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RNA, DNA and total protein levels in subcellular fractions of human brain tumors
Cancer Letters, 34 (1987) 67-71 Elsevier Scientific Publishers Ireland Ltd.
67
RNA, DNA AND TOTAL PROTEIN LEVELS IN SUBCELLULAR FRACTIONS OF HUMAN BRAIN TUMORS
EMINE KiiKOGLIJ Department
of Biochemistry,
Cerrahpya
Medical Faculty,
Istanbul
(Turkey)
(Received 7 July 1986) (Revised version received 25 September 1986) (Accepted 29 September 1986)
SUMMARY
The intracellular distributions of the nucleic acids DNA and RNA and of total proteins were investigated in different types of human brain tumors and were compared with those of normal brain tissue. The tumor types studied were: meningioma, astrocytoma, and glioblastoma multiforme. Nucleic acids and total proteins were found to increase in the nuclear fraction of all tumoral tissues as compared with normal brain tissue. Another important finding is that the rate of the increase of these parameters is not proportional to the degree of malignancy of the tumor. INTRODUCTION
In the present study, we have investigated the subcellular distribution of the nucleic acids, DNA and RNA, and of total proteins, i.e. the final products of the series of events initiated by the nucleic acids, in human brain tumors. The scientific literature contains avery limited number of communications concerning the non-enzyme proteins of the brain tumors. K. Bashar et al. [ 91 have investigated proteins in normal human brain and in brain tumor. Khominsky et al. [ 10,111 have done a histochemical analysis of nucleic acids and proteins in cells of neuroectodermal tumor of different grades of malignancy. Naber et al. have studied the total proteins in the aging human brain [13]. In all these studies, no data have been found concerning the subcellular distribution of total proteins in normal or in tumoral brain tissue. Cuatico et al. [2] have investigated tumor specific DNA sequences in human gliomas, De Reuck et al. [3] have determined DNA cytophotometrically in human oligodendroglial tumors. Miiller [ 121 has measured DNA in various cerebral tumors. Viale et al. [16] have emphasized the transfer RNA in brain tumors. In all these studies, no data have been found concerning the subcellular distribution of nucleic acids in brain tumors. o 1987 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/87/$03.50 Published and Printed in Ireland
68
We have undertaken this study to investigate the total protein, RNA and DNA levels in the subcellular fractions of human brain tumors. The oncotypes studied were: meningioma, astrocytoma and glioblastoma multiforme. MATERIALS
AND METHODS
Tumor tissue samples were obtained from the Department of Neurosurgery within 1 h after surgical intervention by open biopsy. Whole tumor was taken off and then macroscopically homogenous pieces of tumor were selected and one section from each tumor was kept for histological identification. Tissue samples surrounding the tumor and designated to be ‘normal’ in the histological study were used as the normal samples. These were chosen from peritumoral tissues in which there was no cell infiltration. Tissue samples were homogenized in a 0.32 M saccharose medium at 4”C, and at 3000 rev./min for 10 min. Tissue homogenates thus obtained were separated into 5 subcellular fractions using an ultracentrifuge MSE Superspeed 75. The fractions were : P, (nucleus), Pz (mitochondria), P3 (microsomes), P4 (ribosomes), and Ps (supernatant). The technique employed was adopted from Gray and Whittaker [8]. In this method, in order to obtain clear subfractions from brain tissue (free of myelin fragments and cellular debris and synaptosomes), the crude fractions obtained by differential ultracentrifugation were further separated in a density gradient to give pure subfractions. The crude mitochondrial fraction obtained by centrifugation at 17,000 X g for 55 min was further separated into subfractions A, B and C. Fraction A, layer between 0.32 M and 0.8 M sucrose (centrifuged at 10’ X g for 60 min) was found to consist largely of myelin fragments. Fraction B was the layer between 0.8 and 1.2 M sucrose, diluted with equal volume of water, centrifuged at 10’ X gfor 60 min. Fraction C, the pellet below 1.2 M sucrose, was identified as mitochondria and this was the mitochondrial subfraction used in our experiments. Total proteins, DNA and RNA were extracted from the different organelles using the modification by Edelman et al. [4] of the technique Schmidt and Thannhauser [ 151. Total proteins were determined by the biuret reaction [l] using bovine serum albumin as standard, RNA and DNA were assayed, using a PyeUnicam SP 500 [4]. The results were given in mg protein, DNA and RNA per gram of wet tissue. RESULTS
AND DISCUSSION
The subcellular distribution of total proteins in normal and in tumoral tissues is represented in Table 1. It is observed that total protein levels increased in the nuclear fractions of all types of tumoral tissues compared with normals.
