Veterinary Immunology and lmmunopathology, 33 ( 1992 ) 271-277 Elsevier Science Publishers B.V., Amsterdam
271
Induction of the 68 kDa major heat-shock protein in different Theileria annulata- and virustransformed bovine lymphoblastoid cell lines L u t z H e i n e a, D i e r k E. R e b e s k i b, W o l f g a n g L e i b o l d b, K a r l - T . F r i e d h o f f c a n d Eberhard Giintheff'l aAbteilung lmmungenetik der Universitdit, Gosslerstr. 12d, D-3400 G6ttingen, Germany blmmunologie der Tieriirztlichen Hochschule, Bischofsholer Damm 15, D-3000 Hannover, Germany ¢Institut fur Parasitologie der Tiergirztlichen Hochschule, Biinteweg 17, D-3000 Hannover 71, Germany (Accepted 21 October 1991 )
ABSTRACT Heine, L., Rebeski, D.E., Leibold, W., Friedhoff, K.-T. and Giinther, E., 1992. Induction of the 68 kDa major heat-shock protein in different Theileria annulata- and virus-transformed bovine lymphoblastoid cell lines. Vet. Immunol. Immunopathol., 33:271-277. Expression of the major inducible heat-shock protein of 68 kDa (hsp68) has been analyzed in peripheral blood mononuclear cells (PBMC) from cattle and in six Theileria annulata- and two bovine leukemia virus-transformed bovine lymphoblastoid cell lines (BoLCL). By metabolic labeling, hsp68 could be detected in PBMC and BoLCL only after heat-shock, but not under normal culture conditions. Immunoblot analysis with an hsp68 reactive monoclonal antibody similarly revealed a strong hsp68 response after heat-shock in BoLCL, and no hsp68 expression under normal culture conditions. Normally kept PBMC, however, were weakly positive with the antibody. The data are discussed with respect to the constitutive expression of hsp68 seen in several other cell lines.
ABBREVIATIONS BoLCL, bovine lymphoblastoid cell lines; DMEM, Dulbecco's modified Eagle medium; hsp68, heatshock protein of 68 kDa; hsp70, heat-shock protein of 70 kDa; hsps, heat-shock proteins; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulphate-polyacrylamidegel electrophoresis. Correspondence to: Dr. Eberhard Giinther, Abteilung Immungenetik der Universit~it, Gosslerstr. 12d, D-3400 G6ttingen, Germany.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00
272
L. HEINE ET AL.
INTRODUCTION
Heat-shock proteins (hsps) are a heterogeneous group of evolutionally highly conserved proteins that are induced by stress, e.g. elevated temperature. Heat-shock proteins of about 70 kDa (hsp70) are the most prominent family of these proteins and play an essential role in intracellular translocation, folding and renaturation of proteins (Morimoto et al., 1990). Infection with certain viruses, notably adenovirus, herpes virus and paramyxovirus has been shown to lead to hsp70 expression in host cells (Young, 1990). In the case of cells of the 293 line, the adenovirus-encoded E1A gene product has been identified as directly transactivating hsp70 genes (Nevins, 1982). Upon infection with suitable preparations of Theileria annulata sporozoites, bovine lymphoid cells can be transformed into immortal bovine lymphoblastoid cell lines (BoLCL) in vitro (Brown, 1981 ). In contrast to established lymphoblastoid cell lines from other species, Theileria DNA does not integrate into the cellular genome of BoLCL. The immortality rather depends on intracellular regulatory effects of Theileria schizonts situated in the cytoplasm (Dyer and Tait, 1987 ). We have analyzed the expression of the major heat-shock-inducible hsp of the hsp70 family, hsp68, in T. annulata as well as in bovine leukemia virus-transformed cell lines and in peripheral blood mononuclear cells (PBMC) from cattle. MATERIALS AND METHODS
