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Labelling pattern of the nonhistone chromosomal proteins in quiescent and dividing ehrlich ascites tumour cells
0020-711X/83$3.00+.OO Copyright 0 1983Pergamon Press Ltd
Int. J. Biochem.Vol. 15, No. 8, pp. 1093-1094,1983 Printed in Great Britain. All rights reserved
LABELLING PATTERN OF THE NONHISTONE CHROMOSOMAL PROTEINS IN QUIESCENT AND DIVIDING EHRLICH ASCITES TUMOUR CELLS BOYKA ANACHKOVAand GEORGE RUSSEV* Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria (Receiued 15 Nouember 1982)
Abstract-Upon pulse-labelling with [‘%]protein hydrolizate both in dividing and quiescent Ehrlich ascites tumour (EAT) cells the nonhistone chromosomal (NHC) proteins had uniform specific radioac_ tivity with few exceptions: 1. Both in quiescent and dividing EAT cells a polypeptide with molecular weight of about 200 kdalton had specific radioactivity 2-3 times lower than that of the most of the NHC proteins. 2. In the actively proliferating cells a group of proteins with molecular weights between 45 and 65 kdalton had 2-3 times higher specific radioactivity than most of the NHC proteins. 3. In quiescent cells the specific radioactivity of a group of proteins with molecular weights in the range l&25 kdalton was 3-4 times higher than that of the rest of the NHC proteins.
INTRODUCTION In a previous paper (Russev et al., 1975) we have found that in rat liver chromatin there were two groups of NHC proteins with different metabolic rates. The first group was actively metabolized independantly of the cell cycle, while the other one turned over during cell division only. There is increasing evidence that such two groups are present in chromatin from a number of sources (Djondjurov et al., 1979; Tsanev et al., 1974; Stambolova et al., 1979; Koleva & Tsanev, 1978). In the present communication we report on the labelling pattern of the nonhistone chromosomal (NHC) proteins in quiescent and dividing Ehrlich ascites tumour (EAT) cells. In both cases we detected groups of NHC proteins whose specific radioactivities, i.e. rate of labelling differed from those of the remaining NHC proteins. MATERIALS
AND METHODS
Albino mice strain Agnes-Blum, 3 month old and weighing 20-25 g were injected intraperitoneally with 0.3 ml of undiluted ascites liquid of Ehrlich-Lettre hyperdiploid ascites tumour strain. The animals were killed into two groups 5 and 10 days after the inoculation, the tumour cells were collected by a low-speed centrifugation and washed out 2-3 times with cold saline. Chromatin was isolated as previously described (Tsanev & Russev, 1974). Two hours before sacrifice the animals of group “day 5” received intraperitoneally 50 ,uci of C3H]thymidine (11 Ci/mg, DDR) and 50 nCi of [‘%]protein hydrolyzate (2 mCi/mg, CSSR), and the animals of group “day lo”100 pCi of [3H]thymidine and [‘%]protein hydrolyzate. Protein was determined by the method of Lowry et al (1951), and DNA-by the method of Burton (1956). SDSpolyacrylamide electrophoresis in 8.75% slab gel was carried out after Laemmli (1970). The gells were stained with Coomassie Brilliant Blue G250 (Serva), and were scanned * Author to whom correspondence
should be addressed.
at 600nm with a recording spectrodensitometer constructed in this laboratory. Then the individual lanes were cut into 2-mm strips and, dissolved in 0.4ml 30% H,O,-0.1 ml 25% NH,OH at 60°C for several hours. These were mixed with 5 ml of toluene-Triton Xl00 (2: 1) scintillation cocktail (Anderson & McClure, 1973) and counted in a LKB 1020 Ultrobeta liquid scintillation spectrometer. RESULTS AND
DISCUSSION
At “day 5” after inoculation about 80% of the EAT cells were in the mitotic cycle. Chromatin isolated from these cells had DNA :histone :NHC protein ratio of 1: 1:1. The specific radioactivity of histones and of total NHC protein was about the same and in our particular experiment the ratio of these specific radioactivities to the specific radioactivity of DNA was 0.2. SDSpolyacrylamide gel electrophoresis revealed a characteristic NHC protein pattern and the specific radioactivities of all protein fractions, including histones did not vary significantly with two exceptions only: the specific radioactivity of a protein fraction with molecular weight of about 200 kdalton was several times lower than those of the other NHC proteins. On the other hand the specific radioactivity of a group of fractions with molecular weights in the range 45-65 kdalton was 2-3 times higher than those of the other NHC proteins (Fig. 1A). The specific radioactivity of DNA isolated from “day 10” EAT cells was 15-20 times lower than that of DNA isolated from “day 5” cells. This indicated that at day 10 only about 5% of the cells yere still entering the mitotic cycle and that this cell population could be considered as quiescent (Srebreva et al., 1979). Chromatin isolated from these cells had an increased NHC protein content, its DNA :histone :NHC protein ratio being 1:1:1.4. The ratio of the specific radioactivity of the histones to that of DNA was 0.3-0.4, while this ratio for the total NHC protein was
