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On the inhibition of protein synthesis in Ehrlich ascites tumor cells by iodinated deoxyribonucleic acid in vitro
838
PRELIMINARY NOTES
PN 6070
On the inhibition of protein synthesis in Ehrlich ascites tumor cells by iodinoted deoxyribonucleic acid in vifro In a previous report 1, the possibility of the incorporation of intact DNA into the nuclei of Ehrlich ascites tumor cells was suggested. This preliminary note reports that the incorporation of iodinated DNA into nuclei of Ehrlich ascites tumor cells causes the inhibition of amino acid incorporation into the protein of the tumor cells. Ehrlich ascites tumor cells taken from dd-mice 5 days after tumor-cell transplantation were incubated with E2-z4Clglycine for I h at 37 ° in the same medium as described previously ~ which contained iodinated DNA or the DNAase digestion product of iodinated DNA. The total volume of the incubation mixture was 2.5 ml. After incubation, the fractionation of cell proteins was carried out employing a slightly modified method of GRIFFIN et al. ~ to obtain the ribo- and deoxyribonucleoprotein fractions and soluble protein fraction. Each protein fraction was extracted with hot and cold trichloroacetic acid to remove nucleic acids, and its radioactivity was measured b y a windowless gas-flow counter. Iodinated DNA (I-DNA) was prepared from liver DNA of dd-mice b y the method of PRUSOFF et al. 3. The DNAase-digestion product was prepared as follows: 4 mg of I-DNA were dissolved in I ml of Robinson's medium and incubated with 3oo/~g of DNAase (Worthington Biochem. Corp.) for 30 rain at 37 °. After incubation, cold HC104 was added to the incubation mixture to a final concentration of 6 ~o- The cold HC104 extract was neutralized in the presence of phenolphthalein with 20 % K O H and adjusted to a final volume of IO ml with saline. The DNAase-digestion product of DNA was prepared in the same way. It was assumed that the concentration of both digestion products was 0. 4 mg/ml. The incorporation of Em4Clglycine into all protein fractions of tumor cells was inhibited b y about 7 ° ~,, b y the addition of I-DNA. The inhibition was diminished by the addition of DNA to the I - D N A (Table I (a)), while the inhibition b y I-DNA was only slightly diminished b y the addition of DNAase-treated DNA together with I-DNA as shown in Table I(b). The incorporation of glycine into the tumor proteins was inhibited b y DNAase-treated I - D N A to the same extent as with I-DNA and the inhibitions were not diminished b y the addition of DNA together with the DNAasetreated I-DNA (Table II). The fact that I-DNA inhibited the glycine incorporation into each protein fraction to the same extent m a y suggest that I-DNA inhibits the cell division of the tumor. The mechanism of the inhibition b y both I-DNA and DNAase-treated I-DNA m a y be different because the inhibition b y I-DNA was not diminished b y the addition of DNAase-treated D N A and the inhibition b y DNAasetreated I-DNA was only partly suppressed b y DNA, while the inhibition b y I-DNA was almost suppressed by DNA as was the inhibition b y DNAase-treated I-DNA in the presence of DNAase-treated DNA. As DNAase-treated I-DNA is a mixture of iodinated oligonucleotides which is considered to contain mainly iodinated di- and trinucleotides, it m a y be supposed that DNAase-treated I-DNA competes with oligonucleotides for incorporation into polynucleotides and thus might inhibit protein Biochim. Biophys. Acta, 61 (1962) 838-839
839
PRELIMINARY NOTES TABLE I EFFECT OF I - D N A ON [2-14C]GLYCINE UPTAKE BY TUMOR-CELL PROTEINS
I m l of t u m o r cells(final c o n c e n t r a t i o n , IO % v/v) w a s i n c u b a t e d in 1.5 m l o f R o b i n s o n ' s m e d i u m w h i c h c o n t a i n e d glucose (final c o n c e n t r a t i o n , 3 m g / m l ) , folic acid (final c o n c e n t r a t i o n , 5" 1°-5 M), 0. 5/~C of ~2-14C~glycine (final con., 5.06. io-* M) a n d f u r t h e r a d d i t i o n s s h o w n a t 37 ° for i h. (a) Protein ]ractions
