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Effect of retinyl acetate on nuclear proteins in rabbit liver
Gen. Pharmac. Vol. 20, No. 5, pp. 601-604, 1989
0306-3623/89 $3.00+ 0.00 Copyright © 1989 PergamonPress plc
Printed in Great Britain.All rights reserved
EFFECT OF RETINYL ACETATE ON NUCLEAR PROTEINS IN RABBIT LIVER JAN PAEYGA,* KAROLINAGROCHAEA,ANDRZEJ KOWALSKI,MAEGORZATASYLWESTRZAK and J^N RAFAYI Department of Genetics, Educational University of Kielce, 25-518 Kielce, Rewolucji Pa~dziernikowej 33, Poland and IResearch Institute of Animal Production. Nitra, Czechoslovakia (Received 16 January 1989)
Abstract--l. Retinyl acetate injected intraperitoneally to adult rabbits fed on standard diet caused detectable changes in the polyacrylamide gel patterns of liver nucleoplasmic and 0.35 M NaCl-soluble chromatin proteins. 2. Both histones and non-histone proteins soluble in 5 M urea were not affected in vitamin A-treated animals. 3. It seems that variations in liver nuclear proteins from retinyl acetate-administered rabbits may reflect retinol-dependent alterations in structure and function of their chromatins.
INTRODUCTION
et al. (1988) have found that in vivo binding of retinol
It is well known (Zile and Cullum, 1983) that vitamin A is required not only for vision but also for reproduction and growth. The vitamin is essential for differentiation and maintenance of epithelial tissues (Sporn and Roberts, 1983). A lack of retinol results in lesions of skin cells (Chytil, 1984) and germinal epithelium of testis (Porter et al., 1986). The depressed levels of R N A synthesis in liver (Tsai and Chytil, 1978) and testis (Porter et al., 1986) nuclei of retinol-deprived rats were observed. The rate of RNA synthesis increased quickly within 4 hr after refeeding of deficient animals with retinyl acetate because of the increased ability of chromatin to serve as a template for polymerase II (Porter et al., 1986). Shortly after administration of retinyl acetate to retinol-deficient rats, an inhibited synthesis of approximately 280 proteins and an activated synthesis of about 100 proteins were detected among cytosolic proteins of rat testes using two-dimensional polyacrylamide gel electrophoresis (ul Haq and Chytil, 1988). Since retinoids were reported to influence gene expression (see Chytil, 1984 and 1986 for review), it is reasonable to assume that they should induce some changes in chromatin. It was shown (Chytil and Ong, 1979) that vitamin A is first bound to specific cellular retinol-binding protein (CRBP) which delivers it to binding sites in chromatin (Liau et al., 1981). Interestingly, a 4-fold increase in level of lung CRBP-specific m R N A was observed within 4 hr after refeeding of retinol-delicient rats (Sherman et al., 1987). As purified D N A or mono- and dinucleosomes did not accept retinol from its complex with CRBP, it was suggested (Liau et al., 1985) that a native higher order structure of chromatin and/or a presence of specific proteins lost during nuclease digestion of chromatin are required for binding of retinol. Recently, Ferrari
*To whom all correspondence should be addressed.