Cel/s The BoLCL, Be-TA-2, Be-T4A-1, Bo-TA-2, Bo-T4A-1, No-T4A-1 and TheoTA-2 were transformed by infecting isolated PBMC derived from four healthy male cattle of German Black Pied strain and have been characterized as monocytic rather than lymphocytic cells as described elsewhere (Rebeski, 1990). The BoLCL, BL-3 (Romano et al., 1989) and SLB-H (Lange et al., 1983) have been derived from cattle infected with bovine leukemia virus and were kindly provided by Dr. H. Lewin, Urbana, IL, USA, and Dr. O.R. Kaaden, Hannover, Germany, respectively. Culture conditions The cell lines were kept in Dulbecco's modified Eagle medium (DMEM, Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (Biochrom) at 37°C in a 10% CO2 atmosphere. Peripheral blood mononuclear cells were prepared from heparinized blood by ficoll-hypaque density gradient centrifugation and used for heat-shock experiments without further culture. Transformed cells and PBMC were heat-shocked by culture in the
HSP68 EXPRESSION 1N BOVINE CELL LINES
273
above medium (10 ml, density 106 m1-1 ) using parafilm-sealed polystyrol petri dishes (9.4 cm diameter, Greiner, Niirtingen, Germany) in a water bath of 41.8 °C for 5 h. For the last hour the medium was replaced by methioninefree DMEM containing 10 pCi ( 370 kBq) m l - 1of 35S_methionine (spec. act. 1000 Ci mmol -I = 37 TBq mmol -~, Amersham Buchler, Braunschweig, Germany). Cells were then washed three times in phosphate-buffered saline (PBS) and lysed in 10 mM Tris-HC1 (pH 7.4)/0.1 mM EDTA by Dounce homogenization.
Analysis of hsp68 expression The supernatant obtained after 650 × g centrifugation for 20 min at 4°C was used for analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); 40 #g of protein (Bradford, 1976 ) were run per lane of 10% acrylamide gels (Laemmli, 1970) and blotted (Towbin et al., 1979) onto nitrocellulose (Schleicher and Schfill, Dassel, Germany) as described (Heine et al., 1991 ). Blots were washed with PBS/0.05% Tween 20 and incubated for 2 h with monoclonal antibody 8F7 supernatant which binds selectively to hsp68 and cross-reacts with the non-heat-shock protein p48 (Heine et al., 1991 ). After two washes, blots were incubated with rabbit anti-rat antiserum (Dakopatts, Hamburg, Germany) and peroxidase-coupled swine antirabbit antiserum (Dakopatts) for 1 h each. The substrate consisted of 25 mg of diaminobenzidine (Sigma, Deisenhofen, Germany) and 50 pl H202 dissolved in 50 ml of PBS. Autoradiographs were obtained from dried immunoblots. RESULTS AND DISCUSSION
Autoradiographic analysis of bovine PBMC reveals two bands in the 70 kDa range after heat-shock, corresponding to hsp68 and hsp70, respectively (Fig. 1 ). Monoclonal antibody 8F7 reacts only with the lower band, i.e. hsp68, as expected from our previous data (Heine et al., 1991 ). Peripheral blood mononuclear ceils (PBMC) kept at 37 °C show a weak antibody staining of hsp68, which is considerably enhanced after heat-shock (Fig. 1 ). Accumulation of hsp68 in non-heat-shocked PBMC is also seen in humans and other species (Heine et al., 1991 ), and does not reflect newly synthesized hsp68 since it is detected only by immunoblot, but not by metabolic labeling analysis (Fig. 1 ). The expression pattern ofhsp68 in the six T. annulata-transformed BoLCL tested was uniform: hsp68 was not detectable under normal culture conditions and became strongly induced after heat-shock. This is demonstrated in detail for cell line Theo-TA-2 in Fig. 2, where the hsp68 band is radioactively labeled and antibody-stained only in heat-shocked cells, and by immunoblot
