodies labeled with
peroxidase (Institut Pasteur, Paris, France). Sections were
washed again (3 times, 20 min each), and the presence of
the peroxidase was revealed by the technique of Graham
and Karnovsky (14). Some sections (LF hepatoma and
normal adult liver) were studied on optical microscopy.
Others (LF hepatoma only) were postfixed in a 1.5% osmium
tetroxide solution buffered with Veronal, pH 7.2-50 mM
buffer for 1 hr and embedded in Epon. Ultrathin sections
were examined in a Siemens Elmiskop IA electron micro
scope without further staining.
Control reactions were prepared as follows. Some sec
tions were incubated with normal rabbit serum and then
incubated with labeled sheep anti-rabbity-globulin antibod
ies. Other sections were incubated in the Graham and
Karnovsky solution only (14), and still others were incu
bated with anti-aldolase antiserum absorbed by the corre
sponding antigen and then incubated with labeled sheep
anti-rabbit -y-globulin antibodies.
Identification of Tumor Aldolases
Tissue aldolases in the supernatant of hepatomas were
detected by immunodiffusion. Enzymes were extracted with
either 0.5 or 4 ml of 150 mM NaCI in water per g of tissue
and centrifuged at 37,000 x g for 30 min; the supernatants
were put into wells around a central well containing monospecific antiserum. After the appearance of precipitation
lines, immunodiffusion
plates were washed and then
stained by the method of Penhoet ef a/. (30).
RESULTS
Morphological Results
Light Microscopy. In LF hepatomas both aldolases A and
C appeared as dark brown deposits located in the cyto
plasm of the cells, whereas the nucleus remained unstained
(Figs. 1 and 2). Almost all the cells contained aldolases A
and C. However, stain intensity varied from cell to cell. No
aldolase was detected in the connective tissue. Similar
results were obtained for the intracellular distribution of
aldolase B; however, the intensity of the staining was
weaker than that observed for aldolases A and C (Fig. 3).
Because of the thickness of the sections (8 ^m) and the
1978
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17
A. Hatzfeld et al.
size of the cells, the superposition of 2 cells cut at different
levels can sometimes give the appearance of a "positive"
nucleus for aldolase; hepatoma cells have an average diam
eter of about 10 ¡j.mand their nucleoplasmatic ratio is
high. The control reactions were negative (Fig. 4).
In normal adult liver, aldolase B was detected in the cyto
plasm of almost all the hepatocytes. The reaction appeared
very strongly as dark brown granulated deposits (Fig. 5).
Aldolase A was also located in the hepatocyte cytoplasm,
but the intensity of the reaction was very much weaker
than that for aldolase B (Fig. 6). By contrast, no reaction
was detectable in the cytoplasm of the hepatocytes with
anti-aldolase C antiserum (Fig. 7). The control reactions
were negative (Fig. 8).
Electron Microscopy. Results for aldolases A, B, and C
in LF hepatomas were similar: the electron-dense products
of the reaction indicating the presence of aldolase were
located in the whole of the cell cytoplasm; a positive
reaction was sometimes observed on the membranes of
the rough endoplasmic reticulum; however, in LF hepato
mas these organelles are not abundant. No reaction was
visible in the nucleus or in the mitochondria, except for a
slight reaction on the external membranes (Figs. 9 to 11).
All the control reactions were negative (Fig. 12).
Immunological
Results
Immunodiffusion plates showed precipitation lines be
tween normal rat tissues and hepatoma aldolases A and C
and the corresponding aldolase antisera. As demonstrated
in a previous study (19), this confirms the identity between
aldolases A and C from LF fast-growing hepatoma and
from normal rat tissue (muscle, brain, and fetal liver). The
precipitation lines given by fetal liver (with 4 ml of 150 mM
NaCI per g of tissue) proved the presence of aldolase A and
C in this tissue. On the contrary, adult liver extract at the
same concentration and at a stronger concentration (0.5
ml of 150 mM NaCI per g of tissue) did not show any
precipitation lines with anti-aldolase A and C antisera (Fig.
13).
With anti-aldolase B antiserum, no precipitation line was
obtained with hepatoma extracts at a dilution identical with
that used for other normal rat tissues (with 4 ml of 150 mM
NaCI per g of tissue) (Fig. 14). A precipitation line was
obtained only with concentrated LF hepatoma supernatant
extracts (with 0.5 ml of 150 mM NaCI per g of tissue).
Between the precipitation lines of normal rat adult liver
and LF hepatoma, a spur was visible. This line could not
be specifically stained by tetrazolium salt reduction (30).