to chromatin is mediated by a lipoprotein associated with nucleosomes. They proposed that the binding sites on DNA are recognized by a protein component while retinol is solubilized in the fatty acid moiety. Introduction o f retinoi to cultured 3T3 cells induces an augmented sensitivity of their chromatin to DNase I digestion, an increase of acetate uptake and turnover on histones, and a disappearance of a 20-kDa chromatin proteins soluble in 0.35 M NaCI that are coextracted with the high mobility group proteins (Ferrari and Vidali, 1985). Thus, chromatin structure, content and metabolism o f chromosomal proteins may strongly depend on a cellular status of vitamin A. In this report we compared the polyacrylamide gel patterns of nuclear proteins, both non-histone proteins and histones, from liver of normal rabbits and experimental ones injected with high doses of retinyl acetate as a source of vitamin A. MATERIALS AND METHODS
New Zealand White rabbits weighing 2.2-2.5 kg fed on a standard chow were used. Animals were injected intraperitoneaUy with 800,000 tU retinyl acetate (Serva, Heidelberg, F.R.G.) a day for 2 consecutive days. On the third day, the experimental and control rabbits were sacrificed by cervical dislocation and bled. A liver perfusion was performed using a chilled solution containing 0.13 M NaCI, 0.008 M MgCI2, 0.005 M KCI and 0.1 mM phenylmethanesuifonyl fluoride (PMSF). The removed livers were quickly frozen in liquid nitrogen and kept at - 2 0 ° C until used. Nuclei were isolated by a slight modification of the method of Medvedev et al. (1982). Briefly, the tissue was homogenized in 50% glycerol in 0.01 M Tris-0.01 M KCI-0.0015M MgCI2-0.1mM PMSF containing 0.5% Triton X-100 at 0-4°C and pelleted at 3000 rpm for 10 min in Janetzki K-23 centrifuge. The nuclear pellet was washed three times with above solution containing decreasing concentrations of Triton X-100. Nuclear proteins were isolated by consecutive extraction of nuclei with: (a) 0.025 M ethylene diamine tetraacetic
601
602
JAN
PAEYGA
acid--O075 M NaCI, pH 7.6; (b) 0.35 M NaCI-0.02 M Tris-HCl, pH 7.6; 5 M urea-0.05 M sodium phosphate, pH 7.6 containing 0.02 M glycine and (d) 0.25 M HCI. All solutions used for extraction of proteins were supplemented with 0. I mM PMSF. Non-histone proteins were precipitated overnight at -20:'C by adding 2 vol of cold 99.8% ethanol containing 2% potassium acetate. Precipitates were first washed with cold absolute ethanol, then resuspended in small volumes of 0.005 M 2-mercaptoethanol and lyophilized. Histones were precipitated overnight with 20% trichloroacetic acid at 0 4 C by adding appropriate volume of 100% trichloroacetic acid. Precipitates were washed twice with acidified acetone (500cm 3 acetone plus 2 cm 3 concentrated HCI) and acetone. Protein samples were dissolved in 2% sodium dodecyl sulphate, 10% glycerol and 0.0625 M Tris-HC1, pH 6.8. Protein content was determined by the method of Lowry et al. (1951). After supplementing with 2-mercaptoethanol to final concentration of 5%, non-histone proteins and histones were separated in 10 and 12.5% polyacrylamide gels, respectively, containing 0. 1% sodium dodecyl sulphate (Laemmli, 1970).
tightly bound with chromatin and histones by using a series of solutions differing in their extraction properties and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The gel patterns of nucleoplasmic proteins from normal and retinol-treated rabbits were shown in Fig. la. Despite the uniformity in distribution and quantity of the majority of proteins, it was found that the intensity of several protein bands (28, 29, 35 and 76 kDa) were significantly altered in rabbits receiving high amounts of retinyl acetate. Similarly, the gel patterns of the 0.35 M NaCI-soluble chromatin proteins (Fig. l b) were also affected in retinol-treated rabbits. The amounts of proteins with molecular weights of approximately 27 and 31 kDa decreased in comparison with their levels in control animals. On the other hand, the content of 36 and 2 0 0 k D a proteins increased in chromatin of retinyl acetate exposed animals. In addition to those major changes, numerous faintly stained nucleoplasmic and chromatin protein bands were also affected in retinolinjected animals. As it can be seen in Figs lc and d, the gel patterns of 5 M urea-soluble chromatin proteins and histones, respectively, were practically identical in control and retinyl acetate-injected animals.
RESULTS AND DISCUSSION Nuclear proteins from rabbit liver were fractionated into nucleoplasmic proteins, proteins loosely and A A
et al.
B
A
A
B
B
B --
200
..
.,
:....:
76
H1
35
--
'i"
36
.L.i~.l~"
--31
..::/:
~29 .28 --
27
H3 H2B
H2A H4
.....
[a]
(b)
(c)
(d)
Fig. 1. Electrophoretic patterns of nucleoplasmic proteins (a), 0.35 M NaCl-soluble chromatin proteins (b), 5 M urea-soluble chromatin proteins (c) and histones (d) from liver of control (A) and retinyl acetate-injected rabbits (B). The numbers indicate the approximate molecular weights of protein bands in kilodalton. LSP--liver specific proteins (Medvedev et al., 1982).
