- Email: [email protected]
streptomycin
The International Journal of Biochemistry & Cell Biology 31 (1999) 861±868 www.elsevier.com/locate/ijbcb
A comprehensive analysis of heat shock protein synthesis in human peripheral lymphocytes: the eect of penicillin/ streptomycin D. Visala Rao, Graham L. Jones, Kenneth Watson* Human Biology, School of Biological Sciences, University of New England, Armidale, NSW 2351, Australia Received 8 March 1999; received in revised form 19 April 1999; accepted 21 April 1999
Abstract A reliable experimental procedure is described for the simultaneous characterisation of a comprehensive range of heat shock proteins (hsps) in human peripheral lymphocytes. In this system, a mild heat shock from 37 to 428C for 1 h induced the synthesis of hsps 105, 90, 70, 60, 57, 47, 40, 27 and 16. Densitometric analyses of 35[S]-methionine labelled protein gels indicated that levels of these hsps peaked at 3 to 4 h, following post-heat shock recovery at 378C. The presence of penicillin and streptomycin in the cell culture medium, appeared to have little eect on the kinetics of hsp synthesis. The present method can be used for relatively small blood samples and its relative ease of application and reproducibility make it appropriate for screening the expression of hsps in human lymphocytes from a range of individuals. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Heat shock proteins; Human lymphocytes; Antibiotics; Penicillin/streptomycin
1. Introduction Almost all organisms from bacteria to humans respond to a mild heat shock by inducing the synthesis of a set of highly conserved proteins, termed the heat shock proteins (hsps) [1]. The seminal work of Ritossa [2] on heat shock induced pung in the polytene chromosomes of
* Corresponding author. Tel.: +61-2-6773-3125; fax: +612-6773-3267. E-mail address: [email protected] (K. Watson)
Drosophila busckii, led towards later research on the expression and characterisation of hsps. Hsps are not only induced in response to various environmental stressors such as hyperthermia, transition heavy metals, amino acid analogues and chemotherapeutic agents [1,3,4], but are also associated with several pathological conditions including autoimmune diseases and age-related disorders [5±8]. The role of hsps in the immune response to cancer and in antigen presentation has also been recently demonstrated [9,10]. Furthermore, it has been shown that heatinduced apoptosis in transfected human cell lines
1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 3 7 - 0
862
D.V. Rao et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 861±868
is blocked by constitutively elevated or stress inducible levels of hsp70 [11,12]. The heat shock response is a well documented cellular phenomenon in which prior exposure to a mild heat shock induces a transient resistance to a subsequent, normally lethal, heat stress. Heat shock induced thermotolerance, which is believed to be facilitated by hsps, is a characteristic feature of the heat shock response [1,3]. Many hsps are expressed constitutively in cells under normal physiological conditions with synthesis enhanced after stress while others are produced only under stress conditions. In the unstressed cell, many hsps perform essential functions. These include protein folding and assembly and the targeting of proteins to sub-cellular organelles. The term molecular chaperone has, thus, been applied to this class of hsps [13,14]. Hsps are also involved in the degradation of damaged proteins [15]. Of all the hsps, the hsp 70 family has been the most widely studied and well characterised due to its critical and protective role as a molecular chaperone system. In humans, most of the hsp 70 studies have been at the transcriptional level and these include the use of ®broblasts [16,17] and lymphocytes for studies on hsp expression in relation to changes with age and activation by mitogens [18±21]. On the other hand, there have been relatively few studies on hsp expression at the translational level, including hsp 70, in human lymphocytes. Some ®ndings suggest that even though hsp 70 expression was detectable at the transcriptional level, it was decreased at the translational level [22]. We have chosen human lymphocytes as our experimental model for studies on hsp expression for a number of reasons. Lymphocytes are involved in the production of antibodies and are an integral part of the body's defence system. Furthermore, hsps are known to be associated with human disorders linked with the immune system [7]. In this study our major aim was to develop a sensitive and reliable method, using a single venous blood sample, to comprehensively and simultaneously examine the expression of a large number of hsps at the translational level. In ad-
dition, we examined the possible eects of penicillin and streptomycin, the most widely used antibiotics in mammalian cell culture studies, on hsp expression in human peripheral lymphocytes since hsp expression can be aected by a wide variety of stressors. 2. Materials and methods 2.1. Study population and blood sample collection Young adults, aged between 16±29 participating in the present study, were known to be healthy, not to have taken any medication for two weeks prior to blood donation and also not to have undertaken vigorous exercise prior to donating blood samples. Peripheral blood (18±20 ml) was collected by venipuncture into acid citrate dextrose-coated vacutainers (Becton and Dickinson, Sydney, Australia). 2.2. Isolation of lymphocytes Blood samples were diluted with equal volumes of 1 Hank's balanced salt solution (HBSS) (Gibco-BRL, Melbourne, Australia) and layered carefully onto Ficoll-Paque (Pharmacia Biotech, Sydney) and centrifuged at 400 g for 20 min, in a temperature controlled centrifuge (18±208C). Lymphocytes were washed by suspending in 3±4 volumes of 1 HBSS and centrifuging at 100 g for 10 min at 18±208C. An average of 2 107 cells were obtained from each 18±20 ml blood sample. The viability of the lymphocytes, as determined by trypan blue exclusion, was 90 2 5%. 2.3. Cell culture conditions Lymphocytes were suspended in 6 ml RPM1 1640 (methionine-free) medium (Gibco BRL) supplemented with 10% heat-inactivated foetal calf serum (CSL, Melbourne). An aliquot of 500 ml cell suspension was placed in each well of 12well ¯at-bottomed tissue culture plates. Antibiotic stock solution (CSL, Melbourne) consisting of penicillin (100 units/ml) and streptomy-