TABLE 1
8
10
12
12
Glioblastoma multiforme Meningioma
Astrocytoma
Normal
98.9 + (79.0) 40.8 + (42.1) 55.3 + (43.1) 33.5 + (46.1)
P, (a)
3.4
1.6
2.1
12.5 12.3 + (9.8) 22.4 + (23.1) 25.7 + (20.0) 14.6 + (20.1)
P, (%)
OF TOTAL PROTEINSa
3.2
3.1
3.8
1.2 1.5 + (1.2) 9.4 + (9.7) 5.6 + (4.4) 4.8 f (6.6)
P,(S)
0.7
0.5
2.5
0.9
12
12
Astrocytoma
Normal
(35.3)
(34.7) 1.28 + 0.06
(35.0) 1.78 + 0.06
(18.1) 2.14 + 0.12
1.61 + 0.05
P,(W)
OF RNAa
(8.7) 1.4 * 0.07 (22.9) 0.82 + 0.04 (15.8) 0.59 + 0.03 (16.3)
0.77 + 0.04
P,(S)
(9.1)
(5.9) 0.33 + 0.01
(9.8) 0.31 f 0.02
(4.7) 0.60 + 0.02
0.42 + 0.02
P, (a)
The results are expressed as mg RNA per gram of wet tissue for each subcellular fraction.
10
*
8
Glioblastoma
N
DISTRIBUTION
multiforme Meningioma
Oncotype
SUBCELLULAR
TABLE 2
aTbe results are given in mg protein per gram of wet tissue for each subcellular fraction.
N
DISTRIBUTION
Oncotype
SUBCELLULAR
(2.3) 0.22 f 0.06 (4.2) 0.16 + 0.01 (4.4)
0.14 f 0.05 (1.6) 0.14 f 0.02
P, (%)
-
1.2 + 0.7 (1.0) 2.4 f 0.5 (2.5) 3.2 + 0.8 (2.5) 1.0 + 0.1 (1.4)
p.4(%I
_
5.95 + (66.9) 1.84 + (30.0) 2.05 + (39.4) 1.25 f (34.7) .--
P, (%)
_-
0.05
0.1
0.1
0.09
--
11.3 r 2.1 (9.0) 21.9 Yk3.4 (22.6) 38.5 + 1.9 (30.0) 18.7 + 3.5 (25.8)
p, PJ)
-.
z
70 TABLE 3 SUBCELLULAR Oncotype Glioblastoma multiforme Meningioma Astrocytoma Normal
DISTRIBUTION N 8 10 12 12
I
OF DNAa P,
P,
3.55 2 0.19
--
1.38 ? 0.07 2.40 t 0.11 0.80 + 0.03
---
P,
-
P,
P,
-
-
-
_ -
BThe results are given in mg DNA per gram of wet tissue.