DISCUSSION
This is the first time that the 3 types of aldolase have
been located in the cytoplasm of cells of a fast-growing
hepatoma, the LF hepatoma.
Immunofluorescence (8, 20, 42) and immunoperoxidase
techniques (10, 31, 42) with the use of monospecific anti
bodies to locate cellular antigen are already in use. The
immunoperoxidase technique is very sensitive since it ena
bles detection of an enzyme by electron microscopy,
whereas the histochemical techniques used particularly for
18
aldolase do not (1, 22, 29). However, as pointed out by
several authors (3, 42), one of the main problems of the
immunoperoxidase technique is to preserve protein antigenicity. Indeed, our tests for the imrnunological characteris
tics of the fixed protein showed that antigenicity is con
served.
With optical microscopy, aldolases A, B, and C were
identified in the cytoplasm of almost all the cancerous
cells. By contrast, only aldolases A and B were identified in
normal adult liver; moreover, the reaction for aldolase A is
much weaker than that for aldolase B. The small quantity
of aldolase A in rat adult liver, already described by other
authors (24, 26) and estimated as less than 1%, was not
detected by the immunodiffusion technique. This fact em
phasizes the sensitivity of the immunoperoxidase tech
nique.
Electron microscopy demonstrates clearly that aldolases
A, B, and C are always located in the cytoplasm, not in the
nucleus. The fact that a small amount of aldolase was
found on the endoplasmic reticulum membranes could be
due to its passive absorption on these membranes. The
occasional presence of aldolase on the mito^chondrial
membranes could be similarly explained. Consequently,
our results in LF hepatomas disagree with the results
obtained by Foemmel ef al. (10), who found that aldolase B
was mainly associated with the endoplasmic reticulum in
normal adult rat liver. However, we confirm the more recent
experiments of Arion and Lange (2), who have demon
strated that aldolase B is not associated with the endoplas
mic reticulum but is located in the soluble supernatant
fraction obtained after ultracentrifugation of the rat adult
liver homogenate.
Morphological results indicate that aldolase B is present
probably in a small amount in LF fast-growing hepatomas.
This is shown by the weak labeling with anti-aldolase B
antiserum. Immunodiffusion experiments confirm these re
sults and show the loss of enzymatic activity. The spur
between normal adult liver and hepatoma extract indicates
that some antigenic determinants present in normal adult
liver aldolase B are not present in the hepatoma enzyme. It
seems that a "cross-reacting material" for aldolase B oc
curs in hepatoma tissues. This could be the result of a very
low rate of synthesis. The few synthesized molecules would
be partly degraded before their disappearance and could
be the result of a very low rate of synthesis; they could be
detected because they had kept a part of their antigenic
determinants. Other authors have described the appear
ance of a spur when normal and degraded proteins were
studied with the same antiserum (23, 27, 29).
Our results provide indirect evidence that leads us to
think that some cells contain aldolases A, B, and C at the
same time and that the resurgence of fetal aldolases A and
C is not the consequence of stem cell selection. The
cellular origin of fetal antigens that appear during carcinogenesis has been much disputed especially for «-fetoprotein (4, 6, 12, 28, 29, 40). Using immunofluorescence and
radioactive labeling techniques, some authors have found
a-fetoprotein in specialized cells (6, 40), whereas some
others do not distinguish these cells from hepatocytes (12,
28). The results recently obtained by Kuhlman (21), who
used immunoperoxidase, do not point out any difference
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Location
between the labeled and unlabeled cells.
Are the genes of fetal aldolases expressed in fetal liver
by fetal hepatocytes only? We still cannot completely ex
clude the possibility that fetal aldolases are also synthesized
by hematopoietic cells. Indeed the study of fetal liver
sections by the immunoperoxidase technique is difficult
because of the presence of endogenous peroxidase in the
hematopoietic cells. It has been reported that it is difficult
to inhibit this peroxidase completely (38). The question
could be clarified by separating the different fetal liver cell
types, but this procedure was beyond the aim of this study.
In conclusion, our results support the view that the same
cells synthesize both the fetal types of aldolases A and C
and the normal adult type B; consequently, in hepatoma
there is an abnormal expression of normally "silent" genes.
Thus our findings provide new arguments for a "dysfunc
tion of gene control" in cancer (41).
REFERENCES
1. Abe. T., and Shimizu, N. Histochemical Method for Demonstrating
Aldolase. Histochemie, 4: 209-212, 1964.
2. Arion, W. J., and Lange, A. J. The Infra-cellular Location of Hepatic
Fructose 1,6-Diphosphate Aldolase. Biochem. Biophys. Res. Commun.,
68.' 770-775, 1976.