Retinol and liver nuclear proteins Our results clearly indicate that intraperitoneal injection of retinyl acetate caused profound changes in the composition of nueleoplasmic proteins and proteins loosely bound with chromatin in rabbit liver. The protein fractions more tightly bound with D N A (5 M urea-soluble chromatin proteins and histones) did not change significantly following administration of vitamin A. An increasing body of evidence suggests (Sporn and Roberts, 1983; Chytil, 1984; Chytil, 1986) that retinol and its natural and synthetic derivatives can alter genomic expression. The rate of mRNA synthesis rapidly decreases in liver and testis nuclei of retinol-depleted rats, but replenishing the animals with retinol results in an increasing rate of RNA synthesis (Tsai and Chytil, 1978; Porter et al., 1986). It was observed (Omori and Chytil, 1982; ul Haq and Chytil, 1988) that animal genome rapidly responds to vitamin A compounds by suppression or activation the synthesis of specific proteins. Ferrari and Vidali (1985) have shown that retinol induces an augmented sensitivity of chromatin of cultured mouse 3T3 cells to DNase I digestion which is accompanied with a higher rate of acetate uptake and turnover on histones and a decrease in the level of a chromosomal protein of 20 kDa. Another chromatin protein, a testis-specific transitional protein (TP) which is synthesized temporarily at the midspermatid stage (Grimes, 1986), is markedly reduced in the testes of vitamin A-deficient rats (Rao et al., 1980). It is worth noting that retinol-mediated changes in 20 kDa protein of 3T3 cells (Ferrari and Vidali, 1985) and in TP (Rao et al., 1980) are reversible upon returning to normal status. Interestingly, retinoic acid-induced differentiation of retinoicacid-sensitive human leukemia (HL60) cells to mature-like granulocytes is accompanied with appearance of nucleosoma160-kDa protein (Chou et al., 1984). Thus, retinol and other retinoids can influence chromatin structure and function and its protein components. It appears that changes in nucleoplasmic and loosely bound chromatin proteins from rabbit liver following injection of retinyl acetate in our gel patterns may also reflect a modification of chromatin structure and function. It is well known (Thakur, 1984; Patyga, 1984; Palyga et al., 1984) that certain components of nuclear protein fractions undergo changes during cell differentiation and tissue development. Although retinol is believed to play a significant role in growth and differentiation (Sporn and Roberts, 1983), its effect on differentiation- or development-associated nuclear proteins is poorly explored (Rao et al., 1980). High doses of vitamin A induce toxic lesions in cell membranes (Zile and Cullum, 1983). This may lead to greater permeability of certain proteins from nucleus (e.g. those whose amounts decrease following treatment with retinyl acetate; Figs la and b) or to releasing of proteases from damaged lysosomes which may degrade some nuclear proteins. Moreover, retinol-dependent activation of endogenous chromatin proteases cannot be excluded. Our results indicate that vitamin A-mediated alterations in nuclear proteins have preferentially appeared among nucleoplasmic proteins and loosely bound chromatin proteins soluble in 0.35 M NaCI GP 20/~-~E
603
solution that undoubtedly contain components shuttling between cytoplasm and chromatin. It is very likely that prolonged action of retinyl acetate could lead to the alterations of certain chromatin proteins firmly bound with DNA.
REFERENCES
Chou R. H., Chervenick P. A. and Barch D. R. (1984) Appearance of a new nucleosomal protein during differentiation of human leukemia (HL-60) cells. Science 223, 1420-1423. Chytil F. (I 984) Retinoic acid: biochemistry, pharmacology, and therapeutic use. Pharmac. Rev. 36, 93S-100S. Chytil F. (1986) Retinoic acid: biochemistry and metabolism. J. Am. Acad. Dermatol. 15, 741-747. Chytil F. and Ong D. E. (1979) Cellular retinol- and retinoic acid-binding proteins in vitamin A action. Fedn Proc. Fedn Am. Socs exp. Biol. 38, 2510-2514. Ferrari N. and Vidali G. (1985) Effects of retinol on chromatin structure. Eur. J. Biochem. 151, 305-310. Ferrari N., Pfeffer U. and Vidali G. (1988) In vivo binding of retinol to chromatin. The binding is mediated by a lipoprotein. J. biol. Chem. 263, 448-453. Grimes S. R. (1986) Nuclear proteins in spermatogenesis. Comp. Biochem. Physiol. 83B, 495-500. ul Haq R. and Chytil F. (1988) Early effects of retinol and retinoic acid on protein synthesis in retinol deficient rat testes. Biochem. biophys, Res. Commun. 151, 53-60. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 473-483. Liau G., Ong D. E. and Chytil F. (1981) Interaction of the retinol/cellular retinol-binding protein complex with isolated nuclei and nuclear components. J. cell, Biol. 91, 63-68. Liau G., Ong D. E. and Chytil F. (1985) Partial characterization of nuclear binding sites for retinol delivered by cellular retinol binding protein. Arch. biochem. Biophys. 