274 1
L. HEINE ET AL.
2
3
4
Fig. 1. Expressionof hsp68 in bovine PBMC. Peripheral blood mononuclear cells were either kept at 37°C (lanes 1,3) or heat-shocked (lanes 2,4) and analyzedby autoradiography (lanes 1,2) and immunoblot (lanes 3,4). Positions of hsp70 and hsp68 are indicated. analysis for three further cell lines in Fig. 2. It is noteworthy that in contrast to PBMC, the BoLCL were negative for hsp68 under normal culture conditions (see Fig. 1 ). Each of the BoLCL showed a clear reaction of the antibody with p48, which is a heat-shock-independent protein. This protein is observed also in cell lines from other species and becomes detectable in h u m a n PBMC after mitogen stimulation; it thus might be proliferation-associated (Heine et al., 1991 ). Line No-T4A-1 is conspicuous because a further band of about 30 kDa (p30) is detected by monoclonal antibody 8F7 (Fig. 2 ). This type of cross-reactivity has been observed in other species only with red blood ceils (L. Heine et al., unpublished data, 1990). It may be speculated that the No-T4A-1 line is derived from the red blood cell lineage, considering the report of the transformation of red blood cells by T. sergenti (Yagi et al., 1989), or that T. annulata
HSP68EXPRESSIONIN BOVINECELLLINES
1
hsp~9= hsp~
p48-
2
3
4
5
275
6
7
8
9
10
~ -hsp68
1-
~-~
-p48
-p30
Fig. 2. Expression ofhsp68 in T. annulata-transformed bovine cell lines. Lines Theo-TA-2 (lanes 1-4), Bo-TA-2 (lanes 5,6), Be-T4A-1 (lanes 7,8) and No-T4A-1 (lanes 9,10) were analyzed by autoradiography (lanes 1,2 ) and immunoblot (lanes 3-10) before (lanes 1,3,5,7,9 ) or after (lanes 2,4, 6, 8,10) heat-shock. Positions of hsp70, hsp68, p48 and p30 are indicated.
transformation has led to the activation of genes otherwise expressed in the red blood cell lineage. It could be argued that the proteins detected by metabolic labeling and antibody are synthesized by the Theileria schizonts, but the fact that the parasites' contribution to the cellular protein is only about 1% makes this possibility very unlikely. The two virus-transformed BoLCL, which were studied for hsp68 expression, gave results similar to the Theileria-transformed lines. They were hsp68negative under normal culture conditions and expressed hsp68 after heatshock treatment, as is shown in Fig. 3 by immunoblot analysis. Our results demonstrate that T. annulata- and bovine leukemia virus-transformed cell lines of bovine origin do not express hsp68 under normal culture conditions. These bovine cell lines behave differently from many human cell lines which express hsp68 when cultured at 37°C, e.g. 293 cells (Nevins, 1982). HeLa or the Burkitt lymphoma lines BJAB, Daudi and Raji (Heine et al., 1991 ). The question of whether intracellular parasites induce hsp expression in host cells has been studied here for hsp68 in established BoLCL which either carry T. annulata schizonts or bovine leukemia virus. The intracellular para-
276
L. HEINE ET AL.
~i!~!~qi~ ¸
-hsp68
-p48
Fig. 3. Expression of hsp68 in virus-transformed bovine cell lines. Lines BL-3 (lanes 1,2 ) and SBL-H (lanes 3,4) were analyzed by immunoblot before (lanes 1,3) and after (lanes 2,4) heatshock. Positions ofhsp68 and p48 are indicated.
sites failed to induce a constitutive expression of hsp68, which could be due to a direct effect on hsp genes (Nevins, 1982), or could occur as a consequence of damage to intracellular proteins which in turn could lead to hsp expression. The fact that the BoLCL studied here are hsp68-negative and show a clear hsp68-response after heat-shock could render them a useful model of permanently available cells for studying the regulation of hsp68 expression.