D.V. Rao et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 861±868
cin (100 mg/ml) was added to one half of the sample wells and equal amounts of RPM1 1640 added to the other half. One plate with 3 wells containing lymphocyte cell culture with antibiotics and 3 wells without antibiotics was designated for control samples and another plate (6 wells) for heat shock samples. The cells were equilibrated in an incubator at 378C under 5% CO2 and 95% humidity for 1 h. Heat shock treatment was performed for 1 h by placing the plate, designated for heat shock treatment, in a dry block heater, that was previously set to 428C and situated in the same incubator. Temperatures were monitored both in the dry block heater and in the incubator. 35[S]-methionine (100 mCi/ml) was added to both control and heat shocked cells 2 h prior to harvesting. Following initial experiments [23] where lymphocytes were left to recover after heat shock treatment for various recovery periods up to 24 h, it was decided to observe the kinetics of hsp expression in a 2 to 4 h recovery window. 2.4. Protein extraction Cells were harvested by washing the wells thoroughly with warm (378C) RPMI medium and collecting into sterile Eppendorf tubes. Lymphocyte pellets were obtained by centrifuging at 13,000 rpm for 5 min in a microcentrifuge. The pellets were washed twice with 1 HBSS and lysed in ice-cold lysis buer (0.1% Triton X100, 100 mM KCl, 8 mM MgCl2, 150 mM NaCl, 20 mM Tris±HCl, pH 7.4 and 1 mM phenylmethyl sulfonyl ¯uoride). The lysed cells were centrifuged and the supernatant containing proteins from the cytoplasmic fraction was recovered. Protein extractions were carried out at 4± 68C to minimise proteolysis and then stored at ÿ708C until needed. Protein concentrations were determined according to a modi®ed Bradford Coomassie dye binding colorimetric method for total protein concentration (Pierce, Rockford, IL). Equal volumes of 3 SDS-gel loading buer (150 mM Tris±HCl, pH 6.8, 300 mM dithiothreitol, 6% SDS, 0.3% bromophenol blue, 30% glycerol) was added and samples boiled for 3 min to denature the proteins.
863
2.5. Analysis of protein synthesis 1D PAGE was performed using the standard procedures of Laemmli [24]. In brief, 10% SDSacrylamide gels were prepared for resolving the proteins with a 4% stacking gel. Protein standards and equal amounts of protein (10 mg) from each sample were boiled for 3 min and loaded using a 100 ml Hamilton syringe. Gels were run at 30 mA and 108C for about 4 h until the dye migrated to the bottom of the gel. Gels were silver stained (Bio-Rad silver stain kit), dried, exposed to X-ray ®lm (Kodak Biomax) at ÿ708C for 7±10 days and developed. The proteins were identi®ed according to their molecular weights by comparing their migration with that of standards of known molecular weight (Bio-Rad). 35 [S]-methionine labelled protein autoradiographs were scanned using a Phoretix 1D-densitometry software package, Ver. 3.0 (Phoretix International, Newcastle, UK) and a gel documentation system (GDS7600, UV-products, Cambridge, UK). Peak heights of relevant bands and actin (43 kDa) were recorded. Quanti®cation of preferentially synthesised hsp bands was achieved by calculating the ratio of respective band intensities (peak heights) to that of actin, a non-heat shock inducible protein [25]. Relative increase in protein synthesis was expressed as the ratio of normalized (with respect to actin) pixel intensity of hsp after heat shock to the intensity of hsp in the respective non-heat shock control. 2.6. Immunoblotting Following 1D-PAGE, the resolved proteins were transferred [26] to Hybond C-super nitrocellulose membranes (Amersham, Sydney) using a semi-dry Novablot system (Pharmacia). The membrane strips were blocked in PBS containing 0.05% Tween 20 (PBST) and 5% skim milk powder overnight at 48C. The membranes were washed in PBST and incubated with gentle shaking for 1 h with speci®c primary antibodies. Primary antibodies used in this study were: antihsp 105 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-hsp 90, 70, 60 and 40 (Stressgen, Victoria, Canada) and the appropriate dilutions
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Fig. 1. Silver stained gel of human heat shock proteins in peripheral lymphocytes. Heat shock treatment was performed at 428C for 1 h and cells allowed to recover at 378C for 2, 3 or 4 h. Equal amounts (10 mg) of protein extracted from one individual's blood sample were loaded on to 10% SDS-gel. Lanes 1, 4 and 6: controls without antibiotics; lanes 2, 5 and 7: 2, 3 and 4 h recovery after heat shock without antibiotics; lanes 9, 11 and 13: controls with antibiotics; lanes 10, 12 and 14: 2, 3 and 4 h recovery after heat shock with antibiotics; lanes 3 and 8: molecular weight standards. Positions where hsps that are up-regulated as well as actin (43 kDa) are denoted on the right and down-regulated proteins are shown by arrows without numbers (see Fig. 2 for the corresponding autoradiograph). Molecular weight standards (kDa) are shown on the left.
were determined according to manufacturers' instructions. The proteins were probed with appropriate horseradish peroxidase-conjugated secondary antibodies and subsequently detected using an enhanced chemiluminescence detection system (Amersham).
heat shocked samples, with and without antibiotics in the culture medium. Dierences were considered to be signi®cant for values of p < 0.05 and considered to be highly signi®cant for p < 0.01.