237, 354-360. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Medvedev Zh. A., Buchanan J. H., Medvedeva M. N. and Crowne H. M. (1982) The characterization of non-historic proteins whose amounts increase in chromatin from mouse hepatocarcinomas. Int. J. Cancer 30, 87-92. Omori M. and Chytil F. (1982) Mechanism of vitamin A action: Gene expression in retinol-deficient rats. J. biol. Chem. 257, 14370-14374. Patyga J. (1984) Changes in non-histone chromatin proteins from liver of young and adult chickens following ~galactosamine administration. Comp. Biochem. Physiol. 79C, 389-393. Palyga J., Lubofi H. and Tyrawska-Spychalowa D. (1984) Changes in liver nuclear sap proteins during chick embryo development. Roux's Arch. Dev. Biol. 193, 57-59. Porter S. B., Ong D. E. and Chytil F. (1986) Vitamin A status affects chromatin structure. Int. J. Vitam. Nutr. Res. 56, 11-20. Rao M. R. S., Singh J. and Ganguly J. (1980) Effect of deprivation of vitamin A on the basic proteins of the nuclei of rat testes. Biochem. biophys. Res. Commun. 94, 1-8. Sherman D. R., Lloyd R. S. and Chytil F. (1987) Rat cellular retinol-binding protein: eDNA sequence and rapid retinol-dependent accumulation of mRNA. Proc. natn. acad. Sci. U.S.A. 84, 3209-3213. Sporn M. B. and Roberts A. B. (1983) Role of retinoids in differentiation and carcinogenesis. Cancer Res. 43, 3034-3040.
604
JAN PAEYGA et al.
Thakur M. K. (1984) Age-related changes in the structure and function ofchromatin: A review. Mech. Ageing Dev. 27, 263-286. Tsai C. H. and Chytil F. (1978) Effect of vitamin A
deficiency on RNA synthesis in isolataed rat liver nuclei. Life Sci. 23, 1461-1472. Zile M. H. and Cullum M. C. (1983) The function ofvitamin A: Current concepts. Proc. Soc. exp. Biol. Med. 172, 139-152.
0306-3623/89 $3.00+ 0.00 Copyright © 1989 PergamonPress plc
Printed in Great Britain.All rights reserved
EFFECT OF RETINYL ACETATE ON NUCLEAR PROTEINS IN RABBIT LIVER JAN PAEYGA,* KAROLINAGROCHAEA,ANDRZEJ KOWALSKI,MAEGORZATASYLWESTRZAK and J^N RAFAYI Department of Genetics, Educational University of Kielce, 25-518 Kielce, Rewolucji Pa~dziernikowej 33, Poland and IResearch Institute of Animal Production. Nitra, Czechoslovakia (Received 16 January 1989)
Abstract--l. Retinyl acetate injected intraperitoneally to adult rabbits fed on standard diet caused detectable changes in the polyacrylamide gel patterns of liver nucleoplasmic and 0.35 M NaCl-soluble chromatin proteins. 2. Both histones and non-histone proteins soluble in 5 M urea were not affected in vitamin A-treated animals. 3. It seems that variations in liver nuclear proteins from retinyl acetate-administered rabbits may reflect retinol-dependent alterations in structure and function of their chromatins.
INTRODUCTION
et al. (1988) have found that in vivo binding of retinol
It is well known (Zile and Cullum, 1983) that vitamin A is required not only for vision but also for reproduction and growth. The vitamin is essential for differentiation and maintenance of epithelial tissues (Sporn and Roberts, 1983). A lack of retinol results in lesions of skin cells (Chytil, 1984) and germinal epithelium of testis (Porter et al., 1986). The depressed levels of R N A synthesis in liver (Tsai and Chytil, 1978) and testis (Porter et al., 1986) nuclei of retinol-deprived rats were observed. The rate of RNA synthesis increased quickly within 4 hr after refeeding of deficient animals with retinyl acetate because of the increased ability of chromatin to serve as a template for polymerase II (Porter et al., 1986). Shortly after administration of retinyl acetate to retinol-deficient rats, an inhibited synthesis of approximately 280 proteins and an activated synthesis of about 100 proteins were detected among cytosolic proteins of rat testes using two-dimensional polyacrylamide gel electrophoresis (ul Haq and Chytil, 1988). Since retinoids were reported to influence gene expression (see Chytil, 1984 and 1986 for review), it is reasonable to assume that they should induce some changes in chromatin. It was shown (Chytil and Ong, 1979) that vitamin A is first bound to specific cellular retinol-binding protein (CRBP) which delivers it to binding sites in chromatin (Liau et al., 1981). Interestingly, a 4-fold increase in level of lung CRBP-specific m R N A was observed within 4 hr after refeeding of retinol-delicient rats (Sherman et al., 1987). As purified D N A or mono- and dinucleosomes did not accept retinol from its complex with CRBP, it was suggested (Liau et al., 1985) that a native higher order structure of chromatin and/or a presence of specific proteins lost during nuclease digestion of chromatin are required for binding of retinol. Recently, Ferrari
*To whom all correspondence should be addressed.