REFERENCES Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Brown, C.G.D., 1981. Application of in vitro techniques to vaccination against theileriosis. In:
HSP68 EXPRESSION IN BOVINE CELL LINES
277
A.D. Irvin, M.P. Cunningham and A.S. Young (Editors), Advances in the Control of Theileriosis. Martinus Nijhoff, The Hague, pp. 104-119. Dyer, M. and Tait, A., 1987. Control of lymphoproliferation by Theileria annulata. Parasitol. Today, 3:309-311. Heine, L., Drabent, B., Benecke, B.-J. and GiJnther, E., 1991. A novel monoclonal antibody directed against the heat-inducible 68 kDa heat shock protein. Hybridoma, 10:721-730. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Lange, S., Frenzel, B., Kaaden, O.R., Marschall, H.J. and Moenning, V., 1983. Establishment of a cell line from bovine skin leukosis tumor and detection of retrovirus activities. Zentralbl. Veterinaermed. Reihe B, 30:371-381. Morimoto, R., Tissieres, A. and Georgopoulos, C. (Editors), 1990. Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 1-450. Nevins, J.E., 1982. Induction of the synthesis of a 70.000 dalton mammalian heat shock protein by the adenovirus E 1A gene product. Cell, 29:913-919. Rebeski, D.E., 1990. Charakterisierung theilerientransformierter (Theileria annulata) Subpopulationen lymphoider Rinderzellen. Thesis, Veterinary School, Hannover, Germany. Romano, M.J., Stuart, J.A. and Lewin, H.A., 1989. Phenotypic characterization of bovine lymphoblastoid cell lines. Vet. Immunol. Immunpathol., 23: 293-307. Towbin, H., Staehlin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Yagi, Y., Furuuchi, S., Takahashi, H. and Koyama, H., 1989. Abnormality of osmotic fragility and morphological disorder of bovine erythrocytes infected with Theileria sergenti. Jap. J. Vet. Sci., 51: 389-395. Young, R.A., 1990. Stress proteins and immunology. Annu. Rev. Immunol., 8:401-420.
271
Induction of the 68 kDa major heat-shock protein in different Theileria annulata- and virustransformed bovine lymphoblastoid cell lines L u t z H e i n e a, D i e r k E. R e b e s k i b, W o l f g a n g L e i b o l d b, K a r l - T . F r i e d h o f f c a n d Eberhard Giintheff'l aAbteilung lmmungenetik der Universitdit, Gosslerstr. 12d, D-3400 G6ttingen, Germany blmmunologie der Tieriirztlichen Hochschule, Bischofsholer Damm 15, D-3000 Hannover, Germany ¢Institut fur Parasitologie der Tiergirztlichen Hochschule, Biinteweg 17, D-3000 Hannover 71, Germany (Accepted 21 October 1991 )
ABSTRACT Heine, L., Rebeski, D.E., Leibold, W., Friedhoff, K.-T. and Giinther, E., 1992. Induction of the 68 kDa major heat-shock protein in different Theileria annulata- and virus-transformed bovine lymphoblastoid cell lines. Vet. Immunol. Immunopathol., 33:271-277. Expression of the major inducible heat-shock protein of 68 kDa (hsp68) has been analyzed in peripheral blood mononuclear cells (PBMC) from cattle and in six Theileria annulata- and two bovine leukemia virus-transformed bovine lymphoblastoid cell lines (BoLCL). By metabolic labeling, hsp68 could be detected in PBMC and BoLCL only after heat-shock, but not under normal culture conditions. Immunoblot analysis with an hsp68 reactive monoclonal antibody similarly revealed a strong hsp68 response after heat-shock in BoLCL, and no hsp68 expression under normal culture conditions. Normally kept PBMC, however, were weakly positive with the antibody. The data are discussed with respect to the constitutive expression of hsp68 seen in several other cell lines.