2.7. Statistical analysis
3. Results and discussion
Statistical analyses were conducted using paired Student's t-tests for comparing protein synthesis after heat shock and in control non-
The electrophoretic separation of total proteins from control and heat shock lymphocytes, in the presence or absence of antibiotics, is shown in
D.V. Rao et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 861±868
865
Fig. 2. Autoradiograph of 35[S]-labelled heat shock protein expression. Lanes 1, 3 and 5: controls after 2, 3 and 4 h recovery without antibiotics; lanes 2, 4 and 6: 2, 3 and 4 h recovery after heat shock without antibiotics; lanes 7, 9 and 11: controls after 2, 3 and 4 h recovery with antibiotics; lanes 8, 10 and 12: 2, 3 and 4 h recovery after heat shock with antibiotics. Hsps that are up-regulated as well as the 43 kDa actin are denoted on the right and down-regulated proteins are shown by arrows without numbers. Molecular weight standards (kDa) are shown on the left.
Fig. 1 and 2. Fig. 1 shows a silver nitrate-stained gel and Fig. 2 shows an autoradiogram of the same gel. Although in the silver nitrate-stained gel the induction of heat shock proteins is not clearly observed, the gel does illustrate the success of our procedure in the extraction and separation of human lymphocyte proteins. Moreover, it serves as a control for the equal loading of protein in each sample well. On the other hand, the 35[S]-methionine labelling clearly illustrates the de novo synthesis of a
large number of heat shock proteins (Fig. 2). Obviously, proteins with a high rate of synthesis show a more intense band. In particular, upregulation following a 1 h heat shock at 428C, is evident for hsps 105, 90 and 70. In addition, bands corresponding to hsps 60, 56, 47, 40, 27 and 16 are clearly observed following heat shock. All these proteins were up-regulated upon a heat shock and recovery. By contrast, down-regulation was observed for proteins around 38 and 18 kDa (arrows). The synthesis of actin (43 kDa)
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Fig. 3. Western blot analyses of hsps (a) 105, (b) 90, (c) 70, (d) 60 and (e) 40. Lanes 1, 3 and 5: controls without antibiotics; lanes 2, 4 and 6: 2, 3 and 4 h recovery after heat shock without antibiotics; lanes 7, 9 and 11: controls with antibiotics; lanes 8, 10 and 12: 2, 3 and 4 h recovery after heat shock with antibiotics. Equal amounts of protein (10 mg) extracted from one individual's blood sample were loaded per each immunoblotting experiment. The Western blots of dierent hsps were performed on dierent samples.
was largely unaected by a mild heat shock and was therefore used as an internal control in our densitometric analyses to normalise pixel intensities, between lanes as well as between samples, adopting a practice previously reported [25]. The identity of the key heat shock proteins at 105, 90, 70, 60 and 40 was con®rmed by Western blotting with speci®c antibodies, as shown in Fig. 3. It should be noted that 35[S]-methionine identi®es proteins that are actively synthesized during the labelling period whilst immunoblotting identi®es the accumulated presence, both intrinsic and
induced, of a speci®c protein. The Western blots thus reveal that hsp 105, 70 and 40 are especially induced during the recovery phase after heat shock. On the other hand, although increased synthesis of hsp 90 and 60 is evident, the two hsps are also present in the non-heat shocked control samples, indicative of constitutive synthesis. This latter observation was particularly noticeable for hsp 60 in which induced synthesis, as observed by Western blots, was only clearly seen in lanes 1 and 2 (Fig. 3). We were also interested in the kinetics of hsp expression in human lymphocytes. In the present studies, we examined the kinetics of hsp expression over a 4 h recovery period at 378C, immediately following a heat shock at 428C for 1 h. The results, with pixel intensities of hsp protein synthesis normalized against actin, are summarised in Table 1. In most cases the increase in hsp synthesis relative to that of the respective control at all times was highly signi®cant, as revealed by the P-values for paired t-tests. On the other hand, determination of the optimal recovery time window to observe maximal hsp synthesis was equivocal, given the lack of statistical signi®cance of the dierences in heat shock expression at dierent time points. Nevertheless, it does appear that hsp synthesis peaked between 3 and 4 h post heat shock, as recovery beyond 4 h lead to a decrease in hsp expression (results not shown). These observations are in broad agreement with previous studies on the kinetics of hsp 70-mRNA transcription in human lymphocytes that have shown peak levels 1 to 2 h post heat shock, followed by subsequent decay by 4 to 6 h [18,27].