3. Avrameas, S. Enzyme Markers: Their Linkage with Proteins and Use in
Immuno-chemistry. In: P. J. Stoward (ed.), Fixation in Histochemistry,
pp. 183-192. London: Chapman and Hall, Ltd., 1973.
4. Cauchi, M. N., Halley. J. B. W., Irving, M. G., and Williams, J. F.
Hepatocellular Localization of Fetal Antigens during Induction of Rat
Liver Tumors by 3'-Methyl-4-dimethylaminoazobenzene. Cancer Res.,
34: 1808-1812, 1974.
5. Cazillis, M., De Recondo, A. M., and Frayssinet, C. High Molecular
Weight Deoxyribonucleic Acid Polymerase of LF Hepatoma. Biochim.
Biophys. Acta, 407: 133-146, 1975.
6. Dempo. K., Chisaka, N., Yoshida, Y., Kaneko, A., and Onoé,T. Immunofluorescent Study on a-Fetoprotein-producing Cells in the Early Stage
of 3'-Methyl-4-dimethylaminoazobenzene Carcinogenesis. Cancer Res.,
35. 1282-1287, 1975.
7. Eisen. H. N. Antibody-Antigen Reactions. In- Immunology, an Introduc
tion to Molecular and Cellular Principles of the Immune Responses.
Reprinted from Davis, Dulbecco, Eisen, Ginsberg and Wood's Microbi
ology, pp. 359-405. Hagerstown Md., Harper and Row Publishers, 1974.
8. Eng, L. F., Uyeda, C. T., Chao, L. P., and Wolfgram, F. Antibody to
Bovine Choline Acetyltransferase and Immunofluorescent Localization
of the Enzyme in Neurones. Nature, 250. 243-245, 1974.
9. Feldmann, G., Penaud-Laurencin, J., Crassous, J., and Benhamou. J.
P. Albumin Synthesis by Human Liver Cells: Its Morphological Demon
stration. Gastroenterology, 63. 1036-1048, 1972.
10. Foemmel, R. S., Gray. R. H., and Bernstein, I. A. Intracellular Localiza
tion of Fructose 1,6-Diphosphate Aldolase. J. Biol. Chem.. 250. 18921897, 1975.
11. Frayssinet, C., and Lafarge, C. Inaptitude d'un HépatomeGreffédu Rat
à Incorporer l'Acide Orotique dans Ses Acids Ribonucléiques.Compa
raison avec le Foie Foetal. J. Physiol. Paris, 58: 520, 1966.
12. Goussev, A. I., Engelhardt, N. V., Masseyeff, R., Camain, R., and
Basteris, B. Immunofluorescent Study of Alpha-foetoprotein (uFp) in
Liver and Liver Tumours. II. Localization of aFp in the Tissues of
Patients with Primary Liver Cancer (PLC). Intern. J. Cancer, 7. 207-217.
1971.
13. Gracy, R. W.. Lacko, A. G., Brox, L. W., Adelman, R. C., and Horecker,
B. L. Structural Relations in Aldolases Purified from Rat Liver and
Muscle and Novikoff Hepatoma. Arch. Biochem. Biophys.. 736: 480490. 1970.
14. Graham, R. C., Jr., and Karnovsky, M. J. The Early Stages of Absorption
of Injected Horseradish Peroxidase in the Proximal Tubules of Mouse
Kidney: Ultrastructural Cytochemistry by a New Technique. J. Histochem. Cytochem., 14: 291-302. 1966.
15. Hatzfeld, A., Elion, J., Mennecier, F.. and Schapira, F. Purification of
Aldolase C from Rat Brain and Hepatoma. European J. Biochem., 77.
37-43. 1977.
JANUARY
of A/dolases A, B, and C by Immunoperoxidase
Method
16. Hatzfeld, A., Feldman, G., Frayssinet, C., and Schapira, F. Cellular
Localization of a Carcinofetal Enzyme (Aldolase C) and of Normal
Adult Enzyme (Aldolase B) in Fast-growing Rat LF Hepatomas. In:
W. H. Fishman and S. Sell (eds.), Onco-Development Gene Expression,
pp. 185-190. New York, Academic Press, Inc., 1976.
17. Hatzfeld, A., Feldman. G., and Schapira, F. Properties and Localization
of Aldolase C in Fast-growing Hepatomas. In: H. Peelers (ed.), Twentyfourth Annual Colloquium on Protides of the Biological Fluids, pp.
553-566. Oxford, England: Pergamon Press, 1976.