Table 2 represents the subcellular distribution of RNA. Total RNA levels showed elevations in the nuclear and microsomal fractions of all the tumoral tissues. The important point in the distribution of DNA, as shown in Table 3 is that DNA, also, showed increases in the tumoral tissues in the nuclear fraction. In view of our results, a common property observed for all tumor types is that nucleic acids which function in the deposition, transmission and translation of genetic information and the cellular synthesis products, total proteins, are increased in the nuclear fraction of all tumoral tissues as compared with normal brain tissue. The percentages of total protein, DNA and RNA contents for the different organelles showed differences in the different tumor types. This observation is evidence against metabolic uniformity in tumoral tissue. Another important finding is that the rate of increase of these parameters is not proportional to the degree of malignancy of the tumor. As an example, the total RNA level of the nuclear fraction was found to be lower for glioblastoma multiforme than the other tumoral tissues. In a study on gliomas, Nayar [14] has stated that the DNA level of the glioma tissue was 2-8 times that of normal brain tissue. Our data show that the various tumoral tissues contain 1.7-4.5 times higher DNA than normal tissue. These results suggest that the subcellular structures of tumor cells do not have the capacity to control the various processes which permit a synthesis of proteins at the normal cellular level. The results of this investigation are in keeping with our previous studies on human brain tumor subcellular fractions [5-71 providing evidence against metabolic uniformity in the tumor cell. REFERENCES 1 Bauer, J.D., Ackermann, P.G. and Toro, G. (1968) Bray’s Clinical Laboratory Methods, 7th edn., pp. 3 20-322. The C.V. Mosby Company, Saint Louis. 2 Cuatico, W. and Cho, J.R. (1979) Tumorspecific DNA sequences in human gliomas. Cancer, 44,1309-1314.
71 3 De Reuck, J., Sieben, C., De Coster, W., Roels, H. and Eecken, H.V. (1980) Cytophotometric DNA determination in human oligodendroglial tumors. Histopathology, 4,225-232. 4 Edelman, M., Hirsch, C.A., Hiatt, H.M. and Fox, M. (1969) Apparent changes in mouse liver DNA content due to interference by non-DNA diphenylamine-reacting cytoplasmic material. Biochim. Biophys. Acta, 1979, 172-178. 5 Guner, G., Koko$jlu, E. and Giiner, A. (1985) Hydrogen peroxide detoxication by catalase in subcellular fraction of human brain tumors and normal brain tissues. Cancer Letters, 27, 221-224. 6 Giiner, G., Kokoglu, E. and Giiner, A. (1985) Subcellular distribution of GOT and GPT in human brain tumors. IRCS Med. Sci. 13, 1095. 7 Guner, G., Kokoglu, E. and Giiner, A. (1985) Acid and alkaline phosphatase activities in homogenates and subcellular fractions of human brain tumors. Cancer Letters, 29, 339-343. 8 Gray, E.G. and Whittaker, V.P. (1962) The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat., 96, 79-87. 9 Khairul-Bashar, S.A. and Ahmad, F. (1977) Proteins in normal human brain and brain tumor. Indian J. Med. Res., 66, 820-823. 10 Khominsky, B.S. and Brodskaia, I.A. (1973) Histochemistry of nucleic acids and proteins in cells of neuroectodermal tumor of different grades of malignancy. Neoplasma, 20, 281-296. 11 Khominsky, B.S., Brodskaia, I.A. and Verkhogliadova, T.P. (1976) In vivo and in vitro cytospectrophotometric studies of DNA in the cells of human neuroectodermal brain tumors. Tsitologiia, 18,301-306. 12 Miiller, W. (1972) DNA estimations in cerebral tumors of man. Neuropathol. Pol., 10, 121-128. 13 Naber, D., Korte, U. and Krack, K. (1979) Content of water soluble and total proteins in the aging human brain. Exp, Gerontol., 14, 5933. 14 Nayyar, S.N. (1963) A study of phosphate, deoxyribonucleic acid, and phospholipid fractions in neural tumors. Neurology, 13, 287-291. 15 Schmidt, G. and Thannhauser, S.J. (1945) A method for the determination of deoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem., 161,83-89. 16 Viale, G.L., Kroh, H. and Grosso, G. (1971) Transfer RNA and transfer RNA methylase in human brain tumors. Cancer Res., 31, 605-608.