Table 1 Relative protein synthesis without and with antibiotics in human lymphocytes. (ÿ) without antibiotics. (+) with antibiotics. Relative protein synthesis is the ratio of normalised pixel intensity of the heat-shock band to the control band. Figures in the table represent the mean values for data obtained from 8 young adults aged 16±29 with an average age of 24. Figures in parentheses show p-values for respective paired t-tests between protein synthesis after heat-shock and in control lymphocytes Recovery Time
2h 3h 4h
HSP 105
HSP 90
HSP 70
HSP 60
(ÿ)
(+)
(ÿ)
(+)
(ÿ)
(+)
(ÿ)
(+)
8.2 (0.006) 12.7 (0.02) 6.3 (0.01)
12.2 (0.03) 12.1 (0.05) 8.2 (0.05)
3.9 (0.005) 8.1 (0.006) 9.3 (0.005)
5.0 (0.001) 10.2 (0.001) 11.5 (0.005)
7.2 (0.002) 19.4 (0.01) 28.1 (0.002)
6.5 (0.002) 14.2 (0.001) 28.3 (0.002)
6.3 (0.005) 15.1 (0.045) 8.7 (0.02)
11.3 (0.02) 21.7 (0.002) 17.1 (0.01)
D.V. Rao et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 861±868
Similarly, in rat lymphocytes the synthesis of hsp 70 declined rapidly between 4 and 6 h following heat shock [28]. It is dicult to compare our results for the other heat shock proteins (hsp 105, 90 and 60) as we are not aware of any previous studies on the kinetics of induction of these hsps in human lymphocytes. One of the aims of the present studies was to examine the eects of the antibiotics, penicillin and streptomycin (routinely added to culture medium to inhibit bacterial growth), on hsp expression in human lymphocytes. Under our experimental conditions, we did not observe any statistically signi®cant eect of these antibiotics on hsp expression, at least over the 4 h recovery period at 378C. However, this observation does not exclude the possibility that long term exposure to these antibiotics may be detrimental to cell physiology. In this respect altered calcium homeostasis in pig kidney cells, chronically exposed to penicillin and streptomycin, has been reported [29]. On the other hand, only moderate changes in some enzymatic properties in melanoma cells in culture (24 to 48 h) exposed to these antibiotics have been observed [30]. In summary, we describe a reliable experimental procedure for the characterization of a comprehensive range of hsps in human lymphocytes. The procedure can be suitably scaled down for application to smaller blood samples. With a blood sample of 18±20 ml we were able to follow the kinetics of hsp expression with and without antibiotics over a number of time points as well as run repeat protein gels for immunoblotting analysis providing a suitable con®rmation of the isotope labelling results. In short term tissue culture, the eects of penicillin and streptomycin are minimal in human lymphocytes, at least with respect to hsp synthesis and expression. We therefore feel con®dent comparing and contrasting our results on short term culture with other results using long term lymphocyte culture where antibiotic inclusion is routine and indeed mandatory. Given the known role of hsps in modulation of the immune response [7], we are now particularly interested in examining possible eects of antibiotic use in vivo on the expression of hsps in peripheral blood lymphocytes.
867
Acknowledgements We thank the donors for their participation in these studies and the sta of the Blood Bank and Pathology units of the Armidale and New England Hospital. This work was supported by grants from Blackmores (Australia), the Australian Research Council and internal research grants from the University of New England. D.V.R. is the recipient of an Australian postgraduate research award.
References [1] S. Lindquist, E.A. Craig, The heat-shock proteins, Ann. Rev. Genet. 22 (1988) 631±677. [2] F.M. Ritossa, A new pung pattern induced by a temperature shock and DNP in Drosophila, Experientia 18 (1962) 571±573. [3] K. Watson, Microbial stress proteins, Adv. Microb. Physiol. 31 (1990) 183±223. [4] R.I. Morimoto, A. Tissieres, C. Georgopoulos (Eds.), The Biology of Heat Shock Proteins and Molecular Chaperones, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1994. [5] R. Omar, Heat shock proteins in autoimmune disease, J. Clin. Immunoassay 17 (2) (1994) 129±132. [6] K. Renawek, C.E.M. Voorter, G.J.C.G.M. Boosman, F.P.A. van Woorkum, W.W. de Jong, Expression of Bcrystallin in Alzheimer's disease, Acta Neuropathol. 87 (1994) 155±160. [7] W. van Eden, D.B. Young (Eds.), Stress Proteins in Medicine, Marcel Dekker, New York, 1996. [8] G. Pawelec, M. Adibzadeh, R. Solana, I. Beckman, The T-cell in the ageing individual, Mech. Ageing Dev. 93 (1997) 35±45. [9] K. Suzue, X. Zhou, H.N. Eisen, R.A. Young, Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class 1 presentation pathway, Proc. Natl. Acad. USA 94 (1997) 13146±13151. [10] Y. Tamura, P. Peng, K. Liu, M. Daou, P.K. Srivastava, Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations, Science 278 (1997) 117±120. [11] A. Samali, T.G. Cotter, Heat shock proteins increase resistance to apoptosis, Exp. Cell Res. 223 (1996) 163± 170. [12] D.D. Mosser, A.W. Caron, L. Bourget, C. DenisLarose, B. Massie, Role of the human heat shock protein hsp 70 in protection against stress induced apoptosis, Mol. Cell. Biol. 17 (9) (1997) 5317±5327. [13] J. Ananthan, A.L. Goldberg, R. Voellmy, Abnormal