I-DNA (x.6mg/dl) (counts/rain]rag)
DN-Pr* RN-Pr** Sol-Pr***
613 408 127
I-DNA (x,6mg/dl)+ + DNA (1.6 mg/dl) (counts[rain/rag)
433(71%) 288(71%) 88(69 %)
553(90 %) 367(9 ° %) 126(lO 7 %)
(b) Protein ]factions
DN-Pr* RN-Pr** Sol-Pr***
I-DNA (1.6 mg/dl) (counts]rain/rag)
I-DNA (i.6 mg/dl) + + DNAase-treated DNA (1.6 mgldl) (counts/rain~rag)
43o(69 %) 359(75 %) 212(81%
5o8(82 %) 380(79 %) 222(85 °/o)
621 482 263
* D N - P r : P r o t e i n m o i e t y of d e o x y r i b o n u c l e o p r o t e i n fraction *" R N - P r : P r o t e i n m o i e t y of r i b o n u c l e o p r o t e i n fraction. *** Sol-Pr: soluble p r o t e i n fraction. T A B L E II EFFECT OF DNAASE-TREATED I - D N A ON [2-14C~GLYCINE UPTAKE BY TUMOR~CELL PROTEINS I n c u b a t i o n c o n d i t i o n s were t h e s a m e as g i v e n in Table I. Protein ]ractions
D N - P r .1 RN-Pr** Sol-Pr *a
DNA-treated I-DNA (1.6 mg[dl) (counts/rain[rag)
729 704 446
531(73 %) 478(68 %) 284(64 %)
DNAase4reated I-DNA (1.6 mg]dl) + (counts]min]mg) DNA (1.6 mg/dl)
DNAase-treated I-DNA (1.6 mg /dl) + DNAase-treated DNA (1.6 mg dl) (counts/rain/rag)
51o(7o %) 488(69 %) 297(67 %)
66o(91%) 669(95 %) 429(96 %)
,1 D N - P r : P r o t e i n m o i e t y of d e o x y r i b o n u c l e o p r o t i n fraction. *~ R N - P r : P r o t e i n m o i e t y of r i b o n u c l e o p r o t e i n fraction. *~ Sol-Pr: Soluble p r o t e i n fraction.
synthesis. On the other hand, I-DNA might compete with DNA and inhibit its replication, thus causing an inhibition of cell division in these tumor cells.
Laboratory o/ Radioisotope Research, Niigata University, Department o/ Surgery, Niigata University School o/Medicine, Niigata (Japan)
TAIjI SCHIMIZU MAKOT0 IWA~UCnI
1 T. SCHIMIZU, S. KOYAMA AND M. IWAFUCHI, B i o c h i m . B i o p h y s . A c t a , 55 (1962) 7952 S. C. GRIFFIN, W. H. NYLE, L. NODA AND J. M. LUCK, J . Gen. P h y s i o l . , 31 (1947) 1225. W. H. PRUSOFF, W. L. HOLMES AND A. D. WELCH, C a n c e r R e s e a r c h , 13 (1953) 221.
Received August 2ist, 1962 Biochim.
Biophys.
A c t a , 61 (1962) 8 3 8 - 8 3 9
PRELIMINARY NOTES
PN 6070
On the inhibition of protein synthesis in Ehrlich ascites tumor cells by iodinoted deoxyribonucleic acid in vifro In a previous report 1, the possibility of the incorporation of intact DNA into the nuclei of Ehrlich ascites tumor cells was suggested. This preliminary note reports that the incorporation of iodinated DNA into nuclei of Ehrlich ascites tumor cells causes the inhibition of amino acid incorporation into the protein of the tumor cells. Ehrlich ascites tumor cells taken from dd-mice 5 days after tumor-cell transplantation were incubated with E2-z4Clglycine for I h at 37 ° in the same medium as described previously ~ which contained iodinated DNA or the DNAase digestion product of iodinated DNA. The total volume of the incubation mixture was 2.5 ml. After incubation, the fractionation of cell proteins was carried out employing a slightly modified method of GRIFFIN et al. ~ to obtain the ribo- and deoxyribonucleoprotein fractions and soluble protein fraction. Each protein fraction was extracted with hot and cold trichloroacetic acid to remove nucleic acids, and its radioactivity was measured b y a windowless gas-flow counter. Iodinated DNA (I-DNA) was prepared from liver DNA of dd-mice b y the method of PRUSOFF et al. 3. The DNAase-digestion product was prepared as follows: 4 mg of I-DNA were dissolved in I ml of Robinson's medium and incubated with 3oo/~g of DNAase (Worthington Biochem. Corp.) for 30 rain at 37 °. After incubation, cold HC104 was added to the incubation mixture to a final concentration of 6 ~o- The cold HC104 extract was neutralized in the presence of phenolphthalein with 20 % K O H and adjusted to a final volume of IO ml with saline. The DNAase-digestion product of DNA was prepared in the same way. It was assumed that the concentration of both digestion products was 0. 