ABBREVIATIONS BoLCL, bovine lymphoblastoid cell lines; DMEM, Dulbecco's modified Eagle medium; hsp68, heatshock protein of 68 kDa; hsp70, heat-shock protein of 70 kDa; hsps, heat-shock proteins; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulphate-polyacrylamidegel electrophoresis. Correspondence to: Dr. Eberhard Giinther, Abteilung Immungenetik der Universit~it, Gosslerstr. 12d, D-3400 G6ttingen, Germany.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00
272
L. HEINE ET AL.
INTRODUCTION
Heat-shock proteins (hsps) are a heterogeneous group of evolutionally highly conserved proteins that are induced by stress, e.g. elevated temperature. Heat-shock proteins of about 70 kDa (hsp70) are the most prominent family of these proteins and play an essential role in intracellular translocation, folding and renaturation of proteins (Morimoto et al., 1990). Infection with certain viruses, notably adenovirus, herpes virus and paramyxovirus has been shown to lead to hsp70 expression in host cells (Young, 1990). In the case of cells of the 293 line, the adenovirus-encoded E1A gene product has been identified as directly transactivating hsp70 genes (Nevins, 1982). Upon infection with suitable preparations of Theileria annulata sporozoites, bovine lymphoid cells can be transformed into immortal bovine lymphoblastoid cell lines (BoLCL) in vitro (Brown, 1981 ). In contrast to established lymphoblastoid cell lines from other species, Theileria DNA does not integrate into the cellular genome of BoLCL. The immortality rather depends on intracellular regulatory effects of Theileria schizonts situated in the cytoplasm (Dyer and Tait, 1987 ). We have analyzed the expression of the major heat-shock-inducible hsp of the hsp70 family, hsp68, in T. annulata as well as in bovine leukemia virus-transformed cell lines and in peripheral blood mononuclear cells (PBMC) from cattle. MATERIALS AND METHODS
Cel/s The BoLCL, Be-TA-2, Be-T4A-1, Bo-TA-2, Bo-T4A-1, No-T4A-1 and TheoTA-2 were transformed by infecting isolated PBMC derived from four healthy male cattle of German Black Pied strain and have been characterized as monocytic rather than lymphocytic cells as described elsewhere (Rebeski, 1990). The BoLCL, BL-3 (Romano et al., 1989) and SLB-H (Lange et al., 1983) have been derived from cattle infected with bovine leukemia virus and were kindly provided by Dr. H. Lewin, Urbana, IL, USA, and Dr. O.R. Kaaden, Hannover, Germany, respectively. Culture conditions The cell lines were kept in Dulbecco's modified Eagle medium (DMEM, Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (Biochrom) at 37°C in a 10% CO2 atmosphere. Peripheral blood mononuclear cells were prepared from heparinized blood by ficoll-hypaque density gradient centrifugation and used for heat-shock experiments without further culture. Transformed cells and PBMC were heat-shocked by culture in the
HSP68 EXPRESSION 1N BOVINE CELL LINES
273
above medium (10 ml, density 106 m1-1 ) using parafilm-sealed polystyrol petri dishes (9.4 cm diameter, Greiner, Niirtingen, Germany) in a water bath of 41.8 °C for 5 h. For the last hour the medium was replaced by methioninefree DMEM containing 10 pCi ( 370 kBq) m l - 1of 35S_methionine (spec. act. 1000 Ci mmol -I = 37 TBq mmol -~, Amersham Buchler, Braunschweig, Germany). Cells were then washed three times in phosphate-buffered saline (PBS) and lysed in 10 mM Tris-HC1 (pH 7.4)/0.1 mM EDTA by Dounce homogenization.
