Download Intracellular Distribution of 5`-Ribonuclease and 5`

[CANCER
RESEARCH
27, 1722-1723, September
1967]
Brief Communication
Intracellular Distribution of 5'-Ribonuclease
in the Novikoff Hepatoma Ascites Cells1
GASTON
and 5'-Phosphodiesterase
de LAMIRANDE'
Institut du Cancer de Montréal,Laboratoires de Recherche, Hôpital Notre-Dame et Universitéde Montréal,Montréal,Canada
The presence of a 5'-ribonuclease (5'-RNase) in guinea pig and
rat liver has been reported (5, 7) as well as the presence of a 5'phosphodiesterase in rat liver (7, 8). Studies on the intracellular
distribution of these enzymes in rat liver revealed that the 5'RNase was essentially localized in the mitochondrial fraction
whereas the 5'-phosphodiesterase was present in the nuclear and
microsomal fractions (2). It has been shown in addition that the
5'-phosphodiesterase was part of the membrane portion of the
microsomal fraction (3). In view of the lack of information on the
distribution of these enzymes in tumors, their intracellular distri
bution has been studied in Novikoff ascites cells.
Intracelhdar
is mainly due to the almost complete disappearance of the ac
tivity in the mitochondrial fraction which, in the tumor, contains
only 7.1% of the total activity, whereas it contained 64% in
normal liver. The nuclear fraction, on the other hand, retains
two-fifths of the normal nuclear activity and contains the bulk of
the 5'-RNase of the tumor, 67.8%, whereas it contained onlx
23% in normal liver.
The total phosphodiesterase activity of the tumor, 0.43 jumóle,
is reduced to less than 15% that of the normal liver, namely 3.01
/umoles (2). This is due to large decreases in the activity of the
nuclear and microsomal fractions. In fact, only one-sixth and
TABLE 1
Distribution
of Enzymes
distribution6Hc100100N67.8
Enzymes5'
of ex
values"0.28
peri
ments66Absolute
Ribonuclease5'-PhosphodiesteraseNo.
0.090.43
±
3.59.3
±
3.518.6
±
9.04.7
±
28.546.5
±
32.1100.0
±
±9.3Me020.9±11.5s32.1 ±4.7Recovery114.1
±0.16Percentage ±14.0M7.1 ±4.6L7.1
± 11.5
a Absolute values are in ¿oriolesof adenylic acid produced/min/ml
of packed cells for 5'-ribonuclease using an extinction coefficient
of 14.2 X IO6 cm2 mole"1 and in Amólesof p-nitrophenyl 5'-thymidine phosphate hydrolyzed/min/ml
of packed cells for 5'-phosphodiesterase.
* To express the percentage distribution, the absolute values for the homogenate were taken as 100.
c H, homogenate; N, nuclear fraction; M, mitochondrial fraction; L, lysosomal fraction; Me, microsomal
fraction.
The Novikoff hepatoma ascites cells were 7-day-old transplants
which were freed of blood cells, swelled, and broken in an homogenizer consisting of a Teflon pestle fitted into a test tube (4). This
treatment caused a breakage of more than 90% of the tumor cells.
The cellular fractions were obtained according to the method of
de Duve et al. (1), and the enzyme activities were determined as
previously described (2).
The results on the intracellular distribution of the enzymes are
summarized in Table 1. They indicate that the total 5'-RNase
activity of the tumor, 0.28 ¿imole,is reduced to less than 13%
that of the normal liver, namely 2.17 /¿moles(2). This decrease
1This investigation has been supported by a grant from the
National Cancer Institute of Canada to Dr. A. Cantero, director
of the Research Laboratories.
2 Research scient ist of the National Cancer Institute of Canada.
Received May 8, 1967; accepted May 22, 1967.
1722
fraction;
S, supernatant
one-tenth of the nuclear and microsomal activities are retained
in the tumor. However, the percentage of the total activity pres
ent in the nuclear fraction of the hepatoma cell is comparable
with that observed in normal liver. In the microsomal fraction,
only about half the percentage activity is preserved.
The role of 5'-RNase and 5'-phosphodiesterase is not known
presently, and consequently it is not possible to evaluate the
biologic significance of the large losses of activity in the tumor
cells. However, there seems to be a parallel between the loss of
5'-RNase localized in the mitochondrial fraction and the greatly
reduced number of the mitochondria in the Novikoff hepatoma
cell (6). Similarly, the loss of 5'-phosphodiesterase localized in the
membrane portion of the microsomal fraction (3) seems to cor
respond to a loss of ergastoplasmic membranes in the hepatoma
cell (6). The enzymatic variations might then be explained, at
least in part, by the morphologic changes observed in the Novi
koff hepatoma cell.
CANCER
RESEARCH
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VOL. 27
ô'-RNase and S'-Phosphodiesterase in Hepatoma Ascites Cells
REFERENCES
1. De Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R., and
Appelmans, F. Tissue Fractionation Studies. 6. Intracellular
Distribution Patterns of Enzymes in Rat Liver Tissue.
Biochem. J., 60: 604-617, 1955.
2. de Lamirande, G., Moráis,R., and Blackstein, M. Intracellular
Distribution of 5-Ribonuclease and 5'-Phosphodiesterase in
Rat Liver. Arch. Biochem. Biophys., 118: 347-351, 1967.
3. de Lamirande, G., Boileau, S., and Moráis,R. Distribution of
the Nucleases of the Microsomal Fraction of Rat Liver between Ribosomes and Endoplasmic Membranes. Can. J. Bioehem., 44: 273-279, 1966.
4. Hawtree, A. O., and Silk, M. H. Mitochondria of the Ehrlich
Ascites-Tumour Cell. Biochem. J., 74: 21-26, 1960.
5. Heppel, L. A., Ortez, P. J., and Ochoa, S. Small Polyribonucleotides with 5'-Phosphomonoester End-Groups. Science, 123:
415-416, 1956.
6. Howatson, A. F., and Ham, A. W. Electron Microscope Study
of Sections of Two Rat Liver Tumors. Cancer Res., IB: 52-69,
1955.
7. Moráis,R. and de Lamirande, G. Formation of Oligonucleotides
with 5'-Phosphomonoester End Groups by Rat Liver Microsomal Fraction. Biochim. Biophys. Acta, 103: 506-508, 1965.
8. Razzell, W. E. Tissue and Intracellular Distribution of Two
Phosphodiesterases. J. Biol. Chem., 236: 3028-3030, 1961.
SEPTEMBER 1967
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1967 American Association for Cancer Research.
1723
Intracellular Distribution of 5′-Ribonuclease and 5′
-Phosphodiesterase in the Novikoff Hepatoma Ascites Cells
Gaston de Lamirande
Cancer Res 1967;27:1722-1723.
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