1093
1094
BOYKAANACHKOVA and
0
0
2
4
6
o
GEORGE
RUSSEV
2
cm towards
4
6
anode
Fig. 1. SDS-polyacrylamide gel electrophoresis of chromatin isolated from Ehrlich ascites tumour cells 5 (A) and 10 (B) days after inoculation. DNA and the proteins were labelled in oiuo with E3H]tbymidine and [14Clprotein hydrolyzate, respectively, as described in Materials and Methods. The electro~h~resis was carried out in 8.75% polyacrylamide gel according to Laemmli (1970), and the gel was stained, scanned and counted as described in Materials and Methods. Bovine serum albumin (68 kdalton, monomer, and 136 kdalton, dimer), aldolase (40 kdalton) and chymotripsinogen A (25 kdalton) were used to calibrate the gel. -----Coomassie Briliant Blue staining recorded at 600 m; ---[‘4C]radioactivity. 1, which indicated that in the quiescent cells at least part of the NHC proteins were still turning over. The SDS-polyacrylamide gel electrophoresis showed that the NHC protein pattern of chromatin from “day 10’ cells was identical to that of “day 5” cells in respect to both number and abundance of fractions (Fig. IA and B). As in the case of chromatin isolated from dividing cells, the specific radioactivity of the individual NHC proteins from quiescent cells was almost uniform with two exceptions. Here again the specific radioactivity of the 200 kd fraction was much lower than that of the remaining proteins which showed that both in quiescent and prol~fera~ng EAT cells this protein was metabolically the least active. On the other hand there was a distinct group of proteins with molecular weights in the range 18-25 kdalton (Fig. lB), with specific radioactivities 3-4 times higher than those of the rem~ni~g NHC proteins. It is also worthy noting that the specific radioactivity of histone HI in the quiescent cells was about 1.5 times higher than that of the remaining 4 histones, which showed that this histone was turning slowly over even when the synthesis of DNA and of the core histones was inhibited. From the results described above we drew the following conclusions. The SDS-electrophoretical profiles of NHC proteins from quiescent and dividing EAT cells are identical. In both prol~erating and dormant cells there is a high molecular weight protein that turns over much slower than most of the NHC proteins, Both in quiescent and dividing cells there are groups of several NHC proteins turning over at higher rates than bulk NHC protein. In the dividing cells there were proteins with molecular weights between 45 and 65 kdalton, and in quiescent cellsbetween 18 and 25 kdalton.
REFERENCES
Anderson L. A. & McClure W. D. (1973) An improved cocktail of high solubilizinrr power. Annlvt. Biochepa. 51. I-
t73-179.
-
BURTONK. (1956) A study of the conditions and mechanism of the diphenylamine reaction for calorimetric estimation of deoxyribonucleic acid. Biochem. 1. 62, 315-323. DJONDJUROVL., IVANOVA E. & T,YANEVR. (1979) Two chromatin fractions with different metabolic properties of non-!&tone proteins and of newly synthesized RNA. Eur. S. Rio&in. 97, 133-139. KOLEVAS. & TSANEVR. (1978) Metabolically stable nonhistone chromosomal proteins in growing maize roots. Cell LtQ”erentiation 7, 83-88. LAEMMLIU. K. (1970) Cleavage of structural proteins during the assembly of thi head of bacteriophage T4. Nature 22T7,680-685. LOWXY0. H., ROSEBROIJGH N. J., F.QZRA. L. & RZWDAL R. J. (19St) Protein measurement with Folin phenol reagent. d. biol. Chem. 193, 265-275. R&&v G., ANACHKOVA R. & TSANIWR. (1975) Fractionation of rat liver chromatin nonhistone proteins into two groups with ‘different metabolic rates. Eur. J. ~jo~~rn. 58, 253-257. SREBREVAL., RUSSEVG. & TSANEVR. (1979) Metabolic stability of nonhistone chromosomal proteins in Ehrlich ascites tumour cells. Inl. J. Biochem. 10, 253-257. ST~MBO~VAM., ANGELOVA A. & TWNEVR. (1979) Mefabolic stability of nonhistone chromosomal proteins in two different classes of developing rat brain nuclei. Cell Di#efSeren&tion8, 195-202. TSANEVR., DI~NDJUROVL. & IVANO~AE. (1974) Cell cycle dependent turnover of nonhistone proteins in chromatin. Expl Cell Rek 84, 137-142. TSANEVR. & RUSSEVG. (1974) Distribution of newly synthesized histones during DNA replication. Eur. J. Biothem. 43, 257-263.