Analysis of hsp68 expression The supernatant obtained after 650 × g centrifugation for 20 min at 4°C was used for analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); 40 #g of protein (Bradford, 1976 ) were run per lane of 10% acrylamide gels (Laemmli, 1970) and blotted (Towbin et al., 1979) onto nitrocellulose (Schleicher and Schfill, Dassel, Germany) as described (Heine et al., 1991 ). Blots were washed with PBS/0.05% Tween 20 and incubated for 2 h with monoclonal antibody 8F7 supernatant which binds selectively to hsp68 and cross-reacts with the non-heat-shock protein p48 (Heine et al., 1991 ). After two washes, blots were incubated with rabbit anti-rat antiserum (Dakopatts, Hamburg, Germany) and peroxidase-coupled swine antirabbit antiserum (Dakopatts) for 1 h each. The substrate consisted of 25 mg of diaminobenzidine (Sigma, Deisenhofen, Germany) and 50 pl H202 dissolved in 50 ml of PBS. Autoradiographs were obtained from dried immunoblots. RESULTS AND DISCUSSION
Autoradiographic analysis of bovine PBMC reveals two bands in the 70 kDa range after heat-shock, corresponding to hsp68 and hsp70, respectively (Fig. 1 ). Monoclonal antibody 8F7 reacts only with the lower band, i.e. hsp68, as expected from our previous data (Heine et al., 1991 ). Peripheral blood mononuclear ceils (PBMC) kept at 37 °C show a weak antibody staining of hsp68, which is considerably enhanced after heat-shock (Fig. 1 ). Accumulation of hsp68 in non-heat-shocked PBMC is also seen in humans and other species (Heine et al., 1991 ), and does not reflect newly synthesized hsp68 since it is detected only by immunoblot, but not by metabolic labeling analysis (Fig. 1 ). The expression pattern ofhsp68 in the six T. annulata-transformed BoLCL tested was uniform: hsp68 was not detectable under normal culture conditions and became strongly induced after heat-shock. This is demonstrated in detail for cell line Theo-TA-2 in Fig. 2, where the hsp68 band is radioactively labeled and antibody-stained only in heat-shocked cells, and by immunoblot
274 1
L. HEINE ET AL.
2
3
4
Fig. 1. Expressionof hsp68 in bovine PBMC. Peripheral blood mononuclear cells were either kept at 37°C (lanes 1,3) or heat-shocked (lanes 2,4) and analyzedby autoradiography (lanes 1,2) and immunoblot (lanes 3,4). Positions of hsp70 and hsp68 are indicated. analysis for three further cell lines in Fig. 2. It is noteworthy that in contrast to PBMC, the BoLCL were negative for hsp68 under normal culture conditions (see Fig. 1 ). Each of the BoLCL showed a clear reaction of the antibody with p48, which is a heat-shock-independent protein. This protein is observed also in cell lines from other species and becomes detectable in h u m a n PBMC after mitogen stimulation; it thus might be proliferation-associated (Heine et al., 1991 ). Line No-T4A-1 is conspicuous because a further band of about 30 kDa (p30) is detected by monoclonal antibody 8F7 (Fig. 2 ). This type of cross-reactivity has been observed in other species only with red blood ceils (L. Heine et al., unpublished data, 1990). It may be speculated that the No-T4A-1 line is derived from the red blood cell lineage, considering the report of the transformation of red blood cells by T. sergenti (Yagi et al., 1989), or that T. annulata
HSP68EXPRESSIONIN BOVINECELLLINES
1
hsp~9= hsp~
p48-
2
3
4
5
275
6
7
8
9
10
~ -hsp68
1-
~-~
-p48
-p30
Fig. 2. Expression ofhsp68 in T. annulata-transformed bovine cell lines. Lines Theo-TA-2 (lanes 1-4), Bo-TA-2 (lanes 5,6), Be-T4A-1 (lanes 7,8) and No-T4A-1 (lanes 9,10) were analyzed by autoradiography (lanes 1,2 ) and immunoblot (lanes 3-10) before (lanes 1,3,5,7,9 ) or after (lanes 2,4, 6, 8,10) heat-shock. Positions of hsp70, hsp68, p48 and p30 are indicated.