67
RNA, DNA AND TOTAL PROTEIN LEVELS IN SUBCELLULAR FRACTIONS OF HUMAN BRAIN TUMORS
EMINE KiiKOGLIJ Department
of Biochemistry,
Cerrahpya
Medical Faculty,
Istanbul
(Turkey)
(Received 7 July 1986) (Revised version received 25 September 1986) (Accepted 29 September 1986)
SUMMARY
The intracellular distributions of the nucleic acids DNA and RNA and of total proteins were investigated in different types of human brain tumors and were compared with those of normal brain tissue. The tumor types studied were: meningioma, astrocytoma, and glioblastoma multiforme. Nucleic acids and total proteins were found to increase in the nuclear fraction of all tumoral tissues as compared with normal brain tissue. Another important finding is that the rate of the increase of these parameters is not proportional to the degree of malignancy of the tumor. INTRODUCTION
In the present study, we have investigated the subcellular distribution of the nucleic acids, DNA and RNA, and of total proteins, i.e. the final products of the series of events initiated by the nucleic acids, in human brain tumors. The scientific literature contains avery limited number of communications concerning the non-enzyme proteins of the brain tumors. K. Bashar et al. [ 91 have investigated proteins in normal human brain and in brain tumor. Khominsky et al. [ 10,111 have done a histochemical analysis of nucleic acids and proteins in cells of neuroectodermal tumor of different grades of malignancy. Naber et al. have studied the total proteins in the aging human brain [13]. In all these studies, no data have been found concerning the subcellular distribution of total proteins in normal or in tumoral brain tissue. Cuatico et al. [2] have investigated tumor specific DNA sequences in human gliomas, De Reuck et al. [3] have determined DNA cytophotometrically in human oligodendroglial tumors. Miiller [ 121 has measured DNA in various cerebral tumors. Viale et al. [16] have emphasized the transfer RNA in brain tumors. In all these studies, no data have been found concerning the subcellular distribution of nucleic acids in brain tumors. o 1987 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/87/$03.50 Published and Printed in Ireland
68
We have undertaken this study to investigate the total protein, RNA and DNA levels in the subcellular fractions of human brain tumors. The oncotypes studied were: meningioma, astrocytoma and glioblastoma multiforme. MATERIALS
AND METHODS
Tumor tissue samples were obtained from the Department of Neurosurgery within 1 h after surgical intervention by open biopsy. Whole tumor was taken off and then macroscopically homogenous pieces of tumor were selected and one section from each tumor was kept for histological identification. Tissue samples surrounding the tumor and designated to be ‘normal’ in the histological study were used as the normal samples. These were chosen from peritumoral tissues in which there was no cell infiltration. Tissue samples were homogenized in a 0.32 M saccharose medium at 4”C, and at 3000 rev./min for 10 min. Tissue homogenates thus obtained were separated into 5 subcellular fractions using an ultracentrifuge MSE Superspeed 75. The fractions were : P, (nucleus), Pz (mitochondria), P3 (microsomes), P4 (ribosomes), and Ps (supernatant). The technique employed was adopted from Gray and Whittaker [8]. In this method, in order to obtain clear subfractions from brain tissue (free of myelin fragments and cellular debris and synaptosomes), the crude fractions obtained by differential ultracentrifugation were further separated in a density gradient to give pure subfractions. The crude mitochondrial fraction obtained by centrifugation at 17,000 X g for 55 min was further separated into subfractions A, B and C. Fraction A, layer between 0.32 M and 0.8 M sucrose (centrifuged at 10’ X g for 60 min) was found to consist largely of myelin fragments. Fraction B was the layer between 0.8 and 1.2 M sucrose, diluted with equal volume of water, centrifuged at 10’ X gfor 60 min. Fraction C, the pellet below 1.2 M sucrose, was identified as mitochondria and this was the mitochondrial subfraction used in our experiments. Total proteins, DNA and RNA were extracted from the different organelles using the modification by Edelman et al. [4] of the technique Schmidt and Thannhauser [ 151. Total proteins were determined by the biuret reaction [l] using bovine serum albumin as standard, RNA and DNA were assayed, using a PyeUnicam SP 500 [4]. The results were given in mg protein, DNA and RNA per gram of wet tissue. RESULTS
AND DISCUSSION
The subcellular distribution of total proteins in normal and in tumoral tissues is represented in Table 1. It is observed that total protein levels increased in the nuclear fractions of all types of tumoral tissues compared with normals.