18. Hatzfeld, A., and Schapira, F. The Ontogeny of Aldolase in Rat Liver
and Brain. Biochimie, 55: 119-125, 1973.
19. Hatzfeld, A.. Weber, A., and Schapira, F. Biochemical and Immunologicai Studies of Some Carcinofetal Enzymes. Ann. N. Y. Acad. Sci.. 259:
287-297, 1975
20. Kitagawa, T., and Pilot. H. C. Immunohistochemical Demonstration of
Serine Dehydratase in Rat Liver. Am. J. Pathol.. 78: 309-318, 1975.
21. Kuhlmann, W. D. Immunocytological Studies on u 1-Fetoprotein-producing cells under Normal and Pathological Conditions. In: H Peelers
(ed.), Twenty-fourth Annual Colloquium on Protides of the Biological
Fluids, pp. 269-276. Oxford: Pergamon Press. 1976.
22. Lake, B. D. The Histochemical Demonstration of Fructose-1-phosphate
Aldolase and Fructose-1-6-diphosphate Aldolase and Application of the
Method to a Case of Fructose Intolerance. J. Roy. Microscop. Soc.. 84:
489-499. 1965.
23. Lapresle, C., Webb, T., Kaminski, M., and Champagne, M. Isolement et
Propriétés
Immunologiques des Produits de Dégradationde la SérumAlbumine Humaine par laTrypsine. Bull. Soc. Chim. Biol., 41: 695-705,
1959.
24. Lebherz, H. G., and Rutter. W. J. Distribution of Fructose Diphosphate
Aldolase Variants in Biological Systems. Biochemistry, 8: 109-121.1969.
25. Linder-Horowitz, M., Knox, W. E.. and Morris. H. P. Glutaminase
Activities and Growth Rates of Rat Hepatomas. Cancer Res , 29: 11951199, 1969.
26. Masters. C. J. The Ontogeny of Mammalian Fructose 1.6-diphosphate
Aldolase. Biochim. Biophys. Acta, 167: 161-171, 1968.
27. Müller-Eberhard. H. J., Nilsson, U., and Aronsson, T. Isolation and
Characterization of Two B, Glycoproteins of Human Serum. J Exptl
Med., 111: 201-215. 1960.
28. Okita. K., Gruenstein. M., Klaiber. M.. and Farber. E. Localization of aFetoprotein by Immunofluorescence in Hyperplasic Nodules during
Hepatocarcinogenesis Induced by 2-Acetylaminofluorene. Cancer Res.,
34: 2758-2763, 1974.
29. Onoé,T., Kaneko, A., Dempo, K., Ogawa, K., and Minase, T. «-Fetoprotein and Early Histological Changes of Hepatic tissues in DABHepato-carcinogenesis. Ann. N. Y. Acad. Sci., 259: 168-180. 1975.
30. Penhoét,E. E., Rajkumar, T., and Rutter, W. J. Multiple Forms of
Fructose Diphosphate Aldolase in Mammalian Tissues. Proc Nati. Acad.
Sci. U. S.,56: 1275-1281, 1966
31. Pickel. V. M.. Joh, T. H., and Reis. D. J. Ultrastructural Localization of
Tyrosine Hydroxylase in Noradrenergic Neurons of Brain. Proc. Nati.
Acad. Sci. U. S., 72. 659-663, 1975.
32. Schapira. F. Isozymes and Cancer. Advan. Cancer Res., 18: 77-153,
1973.
33. Schapira. F., Dreyfus, J. C., and Schapira, G. Anomaly of Aldolase in
Primary Liver Cancer. Nature. 200: 995-997, 1963.
34. Schapira. F., Hatzfeld, A., and Reuber. M. D. Fetal Pattern of Aldolase
in Transplantable Hepatomas. Cancer Res.. 31: 1224-1230. 1971.
35. Shatton. J. B., Morris. H. P., and Weinhouse. S. Kinetic, Electrophoretic
and Chromatographie Studies on Glucose ATP Phosphotransferase in
Rat Hepatomas. Cancer Res., 29: 1161-1172, 1969.
36. Sibley, J. A., and Lehninger, A. D Determination of Aldolase in Animal
Tissues. J. Biochem.. 777: 859-872, 1949.
37. Stefanini. M.. De Martino, C., and Zamboni. L. Fixation of Ejaculated
Spermatozoa for Electron Microscopy. Nature. 276: 173-174, 1967.
38. Streefkerk. J. G., and Van der Ploeg. M. The Effect of Methanol on
Granulocyte and Horseradish Peroxidase Quantitatively Studied in a
Film Model System. Histochemistry. 40: 105-111. 1974.