4 mg/ml. The incorporation of Em4Clglycine into all protein fractions of tumor cells was inhibited b y about 7 ° ~,, b y the addition of I-DNA. The inhibition was diminished by the addition of DNA to the I - D N A (Table I (a)), while the inhibition b y I-DNA was only slightly diminished b y the addition of DNAase-treated DNA together with I-DNA as shown in Table I(b). The incorporation of glycine into the tumor proteins was inhibited b y DNAase-treated I - D N A to the same extent as with I-DNA and the inhibitions were not diminished b y the addition of DNA together with the DNAasetreated I-DNA (Table II). The fact that I-DNA inhibited the glycine incorporation into each protein fraction to the same extent m a y suggest that I-DNA inhibits the cell division of the tumor. The mechanism of the inhibition b y both I-DNA and DNAase-treated I-DNA m a y be different because the inhibition b y I-DNA was not diminished b y the addition of DNAase-treated D N A and the inhibition b y DNAasetreated I-DNA was only partly suppressed b y DNA, while the inhibition b y I-DNA was almost suppressed by DNA as was the inhibition b y DNAase-treated I-DNA in the presence of DNAase-treated DNA. As DNAase-treated I-DNA is a mixture of iodinated oligonucleotides which is considered to contain mainly iodinated di- and trinucleotides, it m a y be supposed that DNAase-treated I-DNA competes with oligonucleotides for incorporation into polynucleotides and thus might inhibit protein Biochim. Biophys. Acta, 61 (1962) 838-839
839
PRELIMINARY NOTES TABLE I EFFECT OF I - D N A ON [2-14C]GLYCINE UPTAKE BY TUMOR-CELL PROTEINS
I m l of t u m o r cells(final c o n c e n t r a t i o n , IO % v/v) w a s i n c u b a t e d in 1.5 m l o f R o b i n s o n ' s m e d i u m w h i c h c o n t a i n e d glucose (final c o n c e n t r a t i o n , 3 m g / m l ) , folic acid (final c o n c e n t r a t i o n , 5" 1°-5 M), 0. 5/~C of ~2-14C~glycine (final con., 5.06. io-* M) a n d f u r t h e r a d d i t i o n s s h o w n a t 37 ° for i h. (a) Protein ]ractions
I-DNA (x.6mg/dl) (counts/rain]rag)
DN-Pr* RN-Pr** Sol-Pr***
613 408 127
I-DNA (x,6mg/dl)+ + DNA (1.6 mg/dl) (counts[rain/rag)
433(71%) 288(71%) 88(69 %)
553(90 %) 367(9 ° %) 126(lO 7 %)
(b) Protein ]factions
DN-Pr* RN-Pr** Sol-Pr***
I-DNA (1.6 mg/dl) (counts]rain/rag)
I-DNA (i.6 mg/dl) + + DNAase-treated DNA (1.6 mgldl) (counts/rain~rag)
43o(69 %) 359(75 %) 212(81%
5o8(82 %) 380(79 %) 222(85 °/o)
621 482 263
* D N - P r : P r o t e i n m o i e t y of d e o x y r i b o n u c l e o p r o t e i n fraction *" R N - P r : P r o t e i n m o i e t y of r i b o n u c l e o p r o t e i n fraction. *** Sol-Pr: soluble p r o t e i n fraction. T A B L E II EFFECT OF DNAASE-TREATED I - D N A ON [2-14C~GLYCINE UPTAKE BY TUMOR~CELL PROTEINS I n c u b a t i o n c o n d i t i o n s were t h e s a m e as g i v e n in Table I. Protein ]ractions
D N - P r .1 RN-Pr** Sol-Pr *a
DNA-treated I-DNA (1.6 mg[dl) (counts/rain[rag)
729 704 446
531(73 %) 478(68 %) 284(64 %)
DNAase4reated I-DNA (1.6 mg]dl) + (counts]min]mg) DNA (1.6 mg/dl)
DNAase-treated I-DNA (1.6 mg /dl) + DNAase-treated DNA (1.6 mg dl) (counts/rain/rag)
51o(7o %) 488(69 %) 297(67 %)
66o(91%) 669(95 %) 429(96 %)
,1 D N - P r : P r o t e i n m o i e t y of d e o x y r i b o n u c l e o p r o t i n fraction. *~ R N - P r : P r o t e i n m o i e t y of r i b o n u c l e o p r o t e i n fraction. *~ Sol-Pr: Soluble p r o t e i n fraction.
synthesis. On the other hand, I-DNA might compete with DNA and inhibit its replication, thus causing an inhibition of cell division in these tumor cells.
Laboratory o/ Radioisotope Research, Niigata University, Department o/ Surgery, Niigata University School o/Medicine, Niigata (Japan)
TAIjI SCHIMIZU MAKOT0 IWA~UCnI
1 T. SCHIMIZU, S. KOYAMA AND M. IWAFUCHI, B i o c h i m . B i o p h y s . A c t a , 55 (1962) 7952 S. C. GRIFFIN, W. H. NYLE, L. NODA AND J. M. LUCK, J . Gen. P h y s i o l . , 31 (1947) 1225. W. H. PRUSOFF, W. L. HOLMES AND A. D. WELCH, C a n c e r R e s e a r c h , 13 (1953) 221.
Received August 2ist, 1962 Biochim.
Biophys.
A c t a , 61 (1962) 8 3 8 - 8 3 9