TABLE 1
8
10
12
12
Glioblastoma multiforme Meningioma
Astrocytoma
Normal
98.9 + (79.0) 40.8 + (42.1) 55.3 + (43.1) 33.5 + (46.1)
P, (a)
3.4
1.6
2.1
12.5 12.3 + (9.8) 22.4 + (23.1) 25.7 + (20.0) 14.6 + (20.1)
P, (%)
OF TOTAL PROTEINSa
3.2
3.1
3.8
1.2 1.5 + (1.2) 9.4 + (9.7) 5.6 + (4.4) 4.8 f (6.6)
P,(S)
0.7
0.5
2.5
0.9
12
12
Astrocytoma
Normal
(35.3)
(34.7) 1.28 + 0.06
(35.0) 1.78 + 0.06
(18.1) 2.14 + 0.12
1.61 + 0.05
P,(W)
OF RNAa
(8.7) 1.4 * 0.07 (22.9) 0.82 + 0.04 (15.8) 0.59 + 0.03 (16.3)
0.77 + 0.04
P,(S)
(9.1)
(5.9) 0.33 + 0.01
(9.8) 0.31 f 0.02
(4.7) 0.60 + 0.02
0.42 + 0.02
P, (a)
The results are expressed as mg RNA per gram of wet tissue for each subcellular fraction.
10
*
8
Glioblastoma
N
DISTRIBUTION
multiforme Meningioma
Oncotype
SUBCELLULAR
TABLE 2
aTbe results are given in mg protein per gram of wet tissue for each subcellular fraction.
N
DISTRIBUTION
Oncotype
SUBCELLULAR
(2.3) 0.22 f 0.06 (4.2) 0.16 + 0.01 (4.4)
0.14 f 0.05 (1.6) 0.14 f 0.02
P, (%)
-
1.2 + 0.7 (1.0) 2.4 f 0.5 (2.5) 3.2 + 0.8 (2.5) 1.0 + 0.1 (1.4)
p.4(%I
_
5.95 + (66.9) 1.84 + (30.0) 2.05 + (39.4) 1.25 f (34.7) .--
P, (%)
_-
0.05
0.1
0.1
0.09
--
11.3 r 2.1 (9.0) 21.9 Yk3.4 (22.6) 38.5 + 1.9 (30.0) 18.7 + 3.5 (25.8)
p, PJ)
-.
z
70 TABLE 3 SUBCELLULAR Oncotype Glioblastoma multiforme Meningioma Astrocytoma Normal
DISTRIBUTION N 8 10 12 12
I
OF DNAa P,
P,
3.55 2 0.19
--
1.38 ? 0.07 2.40 t 0.11 0.80 + 0.03
---
P,
-
P,
P,
-
-
-
_ -
BThe results are given in mg DNA per gram of wet tissue.