Int. J. Biochem.Vol. 15, No. 8, pp. 1093-1094,1983 Printed in Great Britain. All rights reserved
LABELLING PATTERN OF THE NONHISTONE CHROMOSOMAL PROTEINS IN QUIESCENT AND DIVIDING EHRLICH ASCITES TUMOUR CELLS BOYKA ANACHKOVAand GEORGE RUSSEV* Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria (Receiued 15 Nouember 1982)
Abstract-Upon pulse-labelling with [‘%]protein hydrolizate both in dividing and quiescent Ehrlich ascites tumour (EAT) cells the nonhistone chromosomal (NHC) proteins had uniform specific radioac_ tivity with few exceptions: 1. Both in quiescent and dividing EAT cells a polypeptide with molecular weight of about 200 kdalton had specific radioactivity 2-3 times lower than that of the most of the NHC proteins. 2. In the actively proliferating cells a group of proteins with molecular weights between 45 and 65 kdalton had 2-3 times higher specific radioactivity than most of the NHC proteins. 3. In quiescent cells the specific radioactivity of a group of proteins with molecular weights in the range l&25 kdalton was 3-4 times higher than that of the rest of the NHC proteins.
INTRODUCTION In a previous paper (Russev et al., 1975) we have found that in rat liver chromatin there were two groups of NHC proteins with different metabolic rates. The first group was actively metabolized independantly of the cell cycle, while the other one turned over during cell division only. There is increasing evidence that such two groups are present in chromatin from a number of sources (Djondjurov et al., 1979; Tsanev et al., 1974; Stambolova et al., 1979; Koleva & Tsanev, 1978). In the present communication we report on the labelling pattern of the nonhistone chromosomal (NHC) proteins in quiescent and dividing Ehrlich ascites tumour (EAT) cells. In both cases we detected groups of NHC proteins whose specific radioactivities, i.e. rate of labelling differed from those of the remaining NHC proteins. MATERIALS
AND METHODS
Albino mice strain Agnes-Blum, 3 month old and weighing 20-25 g were injected intraperitoneally with 0.3 ml of undiluted ascites liquid of Ehrlich-Lettre hyperdiploid ascites tumour strain. The animals were killed into two groups 5 and 10 days after the inoculation, the tumour cells were collected by a low-speed centrifugation and washed out 2-3 times with cold saline. Chromatin was isolated as previously described (Tsanev & Russev, 1974). Two hours before sacrifice the animals of group “day 5” received intraperitoneally 50 ,uci of C3H]thymidine (11 Ci/mg, DDR) and 50 nCi of [‘%]protein hydrolyzate (2 mCi/mg, CSSR), and the animals of group “day lo”100 pCi of [3H]thymidine and [‘%]protein hydrolyzate. Protein was determined by the method of Lowry et al (1951), and DNA-by the method of Burton (1956). SDSpolyacrylamide electrophoresis in 8.75% slab gel was carried out after Laemmli (1970). The gells were stained with Coomassie Brilliant Blue G250 (Serva), and were scanned * Author to whom correspondence
should be addressed.
at 600nm with a recording spectrodensitometer constructed in this laboratory. Then the individual lanes were cut into 2-mm strips and, dissolved in 0.4ml 30% H,O,-0.1 ml 25% NH,OH at 60°C for several hours. These were mixed with 5 ml of toluene-Triton Xl00 (2: 1) scintillation cocktail (Anderson & McClure, 1973) and counted in a LKB 1020 Ultrobeta liquid scintillation spectrometer. RESULTS AND
DISCUSSION
At “day 5” after inoculation about 80% of the EAT cells were in the mitotic cycle. Chromatin isolated from these cells had DNA :histone :NHC protein ratio of 1: 1:1. The specific radioactivity of histones and of total NHC protein was about the same and in our particular experiment the ratio of these specific radioactivities to the specific radioactivity of DNA was 0.2. SDSpolyacrylamide gel electrophoresis revealed a characteristic NHC protein pattern and the specific radioactivities of all protein fractions, including histones did not vary significantly with two exceptions only: the specific radioactivity of a protein fraction with molecular weight of about 200 kdalton was several times lower than those of the other NHC proteins. On the other hand the specific radioactivity of a group of fractions with molecular weights in the range 45-65 kdalton was 2-3 times higher than those of the other NHC proteins (Fig. 1A). The specific radioactivity of DNA isolated from “day 10” EAT cells was 15-20 times lower than that of DNA isolated from “day 5” cells. This indicated that at day 10 only about 5% of the cells yere still entering the mitotic cycle and that this cell population could be considered as quiescent (Srebreva et al., 1979). Chromatin isolated from these cells had an increased NHC protein content, its DNA :histone :NHC protein ratio being 1:1:1.4. The ratio of the specific radioactivity of the histones to that of DNA was 0.3-0.4, while this ratio for the total NHC protein was