39. Uriel. J. Immunoelectrophoretic Analysis of Enzymes. In: B. Cinader
(ed.). Antibodies to Biologically Active Molecules, pp. 181-196. Oxford:
Pergamon Press. 1967.
40. Uriel. J.. Aussel. C.. Bouillon. D., De Nechaud, B., and Loisillier. F.
Localization of Rat Liver (l-Foetoprotein by Cell Affinity Labelling with
Tritiated Oestrogens. Nature New Biol., 244: 190-192, 1973.
41. Weinhouse, S. Glycolysis. Respiration, and Anomalous Gene Expression
in Experimental Hepatomas: G. H. A. Clowes Memorial Lecture. Cancer
Res..32: 2007-2016, 1972.
42. Weston, P. D., and Poole, A. R. Antibodies to Enzymes and Their Uses.
In: J. T. Dingle (ed.). Lysosomes in Biology and Pathology. Vol. 3. pp
425-464. Amsterdam: North-Holland Publishing Co . 1973.
1978
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A. Hatzfeld et al.
Figs. 1 to 3. Light microscopy; immunoperoxidase; LF hepatomas. The products of the reaction indicating the presence of aldolase are represented by
dark brown deposits located in the cytoplasm of almost all the cancerous cells. Fig. 1. With anti-aldolase A antiserum, x 570. Fig. 2. With anti-aldolase C
antiserum, x 570. Fig. 3. With anti-aldolase B antiserum. The intensity of the reaction is weaker for this isozyme. * 570.
Fig. 4. Light microscopy: immunoperoxidase; LF hepatomas. Control reaction. No dark brown deposits are visible in the cytoplasm of the cells, x 570.
Figs. 5 to 7. Light microscopy; immunoperoxidase; normal adult liver. The products of the reaction indicating the presence of aldolase are visible in
almost all the cells. Fig. 5. With anti-aldolase B antiserum, x 340. Fig. 6. With anti-aldolase A antiserum, x 340. Fig. 7. With anti-aldolase C antiserum. No
reaction is visible in the cytoplasm of the hepatocytes for this isozyme. x 340.
Fig. 8. Light microscopy: immunoperoxidase; normal adult liver. Control reaction. No reaction is visible in the cytoplasm of the hepatocytes. * 340.
Figs. 9 to 11. Electron microscopy: immunoperoxidase; LF hepatomas The products of the reaction indicating the presence of aldolase are represented
by electron-dense deposits, which are exclusively located in the cytoplasm of the cells and not in the nucleus. Fig. 9. With anti-aldolase A antiserum.
x 20,000. Fig. 10. With anti-aldolase B antiserum x 23,000. Fig. 11. With anti-aldolase C antiserum. x 16,000. The reaction is homogeneous for aldolases
A and C and heterogeneous for aldolase B. N, nucleus: M. mitochondria; ER. endoplasmic reticulum.
Fig. 12. Electron microscopy; immunoperoxidase; LF hepatomas. Control reaction No electron-dense products are visible in the cytoplasm of the cell.
N, nucleus; M. mitochondria; ER, endoplasmic reticulum. x 23.000.
Fig. 13. Ouchterlony double-diffusion with anti-aldolase A antiserum (a) and anti-aldolase C antiserum (o). Center well: anti-aldolase A antiserum (a);
anti-aldolase C antiserum (i>). Well 1. adult brain: Well 2. LF fast-growing hepatomas; We// 3. muscle; Well 4, adult liver; Well 5. adult liver: Well 6. fetal liver.
Extracts 1, 2, 3, 5, and 6. 4 ml of 150 mM NaCI in water per g of tissue. Extract 4, 0.5 ml of 150 mM NaCI in water per g of tissue.
Fig. 14. Ouchterlony double-diffusion with anti-aldolase B antiserum. Center well, anti-aldolase B; Wells 1. 3. and 5. normal adult liver; We/I 2. fetal liver;
Well 4, normal adult brain: Well 6. LF fast-growing hepatomas; Extracts 1 to 5, 4 ml of 150 mM NaCI in water per g of tissue: Extract 6. 0.5 ml of 150 mM
NaCI in water per g of tissue.
20
CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
Location
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A. B, and C by Immunoperoxidase
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A. Hatzfeld et al.
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CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
Location of Adult and Fetal Aldolases A, B, and C by
Immunoperoxidase Technique in LF Fast-growing Rat
Hepatomas
Antoinette Hatzfeld, Gérard Feldmann, Jocelyne Guesnon, et al.
Cancer Res 1978;38:16-22.
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