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D.V. Rao et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 861±868 proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes, Science 232 (1986) 252±254. M.J. Gething, J. Sambrook, Protein folding in the cell, Nature 355 (1992) 33±45. F.U. Hartl, Molecular chaperones in cellular protein folding, Nature 381 (1996) 571±580. A.Y.C. Liu, Z. Lin, H. Choi, F. Sorhage, B. Li, Attenuated induction of heat shock gene expression in aging diploid ®broblasts, J. Biol. Chem. 264 (1989) 12037±12045. A.Y.C. Liu, H. Choi, Y. Lee, K.Y. Chen, Molecular events, involved in transcriptional activation of heat shock genes become progressively refractory to heat stimulation during aging of human diploid ®broblasts, J. Cell. Physiol. 149 (1991) 560±566. Y. Deguchi, S. Negoro, S. Kishimoto, Age related changes of heat shock protein gene trnscription in human peripheral blood mononuclear cells, Biochem. Biophys. Res. Commun. 157 (1988) 580±584. R.N. Haire, M.S. Peterson, J.J. O' Leary, Mitogen activation induces the enhanced synthesis of two heat-shock proteins in human lymphocytes, Cell Biol. 106 (1988) 883±891. A.E. Faassen, J.J. O'Leary, K.J. Rodysil, N. Bergh, H.M. Hallgren, Diminished heat-shock protein synthesis following mitogen stimulation of lymphocytes from aged donors, Exp. Cell Res. 183 (1989) 326±334. D.A. Jurivich, L. Qiu, J.F. Welk, Attenuated stress responses in young and old human lymphocytes, Mech. Ageing Dev. 94 (1997) 233±249. T.S. Nowak Jr., H. Abe, Postischemic stress response in brain, in: R.I. Morimoto, A. Tissieres, C. Georgopoulos (Eds.), The Biology of Heat Shock Proteins and
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Molecular Chaperones, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1994, pp. 553±575. D.V. Rao, G.L. Jones, K. Watson, Heat shock protein synthesis in human lymphocytes, in: Proc. 5th International Federation of Teratology Societies Scienti®c Conference, 16±19 Nov., Sydney, Australia, 1997. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680±685. R.B. Eros, X. Zhu, R.L. Walford, Stress response of senescent T lymphocytes: reduced hsp 70 is independent of the proliferative block, J. Geront. 49 (1994) B65±B70. H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. 76 (9) (1979) 4350±4354. S.L. Bratton, D.S. Jardine, P.E. Mirkes, Constitutive synthesis of heat shock protein (72 kDa) in human peripheral blood mononuclear cells: implications for use as a clinical test of recent thermal stress, Int. J. Hyperthermia 13 (2) (1997) 157±168. M.A. Pahlavani, M.D. Harris, S.A. Moore, A. Richardson, Expression of heat shock protein 70 in rat spleen lymphocytes is aected by age but not by food restriction, J. Nutr. 126 (1996) 2069±2075. S.D. Bird, R.J. Walker, M.J. Hubbard, Altered free calcium transients in pig kidney cells (LLC-PK1) cultured with penicillin/streptomycin, In Vitro Cell. Dev. Biol. 30A (1994) 420±424. J.H. Martinez-Liarte, F. Solano, J.A. Lozano, Eect of penicillin-streptomycin and other antibiotics on melanogenic parameters in cultured B16/F10 melanoma cells, Pigment Cell Res. 8 (1995) 83±88.
A comprehensive analysis of heat shock protein synthesis in human peripheral lymphocytes: the eect of penicillin/ streptomycin D. Visala Rao, Graham L. Jones, Kenneth Watson* Human Biology, School of Biological Sciences, University of New England, Armidale, NSW 2351, Australia Received 8 March 1999; received in revised form 19 April 1999; accepted 21 April 1999
Abstract A reliable experimental procedure is described for the simultaneous characterisation of a comprehensive range of heat shock proteins (hsps) in human peripheral lymphocytes. In this system, a mild heat shock from 37 to 428C for 1 h induced the synthesis of hsps 105, 90, 70, 60, 57, 47, 40, 27 and 16. Densitometric analyses of 35[S]-methionine labelled protein gels indicated that levels of these hsps peaked at 3 to 4 h, following post-heat shock recovery at 378C. The presence of penicillin and streptomycin in the cell culture medium, appeared to have little eect on the kinetics of hsp synthesis. The present method can be used for relatively small blood samples and its relative ease of application and reproducibility make it appropriate for screening the expression of hsps in human lymphocytes from a range of individuals. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Heat shock proteins; Human lymphocytes; Antibiotics; Penicillin/streptomycin
1. Introduction Almost all organisms from bacteria to humans respond to a mild heat shock by inducing the synthesis of a set of highly conserved proteins, termed the heat shock proteins (hsps) [1]. The seminal work of Ritossa [2] on heat shock induced pung in the polytene chromosomes of
* Corresponding author. Tel.: +61-2-6773-3125; fax: +612-6773-3267. E-mail address: [email protected] (K. Watson)
Drosophila busckii, led towards later research on the expression and characterisation of hsps. Hsps are not only induced in response to various environmental stressors such as hyperthermia, transition heavy metals, amino acid analogues and chemotherapeutic agents [1,3,4], but are also associated with several pathological conditions including autoimmune diseases and age-related disorders [5±8]. The role of hsps in the immune response to cancer and in antigen presentation has also been recently demonstrated [9,10]. Furthermore, it has been shown that heatinduced apoptosis in transfected human cell lines
1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 3 7 - 0
862
D.V. Rao et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 861±868