1093
1094
BOYKAANACHKOVA and
0
0
2
4
6
o
GEORGE
RUSSEV
2
cm towards
4
6
anode
Fig. 1. SDS-polyacrylamide gel electrophoresis of chromatin isolated from Ehrlich ascites tumour cells 5 (A) and 10 (B) days after inoculation. DNA and the proteins were labelled in oiuo with E3H]tbymidine and [14Clprotein hydrolyzate, respectively, as described in Materials and Methods. The electro~h~resis was carried out in 8.75% polyacrylamide gel according to Laemmli (1970), and the gel was stained, scanned and counted as described in Materials and Methods. Bovine serum albumin (68 kdalton, monomer, and 136 kdalton, dimer), aldolase (40 kdalton) and chymotripsinogen A (25 kdalton) were used to calibrate the gel. -----Coomassie Briliant Blue staining recorded at 600 m; ---[‘4C]radioactivity. 1, which indicated that in the quiescent cells at least part of the NHC proteins were still turning over. The SDS-polyacrylamide gel electrophoresis showed that the NHC protein pattern of chromatin from “day 10’ cells was identical to that of “day 5” cells in respect to both number and abundance of fractions (Fig. IA and B). As in the case of chromatin isolated from dividing cells, the specific radioactivity of the individual NHC proteins from quiescent cells was almost uniform with two exceptions. Here again the specific radioactivity of the 200 kd fraction was much lower than that of the remaining proteins which showed that both in quiescent and prol~fera~ng EAT cells this protein was metabolically the least active. On the other hand there was a distinct group of proteins with molecular weights in the range 18-25 kdalton (Fig. lB), with specific radioactivities 3-4 times higher than those of the rem~ni~g NHC proteins. It is also worthy noting that the specific radioactivity of histone HI in the quiescent cells was about 1.5 times higher than that of the remaining 4 histones, which showed that this histone was turning slowly over even when the synthesis of DNA and of the core histones was inhibited. From the results described above we drew the following conclusions. The SDS-electrophoretical profiles of NHC proteins from quiescent and dividing EAT cells are identical. In both prol~erating and dormant cells there is a high molecular weight protein that turns over much slower than most of the NHC proteins, Both in quiescent and dividing cells there are groups of several NHC proteins turning over at higher rates than bulk NHC protein. In the dividing cells there were proteins with molecular weights between 45 and 65 kdalton, and in quiescent cellsbetween 18 and 25 kdalton.
REFERENCES
Anderson L. A. & McClure W. D. (1973) An improved cocktail of high solubilizinrr power. Annlvt. Biochepa. 51. I-
t73-179.
-
BURTONK. (1956) A study of the conditions and mechanism of the diphenylamine reaction for calorimetric estimation of deoxyribonucleic acid. Biochem. 1. 62, 315-323. DJONDJUROVL., IVANOVA E. & T,YANEVR. (1979) Two chromatin fractions with different metabolic properties of non-!&tone proteins and of newly synthesized RNA. Eur. S. Rio&in. 97, 133-139. KOLEVAS. & TSANEVR. (1978) Metabolically stable nonhistone chromosomal proteins in growing maize roots. Cell LtQ”erentiation 7, 83-88. LAEMMLIU. K. (1970) Cleavage of structural proteins during the assembly of thi head of bacteriophage T4. Nature 22T7,680-685. LOWXY0. H., ROSEBROIJGH N. J., F.QZRA. L. & RZWDAL R. J. (19St) Protein measurement with Folin phenol reagent. d. biol. Chem. 193, 265-275. R&&v G., ANACHKOVA R. & TSANIWR. (1975) Fractionation of rat liver chromatin nonhistone proteins into two groups with ‘different metabolic rates. Eur. J. ~jo~~rn. 58, 253-257. SREBREVAL., RUSSEVG. & TSANEVR. (1979) Metabolic stability of nonhistone chromosomal proteins in Ehrlich ascites tumour cells. Inl. J. Biochem. 10, 253-257. ST~MBO~VAM., ANGELOVA A. & TWNEVR. (1979) Mefabolic stability of nonhistone chromosomal proteins in two different classes of developing rat brain nuclei. Cell Di#efSeren&tion8, 195-202. TSANEVR., DI~NDJUROVL. & IVANO~AE. (1974) Cell cycle dependent turnover of nonhistone proteins in chromatin. Expl Cell Rek 84, 137-142. TSANEVR. & RUSSEVG. (1974) Distribution of newly synthesized histones during DNA replication. Eur. J. Biothem. 43, 257-263.