to chromatin is mediated by a lipoprotein associated with nucleosomes. They proposed that the binding sites on DNA are recognized by a protein component while retinol is solubilized in the fatty acid moiety. Introduction o f retinoi to cultured 3T3 cells induces an augmented sensitivity of their chromatin to DNase I digestion, an increase of acetate uptake and turnover on histones, and a disappearance of a 20-kDa chromatin proteins soluble in 0.35 M NaCI that are coextracted with the high mobility group proteins (Ferrari and Vidali, 1985). Thus, chromatin structure, content and metabolism o f chromosomal proteins may strongly depend on a cellular status of vitamin A. In this report we compared the polyacrylamide gel patterns of nuclear proteins, both non-histone proteins and histones, from liver of normal rabbits and experimental ones injected with high doses of retinyl acetate as a source of vitamin A. MATERIALS AND METHODS
New Zealand White rabbits weighing 2.2-2.5 kg fed on a standard chow were used. Animals were injected intraperitoneaUy with 800,000 tU retinyl acetate (Serva, Heidelberg, F.R.G.) a day for 2 consecutive days. On the third day, the experimental and control rabbits were sacrificed by cervical dislocation and bled. A liver perfusion was performed using a chilled solution containing 0.13 M NaCI, 0.008 M MgCI2, 0.005 M KCI and 0.1 mM phenylmethanesuifonyl fluoride (PMSF). The removed livers were quickly frozen in liquid nitrogen and kept at - 2 0 ° C until used. Nuclei were isolated by a slight modification of the method of Medvedev et al. (1982). Briefly, the tissue was homogenized in 50% glycerol in 0.01 M Tris-0.01 M KCI-0.0015M MgCI2-0.1mM PMSF containing 0.5% Triton X-100 at 0-4°C and pelleted at 3000 rpm for 10 min in Janetzki K-23 centrifuge. The nuclear pellet was washed three times with above solution containing decreasing concentrations of Triton X-100. Nuclear proteins were isolated by consecutive extraction of nuclei with: (a) 0.025 M ethylene diamine tetraacetic
601
602
JAN
PAEYGA
acid--O075 M NaCI, pH 7.6; (b) 0.35 M NaCI-0.02 M Tris-HCl, pH 7.6; 5 M urea-0.05 M sodium phosphate, pH 7.6 containing 0.02 M glycine and (d) 0.25 M HCI. All solutions used for extraction of proteins were supplemented with 0. I mM PMSF. Non-histone proteins were precipitated overnight at -20:'C by adding 2 vol of cold 99.8% ethanol containing 2% potassium acetate. Precipitates were first washed with cold absolute ethanol, then resuspended in small volumes of 0.005 M 2-mercaptoethanol and lyophilized. Histones were precipitated overnight with 20% trichloroacetic acid at 0 4 C by adding appropriate volume of 100% trichloroacetic acid. Precipitates were washed twice with acidified acetone (500cm 3 acetone plus 2 cm 3 concentrated HCI) and acetone. Protein samples were dissolved in 2% sodium dodecyl sulphate, 10% glycerol and 0.0625 M Tris-HC1, pH 6.8. Protein content was determined by the method of Lowry et al. (1951). After supplementing with 2-mercaptoethanol to final concentration of 5%, non-histone proteins and histones were separated in 10 and 12.5% polyacrylamide gels, respectively, containing 0. 1% sodium dodecyl sulphate (Laemmli, 1970).