transformation has led to the activation of genes otherwise expressed in the red blood cell lineage. It could be argued that the proteins detected by metabolic labeling and antibody are synthesized by the Theileria schizonts, but the fact that the parasites' contribution to the cellular protein is only about 1% makes this possibility very unlikely. The two virus-transformed BoLCL, which were studied for hsp68 expression, gave results similar to the Theileria-transformed lines. They were hsp68negative under normal culture conditions and expressed hsp68 after heatshock treatment, as is shown in Fig. 3 by immunoblot analysis. Our results demonstrate that T. annulata- and bovine leukemia virus-transformed cell lines of bovine origin do not express hsp68 under normal culture conditions. These bovine cell lines behave differently from many human cell lines which express hsp68 when cultured at 37°C, e.g. 293 cells (Nevins, 1982). HeLa or the Burkitt lymphoma lines BJAB, Daudi and Raji (Heine et al., 1991 ). The question of whether intracellular parasites induce hsp expression in host cells has been studied here for hsp68 in established BoLCL which either carry T. annulata schizonts or bovine leukemia virus. The intracellular para-
276
L. HEINE ET AL.
~i!~!~qi~ ¸
-hsp68
-p48
Fig. 3. Expression of hsp68 in virus-transformed bovine cell lines. Lines BL-3 (lanes 1,2 ) and SBL-H (lanes 3,4) were analyzed by immunoblot before (lanes 1,3) and after (lanes 2,4) heatshock. Positions ofhsp68 and p48 are indicated.
sites failed to induce a constitutive expression of hsp68, which could be due to a direct effect on hsp genes (Nevins, 1982), or could occur as a consequence of damage to intracellular proteins which in turn could lead to hsp expression. The fact that the BoLCL studied here are hsp68-negative and show a clear hsp68-response after heat-shock could render them a useful model of permanently available cells for studying the regulation of hsp68 expression.
REFERENCES Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Brown, C.G.D., 1981. Application of in vitro techniques to vaccination against theileriosis. In:
HSP68 EXPRESSION IN BOVINE CELL LINES
277
A.D. Irvin, M.P. Cunningham and A.S. Young (Editors), Advances in the Control of Theileriosis. Martinus Nijhoff, The Hague, pp. 104-119. Dyer, M. and Tait, A., 1987. Control of lymphoproliferation by Theileria annulata. Parasitol. Today, 3:309-311. Heine, L., Drabent, B., Benecke, B.-J. and GiJnther, E., 1991. A novel monoclonal antibody directed against the heat-inducible 68 kDa heat shock protein. Hybridoma, 10:721-730. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Lange, S., Frenzel, B., Kaaden, O.R., Marschall, H.J. and Moenning, V., 1983. Establishment of a cell line from bovine skin leukosis tumor and detection of retrovirus activities. Zentralbl. Veterinaermed. Reihe B, 30:371-381. Morimoto, R., Tissieres, A. and Georgopoulos, C. (Editors), 1990. Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 1-450. Nevins, J.E., 1982. Induction of the synthesis of a 70.000 dalton mammalian heat shock protein by the adenovirus E 1A gene product. Cell, 29:913-919. Rebeski, D.E., 1990. Charakterisierung theilerientransformierter (Theileria annulata) Subpopulationen lymphoider Rinderzellen. Thesis, Veterinary School, Hannover, Germany. Romano, M.J., Stuart, J.A. and Lewin, H.A., 1989. Phenotypic characterization of bovine lymphoblastoid cell lines. Vet. Immunol. Immunpathol., 23: 293-307. Towbin, H., Staehlin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Yagi, Y., Furuuchi, S., Takahashi, H. and Koyama, H., 1989. Abnormality of osmotic fragility and morphological disorder of bovine erythrocytes infected with Theileria sergenti. Jap. J. Vet. Sci., 51: 389-395. Young, R.A., 1990. Stress proteins and immunology. Annu. Rev. Immunol., 8:401-420.