is blocked by constitutively elevated or stress inducible levels of hsp70 [11,12]. The heat shock response is a well documented cellular phenomenon in which prior exposure to a mild heat shock induces a transient resistance to a subsequent, normally lethal, heat stress. Heat shock induced thermotolerance, which is believed to be facilitated by hsps, is a characteristic feature of the heat shock response [1,3]. Many hsps are expressed constitutively in cells under normal physiological conditions with synthesis enhanced after stress while others are produced only under stress conditions. In the unstressed cell, many hsps perform essential functions. These include protein folding and assembly and the targeting of proteins to sub-cellular organelles. The term molecular chaperone has, thus, been applied to this class of hsps [13,14]. Hsps are also involved in the degradation of damaged proteins [15]. Of all the hsps, the hsp 70 family has been the most widely studied and well characterised due to its critical and protective role as a molecular chaperone system. In humans, most of the hsp 70 studies have been at the transcriptional level and these include the use of ®broblasts [16,17] and lymphocytes for studies on hsp expression in relation to changes with age and activation by mitogens [18±21]. On the other hand, there have been relatively few studies on hsp expression at the translational level, including hsp 70, in human lymphocytes. Some ®ndings suggest that even though hsp 70 expression was detectable at the transcriptional level, it was decreased at the translational level [22]. We have chosen human lymphocytes as our experimental model for studies on hsp expression for a number of reasons. Lymphocytes are involved in the production of antibodies and are an integral part of the body's defence system. Furthermore, hsps are known to be associated with human disorders linked with the immune system [7]. In this study our major aim was to develop a sensitive and reliable method, using a single venous blood sample, to comprehensively and simultaneously examine the expression of a large number of hsps at the translational level. In ad-
dition, we examined the possible eects of penicillin and streptomycin, the most widely used antibiotics in mammalian cell culture studies, on hsp expression in human peripheral lymphocytes since hsp expression can be aected by a wide variety of stressors. 2. Materials and methods 2.1. Study population and blood sample collection Young adults, aged between 16±29 participating in the present study, were known to be healthy, not to have taken any medication for two weeks prior to blood donation and also not to have undertaken vigorous exercise prior to donating blood samples. Peripheral blood (18±20 ml) was collected by venipuncture into acid citrate dextrose-coated vacutainers (Becton and Dickinson, Sydney, Australia). 2.2. Isolation of lymphocytes Blood samples were diluted with equal volumes of 1 Hank's balanced salt solution (HBSS) (Gibco-BRL, Melbourne, Australia) and layered carefully onto Ficoll-Paque (Pharmacia Biotech, Sydney) and centrifuged at 400 g for 20 min, in a temperature controlled centrifuge (18±208C). Lymphocytes were washed by suspending in 3±4 volumes of 1 HBSS and centrifuging at 100 g for 10 min at 18±208C. An average of 2 107 cells were obtained from each 18±20 ml blood sample. The viability of the lymphocytes, as determined by trypan blue exclusion, was 90 2 5%. 2.3. Cell culture conditions Lymphocytes were suspended in 6 ml RPM1 1640 (methionine-free) medium (Gibco BRL) supplemented with 10% heat-inactivated foetal calf serum (CSL, Melbourne). An aliquot of 500 ml cell suspension was placed in each well of 12well ¯at-bottomed tissue culture plates. Antibiotic stock solution (CSL, Melbourne) consisting of penicillin (100 units/ml) and streptomy-
D.V. Rao et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 861±868
cin (100 mg/ml) was added to one half of the sample wells and equal amounts of RPM1 1640 added to the other half. One plate with 3 wells containing lymphocyte cell culture with antibiotics and 3 wells without antibiotics was designated for control samples and another plate (6 wells) for heat shock samples. The cells were equilibrated in an incubator at 378C under 5% CO2 and 95% humidity for 1 h. Heat shock treatment was performed for 1 h by placing the plate, designated for heat shock treatment, in a dry block heater, that was previously set to 428C and situated in the same incubator. Temperatures were monitored both in the dry block heater and in the incubator. 35[S]-methionine (100 mCi/ml) was added to both control and heat shocked cells 2 h prior to harvesting. Following initial experiments [23] where lymphocytes were left to recover after heat shock treatment for various recovery periods up to 24 h, it was decided to observe the kinetics of hsp expression in a 2 to 4 h recovery window. 2.4. Protein extraction Cells were harvested by washing the wells thoroughly with warm (378C) RPMI medium and collecting into sterile Eppendorf tubes. Lymphocyte pellets were obtained by centrifuging at 13,000 rpm for 5 min in a microcentrifuge. The pellets were washed twice with 1 HBSS and lysed in ice-cold lysis buer (0.1% Triton X100, 100 mM KCl, 8 mM MgCl2, 150 mM NaCl, 20 mM Tris±HCl, pH 7.4 and 1 mM phenylmethyl sulfonyl ¯uoride). The lysed cells were centrifuged and the supernatant containing proteins from the cytoplasmic fraction was recovered. Protein extractions were carried out at 4± 68C to minimise proteolysis and then stored at ÿ708C until needed. Protein concentrations were determined according to a modi®ed Bradford Coomassie dye binding colorimetric method for total protein concentration (Pierce, Rockford, IL). Equal volumes of 3 SDS-gel loading buer (150 mM Tris±HCl, pH 6.8, 300 mM dithiothreitol, 6% SDS, 0.3% bromophenol blue, 30% glycerol) was added and samples boiled for 3 min to denature the proteins.