tightly bound with chromatin and histones by using a series of solutions differing in their extraction properties and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The gel patterns of nucleoplasmic proteins from normal and retinol-treated rabbits were shown in Fig. la. Despite the uniformity in distribution and quantity of the majority of proteins, it was found that the intensity of several protein bands (28, 29, 35 and 76 kDa) were significantly altered in rabbits receiving high amounts of retinyl acetate. Similarly, the gel patterns of the 0.35 M NaCI-soluble chromatin proteins (Fig. l b) were also affected in retinol-treated rabbits. The amounts of proteins with molecular weights of approximately 27 and 31 kDa decreased in comparison with their levels in control animals. On the other hand, the content of 36 and 2 0 0 k D a proteins increased in chromatin of retinyl acetate exposed animals. In addition to those major changes, numerous faintly stained nucleoplasmic and chromatin protein bands were also affected in retinolinjected animals. As it can be seen in Figs lc and d, the gel patterns of 5 M urea-soluble chromatin proteins and histones, respectively, were practically identical in control and retinyl acetate-injected animals.
RESULTS AND DISCUSSION Nuclear proteins from rabbit liver were fractionated into nucleoplasmic proteins, proteins loosely and A A
et al.
B
A
A
B
B
B --
200
..
.,
:....:
76
H1
35
--
'i"
36
.L.i~.l~"
--31
..::/:
~29 .28 --
27
H3 H2B
H2A H4
.....
[a]
(b)
(c)
(d)
Fig. 1. Electrophoretic patterns of nucleoplasmic proteins (a), 0.35 M NaCl-soluble chromatin proteins (b), 5 M urea-soluble chromatin proteins (c) and histones (d) from liver of control (A) and retinyl acetate-injected rabbits (B). The numbers indicate the approximate molecular weights of protein bands in kilodalton. LSP--liver specific proteins (Medvedev et al., 1982).
Retinol and liver nuclear proteins Our results clearly indicate that intraperitoneal injection of retinyl acetate caused profound changes in the composition of nueleoplasmic proteins and proteins loosely bound with chromatin in rabbit liver. The protein fractions more tightly bound with D N A (5 M urea-soluble chromatin proteins and histones) did not change significantly following administration of vitamin A. An increasing body of evidence suggests (Sporn and Roberts, 1983; Chytil, 1984; Chytil, 1986) that retinol and its natural and synthetic derivatives can alter genomic expression. The rate of mRNA synthesis rapidly decreases in liver and testis nuclei of retinol-depleted rats, but replenishing the animals with retinol results in an increasing rate of RNA synthesis (Tsai and Chytil, 1978; Porter et al., 1986). It was observed (Omori and Chytil, 1982; ul Haq and Chytil, 1988) that animal genome rapidly responds to vitamin A compounds by suppression or activation the synthesis of specific proteins. Ferrari and Vidali (1985) have shown that retinol induces an augmented sensitivity of chromatin of cultured mouse 3T3 cells to DNase I digestion which is accompanied with a higher rate of acetate uptake and turnover on histones and a decrease in the level of a chromosomal protein of 20 kDa. Another chromatin protein, a testis-specific transitional protein (TP) which is synthesized temporarily at the midspermatid stage (Grimes, 1986), is markedly reduced in the testes of vitamin A-deficient rats (Rao et al., 1980). It is worth noting that retinol-mediated changes in 20 kDa protein of 3T3 cells (Ferrari and Vidali, 1985) and in TP (Rao et al., 1980) are reversible upon returning to normal status. Interestingly, retinoic acid-induced differentiation of retinoicacid-sensitive human leukemia (HL60) cells to mature-like granulocytes is accompanied with appearance of nucleosoma160-kDa protein (Chou et al., 1984). Thus, retinol and other retinoids can influence chromatin structure and function and its protein components. It appears that changes in nucleoplasmic and loosely bound chromatin proteins from rabbit liver following injection of retinyl acetate in our gel patterns may also reflect a modification of chromatin structure and function. It is well known (Thakur, 1984; Patyga, 1984; Palyga et al., 1984) that certain components of nuclear protein fractions undergo changes during cell differentiation and tissue development. Although retinol is believed to play a significant role in growth and differentiation (Sporn and Roberts, 1983), its effect on differentiation- or development-associated nuclear proteins is poorly explored (Rao et al., 1980). High doses of vitamin A induce toxic lesions in cell membranes (Zile and Cullum, 1983). This may lead to greater permeability of certain proteins from nucleus (e.g. those whose amounts decrease following treatment with retinyl acetate; Figs la and b) or to releasing of proteases from damaged lysosomes which may degrade some nuclear proteins. Moreover, retinol-dependent activation of endogenous chromatin proteases cannot be excluded. Our results indicate that vitamin A-mediated alterations in nuclear proteins have preferentially appeared among nucleoplasmic proteins and loosely bound chromatin proteins soluble in 0.35 M NaCI GP 20/~-~E
603
solution that undoubtedly contain components shuttling between cytoplasm and chromatin. It is very likely that prolonged action of retinyl acetate could lead to the alterations of certain chromatin proteins firmly bound with DNA.