Table 2 represents the subcellular distribution of RNA. Total RNA levels showed elevations in the nuclear and microsomal fractions of all the tumoral tissues. The important point in the distribution of DNA, as shown in Table 3 is that DNA, also, showed increases in the tumoral tissues in the nuclear fraction. In view of our results, a common property observed for all tumor types is that nucleic acids which function in the deposition, transmission and translation of genetic information and the cellular synthesis products, total proteins, are increased in the nuclear fraction of all tumoral tissues as compared with normal brain tissue. The percentages of total protein, DNA and RNA contents for the different organelles showed differences in the different tumor types. This observation is evidence against metabolic uniformity in tumoral tissue. Another important finding is that the rate of increase of these parameters is not proportional to the degree of malignancy of the tumor. As an example, the total RNA level of the nuclear fraction was found to be lower for glioblastoma multiforme than the other tumoral tissues. In a study on gliomas, Nayar [14] has stated that the DNA level of the glioma tissue was 2-8 times that of normal brain tissue. Our data show that the various tumoral tissues contain 1.7-4.5 times higher DNA than normal tissue. These results suggest that the subcellular structures of tumor cells do not have the capacity to control the various processes which permit a synthesis of proteins at the normal cellular level. The results of this investigation are in keeping with our previous studies on human brain tumor subcellular fractions [5-71 providing evidence against metabolic uniformity in the tumor cell. REFERENCES 1 Bauer, J.D., Ackermann, P.G. and Toro, G. (1968) Bray’s Clinical Laboratory Methods, 7th edn., pp. 3 20-322. The C.V. Mosby Company, Saint Louis. 2 Cuatico, W. and Cho, J.R. (1979) Tumorspecific DNA sequences in human gliomas. Cancer, 44,1309-1314.
71 3 De Reuck, J., Sieben, C., De Coster, W., Roels, H. and Eecken, H.V. (1980) Cytophotometric DNA determination in human oligodendroglial tumors. Histopathology, 4,225-232. 4 Edelman, M., Hirsch, C.A., Hiatt, H.M. and Fox, M. (1969) Apparent changes in mouse liver DNA content due to interference by non-DNA diphenylamine-reacting cytoplasmic material. Biochim. Biophys. Acta, 1979, 172-178. 5 Guner, G., Koko$jlu, E. and Giiner, A. (1985) Hydrogen peroxide detoxication by catalase in subcellular fraction of human brain tumors and normal brain tissues. Cancer Letters, 27, 221-224. 6 Giiner, G., Kokoglu, E. and Giiner, A. (1985) Subcellular distribution of GOT and GPT in human brain tumors. IRCS Med. Sci. 13, 1095. 7 Guner, G., Kokoglu, E. and Giiner, A. (1985) Acid and alkaline phosphatase activities in homogenates and subcellular fractions of human brain tumors. Cancer Letters, 29, 339-343. 8 Gray, E.G. and Whittaker, V.P. (1962) The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat., 96, 79-87. 9 Khairul-Bashar, S.A. and Ahmad, F. (1977) Proteins in normal human brain and brain tumor. Indian J. Med. Res., 66, 820-823. 10 Khominsky, B.S. and Brodskaia, I.A. (1973) Histochemistry of nucleic acids and proteins in cells of neuroectodermal tumor of different grades of malignancy. Neoplasma, 20, 281-296. 11 Khominsky, B.S., Brodskaia, I.A. and Verkhogliadova, T.P. (1976) In vivo and in vitro cytospectrophotometric studies of DNA in the cells of human neuroectodermal brain tumors. Tsitologiia, 18,301-306. 12 Miiller, W. (1972) DNA estimations in cerebral tumors of man. Neuropathol. Pol., 10, 121-128. 13 Naber, D., Korte, U. and Krack, K. (1979) Content of water soluble and total proteins in the aging human brain. Exp, Gerontol., 14, 5933. 14 Nayyar, S.N. (1963) A study of phosphate, deoxyribonucleic acid, and phospholipid fractions in neural tumors. Neurology, 13, 287-291. 15 Schmidt, G. and Thannhauser, S.J. (1945) A method for the determination of deoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem., 161,83-89. 16 Viale, G.L., Kroh, H. and Grosso, G. (1971) Transfer RNA and transfer RNA methylase in human brain tumors. Cancer Res., 31, 605-608.