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2.5. Analysis of protein synthesis 1D PAGE was performed using the standard procedures of Laemmli [24]. In brief, 10% SDSacrylamide gels were prepared for resolving the proteins with a 4% stacking gel. Protein standards and equal amounts of protein (10 mg) from each sample were boiled for 3 min and loaded using a 100 ml Hamilton syringe. Gels were run at 30 mA and 108C for about 4 h until the dye migrated to the bottom of the gel. Gels were silver stained (Bio-Rad silver stain kit), dried, exposed to X-ray ®lm (Kodak Biomax) at ÿ708C for 7±10 days and developed. The proteins were identi®ed according to their molecular weights by comparing their migration with that of standards of known molecular weight (Bio-Rad). 35 [S]-methionine labelled protein autoradiographs were scanned using a Phoretix 1D-densitometry software package, Ver. 3.0 (Phoretix International, Newcastle, UK) and a gel documentation system (GDS7600, UV-products, Cambridge, UK). Peak heights of relevant bands and actin (43 kDa) were recorded. Quanti®cation of preferentially synthesised hsp bands was achieved by calculating the ratio of respective band intensities (peak heights) to that of actin, a non-heat shock inducible protein [25]. Relative increase in protein synthesis was expressed as the ratio of normalized (with respect to actin) pixel intensity of hsp after heat shock to the intensity of hsp in the respective non-heat shock control. 2.6. Immunoblotting Following 1D-PAGE, the resolved proteins were transferred [26] to Hybond C-super nitrocellulose membranes (Amersham, Sydney) using a semi-dry Novablot system (Pharmacia). The membrane strips were blocked in PBS containing 0.05% Tween 20 (PBST) and 5% skim milk powder overnight at 48C. The membranes were washed in PBST and incubated with gentle shaking for 1 h with speci®c primary antibodies. Primary antibodies used in this study were: antihsp 105 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-hsp 90, 70, 60 and 40 (Stressgen, Victoria, Canada) and the appropriate dilutions
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Fig. 1. Silver stained gel of human heat shock proteins in peripheral lymphocytes. Heat shock treatment was performed at 428C for 1 h and cells allowed to recover at 378C for 2, 3 or 4 h. Equal amounts (10 mg) of protein extracted from one individual's blood sample were loaded on to 10% SDS-gel. Lanes 1, 4 and 6: controls without antibiotics; lanes 2, 5 and 7: 2, 3 and 4 h recovery after heat shock without antibiotics; lanes 9, 11 and 13: controls with antibiotics; lanes 10, 12 and 14: 2, 3 and 4 h recovery after heat shock with antibiotics; lanes 3 and 8: molecular weight standards. Positions where hsps that are up-regulated as well as actin (43 kDa) are denoted on the right and down-regulated proteins are shown by arrows without numbers (see Fig. 2 for the corresponding autoradiograph). Molecular weight standards (kDa) are shown on the left.
were determined according to manufacturers' instructions. The proteins were probed with appropriate horseradish peroxidase-conjugated secondary antibodies and subsequently detected using an enhanced chemiluminescence detection system (Amersham).
heat shocked samples, with and without antibiotics in the culture medium. Dierences were considered to be signi®cant for values of p < 0.05 and considered to be highly signi®cant for p < 0.01.
2.7. Statistical analysis
3. Results and discussion
Statistical analyses were conducted using paired Student's t-tests for comparing protein synthesis after heat shock and in control non-
The electrophoretic separation of total proteins from control and heat shock lymphocytes, in the presence or absence of antibiotics, is shown in
D.V. Rao et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 861±868
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Fig. 2. Autoradiograph of 35[S]-labelled heat shock protein expression. Lanes 1, 3 and 5: controls after 2, 3 and 4 h recovery without antibiotics; lanes 2, 4 and 6: 2, 3 and 4 h recovery after heat shock without antibiotics; lanes 7, 9 and 11: controls after 2, 3 and 4 h recovery with antibiotics; lanes 8, 10 and 12: 2, 3 and 4 h recovery after heat shock with antibiotics. Hsps that are up-regulated as well as the 43 kDa actin are denoted on the right and down-regulated proteins are shown by arrows without numbers. Molecular weight standards (kDa) are shown on the left.
Fig. 1 and 2. Fig. 1 shows a silver nitrate-stained gel and Fig. 2 shows an autoradiogram of the same gel. Although in the silver nitrate-stained gel the induction of heat shock proteins is not clearly observed, the gel does illustrate the success of our procedure in the extraction and separation of human lymphocyte proteins. Moreover, it serves as a control for the equal loading of protein in each sample well. On the other hand, the 35[S]-methionine labelling clearly illustrates the de novo synthesis of a
large number of heat shock proteins (Fig. 2). Obviously, proteins with a high rate of synthesis show a more intense band. In particular, upregulation following a 1 h heat shock at 428C, is evident for hsps 105, 90 and 70. In addition, bands corresponding to hsps 60, 56, 47, 40, 27 and 16 are clearly observed following heat shock. All these proteins were up-regulated upon a heat shock and recovery. By contrast, down-regulation was observed for proteins around 38 and 18 kDa (arrows). The synthesis of actin (43 kDa)
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Fig. 3. Western blot analyses of hsps (a) 105, (b) 90, (c) 70, (d) 60 and (e) 40. Lanes 1, 3 and 5: controls without antibiotics; lanes 2, 4 and 6: 2, 3 and 4 h recovery after heat shock without antibiotics; lanes 7, 9 and 11: controls with antibiotics; lanes 8, 10 and 12: 2, 3 and 4 h recovery after heat shock with antibiotics. Equal amounts of protein (10 mg) extracted from one individual's blood sample were loaded per each immunoblotting experiment. The Western blots of dierent hsps were performed on dierent samples.