REFERENCES
Chou R. H., Chervenick P. A. and Barch D. R. (1984) Appearance of a new nucleosomal protein during differentiation of human leukemia (HL-60) cells. Science 223, 1420-1423. Chytil F. (I 984) Retinoic acid: biochemistry, pharmacology, and therapeutic use. Pharmac. Rev. 36, 93S-100S. Chytil F. (1986) Retinoic acid: biochemistry and metabolism. J. Am. Acad. Dermatol. 15, 741-747. Chytil F. and Ong D. E. (1979) Cellular retinol- and retinoic acid-binding proteins in vitamin A action. Fedn Proc. Fedn Am. Socs exp. Biol. 38, 2510-2514. Ferrari N. and Vidali G. (1985) Effects of retinol on chromatin structure. Eur. J. Biochem. 151, 305-310. Ferrari N., Pfeffer U. and Vidali G. (1988) In vivo binding of retinol to chromatin. The binding is mediated by a lipoprotein. J. biol. Chem. 263, 448-453. Grimes S. R. (1986) Nuclear proteins in spermatogenesis. Comp. Biochem. Physiol. 83B, 495-500. ul Haq R. and Chytil F. (1988) Early effects of retinol and retinoic acid on protein synthesis in retinol deficient rat testes. Biochem. biophys, Res. Commun. 151, 53-60. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 473-483. Liau G., Ong D. E. and Chytil F. (1981) Interaction of the retinol/cellular retinol-binding protein complex with isolated nuclei and nuclear components. J. cell, Biol. 91, 63-68. Liau G., Ong D. E. and Chytil F. (1985) Partial characterization of nuclear binding sites for retinol delivered by cellular retinol binding protein. Arch. biochem. Biophys. 237, 354-360. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Medvedev Zh. A., Buchanan J. H., Medvedeva M. N. and Crowne H. M. (1982) The characterization of non-historic proteins whose amounts increase in chromatin from mouse hepatocarcinomas. Int. J. Cancer 30, 87-92. Omori M. and Chytil F. (1982) Mechanism of vitamin A action: Gene expression in retinol-deficient rats. J. biol. Chem. 257, 14370-14374. Patyga J. (1984) Changes in non-histone chromatin proteins from liver of young and adult chickens following ~galactosamine administration. Comp. Biochem. Physiol. 79C, 389-393. Palyga J., Lubofi H. and Tyrawska-Spychalowa D. (1984) Changes in liver nuclear sap proteins during chick embryo development. Roux's Arch. Dev. Biol. 193, 57-59. Porter S. B., Ong D. E. and Chytil F. (1986) Vitamin A status affects chromatin structure. Int. J. Vitam. Nutr. Res. 56, 11-20. Rao M. R. S., Singh J. and Ganguly J. (1980) Effect of deprivation of vitamin A on the basic proteins of the nuclei of rat testes. Biochem. biophys. Res. Commun. 94, 1-8. Sherman D. R., Lloyd R. S. and Chytil F. (1987) Rat cellular retinol-binding protein: eDNA sequence and rapid retinol-dependent accumulation of mRNA. Proc. natn. acad. Sci. U.S.A. 84, 3209-3213. Sporn M. B. and Roberts A. B. (1983) Role of retinoids in differentiation and carcinogenesis. Cancer Res. 43, 3034-3040.
604
JAN PAEYGA et al.
Thakur M. K. (1984) Age-related changes in the structure and function ofchromatin: A review. Mech. Ageing Dev. 27, 263-286. Tsai C. H. and Chytil F. (1978) Effect of vitamin A
deficiency on RNA synthesis in isolataed rat liver nuclei. Life Sci. 23, 1461-1472. Zile M. H. and Cullum M. C. (1983) The function ofvitamin A: Current concepts. Proc. Soc. exp. Biol. Med. 172, 139-152.