was largely unaected by a mild heat shock and was therefore used as an internal control in our densitometric analyses to normalise pixel intensities, between lanes as well as between samples, adopting a practice previously reported [25]. The identity of the key heat shock proteins at 105, 90, 70, 60 and 40 was con®rmed by Western blotting with speci®c antibodies, as shown in Fig. 3. It should be noted that 35[S]-methionine identi®es proteins that are actively synthesized during the labelling period whilst immunoblotting identi®es the accumulated presence, both intrinsic and
induced, of a speci®c protein. The Western blots thus reveal that hsp 105, 70 and 40 are especially induced during the recovery phase after heat shock. On the other hand, although increased synthesis of hsp 90 and 60 is evident, the two hsps are also present in the non-heat shocked control samples, indicative of constitutive synthesis. This latter observation was particularly noticeable for hsp 60 in which induced synthesis, as observed by Western blots, was only clearly seen in lanes 1 and 2 (Fig. 3). We were also interested in the kinetics of hsp expression in human lymphocytes. In the present studies, we examined the kinetics of hsp expression over a 4 h recovery period at 378C, immediately following a heat shock at 428C for 1 h. The results, with pixel intensities of hsp protein synthesis normalized against actin, are summarised in Table 1. In most cases the increase in hsp synthesis relative to that of the respective control at all times was highly signi®cant, as revealed by the P-values for paired t-tests. On the other hand, determination of the optimal recovery time window to observe maximal hsp synthesis was equivocal, given the lack of statistical signi®cance of the dierences in heat shock expression at dierent time points. Nevertheless, it does appear that hsp synthesis peaked between 3 and 4 h post heat shock, as recovery beyond 4 h lead to a decrease in hsp expression (results not shown). These observations are in broad agreement with previous studies on the kinetics of hsp 70-mRNA transcription in human lymphocytes that have shown peak levels 1 to 2 h post heat shock, followed by subsequent decay by 4 to 6 h [18,27].
Table 1 Relative protein synthesis without and with antibiotics in human lymphocytes. (ÿ) without antibiotics. (+) with antibiotics. Relative protein synthesis is the ratio of normalised pixel intensity of the heat-shock band to the control band. Figures in the table represent the mean values for data obtained from 8 young adults aged 16±29 with an average age of 24. Figures in parentheses show p-values for respective paired t-tests between protein synthesis after heat-shock and in control lymphocytes Recovery Time
2h 3h 4h
HSP 105
HSP 90
HSP 70
HSP 60
(ÿ)
(+)
(ÿ)
(+)
(ÿ)
(+)
(ÿ)
(+)
8.2 (0.006) 12.7 (0.02) 6.3 (0.01)
12.2 (0.03) 12.1 (0.05) 8.2 (0.05)
3.9 (0.005) 8.1 (0.006) 9.3 (0.005)
5.0 (0.001) 10.2 (0.001) 11.5 (0.005)
7.2 (0.002) 19.4 (0.01) 28.1 (0.002)
6.5 (0.002) 14.2 (0.001) 28.3 (0.002)
6.3 (0.005) 15.1 (0.045) 8.7 (0.02)
11.3 (0.02) 21.7 (0.002) 17.1 (0.01)
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Similarly, in rat lymphocytes the synthesis of hsp 70 declined rapidly between 4 and 6 h following heat shock [28]. It is dicult to compare our results for the other heat shock proteins (hsp 105, 90 and 60) as we are not aware of any previous studies on the kinetics of induction of these hsps in human lymphocytes. One of the aims of the present studies was to examine the eects of the antibiotics, penicillin and streptomycin (routinely added to culture medium to inhibit bacterial growth), on hsp expression in human lymphocytes. Under our experimental conditions, we did not observe any statistically signi®cant eect of these antibiotics on hsp expression, at least over the 4 h recovery period at 378C. However, this observation does not exclude the possibility that long term exposure to these antibiotics may be detrimental to cell physiology. In this respect altered calcium homeostasis in pig kidney cells, chronically exposed to penicillin and streptomycin, has been reported [29]. On the other hand, only moderate changes in some enzymatic properties in melanoma cells in culture (24 to 48 h) exposed to these antibiotics have been observed [30]. In summary, we describe a reliable experimental procedure for the characterization of a comprehensive range of hsps in human lymphocytes. The procedure can be suitably scaled down for application to smaller blood samples. With a blood sample of 18±20 ml we were able to follow the kinetics of hsp expression with and without antibiotics over a number of time points as well as run repeat protein gels for immunoblotting analysis providing a suitable con®rmation of the isotope labelling results. In short term tissue culture, the eects of penicillin and streptomycin are minimal in human lymphocytes, at least with respect to hsp synthesis and expression. We therefore feel con®dent comparing and contrasting our results on short term culture with other results using long term lymphocyte culture where antibiotic inclusion is routine and indeed mandatory. Given the known role of hsps in modulation of the immune response [7], we are now particularly interested in examining possible eects of antibiotic use in vivo on the expression of hsps in peripheral blood lymphocytes.
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Acknowledgements We thank the donors for their participation in these studies and the sta of the Blood Bank and Pathology units of the Armidale and New England Hospital. This work was supported by grants from Blackmores (Australia), the Australian Research Council and internal research grants from the University of New England. D.V.R. is the recipient of an Australian postgraduate research award.
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