Review Article

Emerging Role of Heat Shock Proteins in Biology and Medicine

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Marja Jaattela and Dorte Wissing

All cells, procaryotic and eucaryotic, respond to an elevation in temperature by increasing the synthesis of a family of proteins collectively known as heat shock proteins (HSPs). HSPs are among the most highly conserved and abundant proteins in nature. Studies on the regulation of the synthesis of HSPs have for several years shed

light on the mechanisms regulating gene expression. The results from recent years, showing that HSPs play crucial roles in a wide variety of normal cellular processes, has made them an object of even broader interest, first to molecular and cellular biologists and later to specialists in various fields of medicine including oncology, immunology, infectious disease, autoimmunity, embryology, neurology and endocrinology. The aim of this review is to briefly summarize our present knowledge of the regulation of the heat shock response and the structure of the relevant gene products, HSPs. Moreover, some of the exciting associations between HSPs and various fields of medicine will be discussed. Key words: heat shock proteins; stress; chaperone; immunity; autoimmunity; tumour. (Annals of Medicine 24: 249-256,1992)

Introduction Heat shock response was discovered 30 years ago as a new heat-inducible puffing pattern in salivary gland chromosomes of fruit fly (1). The universal nature of the phenomenon was gradually uncovered and it became evident that all organisms respond to a rise in temperature by rapid synthesis of a set of evolutionarily highly conserved proteins, heat shock proteins (HSPs). Research on the heat shock response progressed considerably in the 1980s in the wealth of modern DNA technology. Sequences of several heat-inducible proteins and heat-activated transcription factors were revealed (2, 3). After years of frustration concerning possible roles of HSPs, enormous progress has been made in the last 5 years. HSPs have been shown to play a pivotal role in such crucial phenomena as protein import and assembly, protection from environmental stress, immunity, autoimmunity and cancer. From the Department of Tumor Cell Biology, Fibiger Institute, Danish Cancer Society, Ndr. Frihavnsgade 70, DK-2100 Copenhagen, Denmark. Address and reprint requests: Marja Jaattela, M.D., Dr. M.Sc., Department of Tumor Cell Biology, Fibiger Institute, Danish Cancer Society, Ndr. Frihavnsgade 70, DK-2100 Copenhagen, Denmark.

Families of Heat Shock Proteins HSPs are among the most conserved proteins known in phylogeny with respect to both function and structure. The superfamily of HSPs include a number of different molecular weight class families of which the HSP70 family is the most conserved in evolution, human HSP70 being 72% homologous to fruit fly HSP70 and 47% homologous to Escherichia coli dnaK (4). While E. coli has a single HSP70-like protein, mammalian cells contain a family of HSP70-like proteins, that have different patterns of expression and intracellular locations (2). The other HSP families are nucleolar HSPllO (5),the highly conserved HSPSO family (6), mitochondria1 HSP6O (7), collagen-binding HSP47 (8) and the heterogeneous family of small HSPs with molecular weights ranging from 16 to 40 kD in various species (2). In human cells, only one small HSP is identified and it is referred to as HSP27 or HSP28 (9, 10). Some suggested functions of various HSPs are discussed below and summarized in Table 1. Although they will not be discussed in detail in this review, it should be noted that there is another class of stress-induced proteins closely related to HSPs and known as glucose-regulated proteins (GRP; for review see refs 11, 12). GRPs are induced by glucose deprivation and, as is the case for many HSPs, GRPs are also

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Table 1. Suggested functions of heat shock proteins In mammafian cells. HSP-family

Members'

Expression: normal/ heat-induced

HSP110 HSPSO

HSPlIO HSPBSa,O

4-

+/+

HSP70

f

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HSPBO HSP47 HSP27

Localization: normaVs1ressed

Suggested functions

++/++

Nucleolushucleotus CytosoVnucleus

HSC70

+

+/-

Cy!osol/nucleus

HSP70at

(+I/+ + + +

Cylosol/nucleus

HSP70bt HSP58 HSP47 HSP27

-I+

Protection of nucleoli from heat. Regulation of steroid receptor activity, binding to protein kinases, actin and tubufin Chaperone, uncoating of chlatrin vesicles, binding to oncogenes and anti-oncogenes autoantigen. Chaperone, protection from heat, protection, from TNF cytotoxicity. Chaperone, protection from heal. Mitochondria1chaperone, autoantigen. Collagen-binding. Protection horn heat, TNF signal transduction.

f

f f

+

+ + / + 4- 4+ + / + i 4-k

+I+

-/nucleus (?) Mitochondria/m.ch. Cytosol/cy10sol Cylosollnucleus

*Notall members of each family are jisted. tHeat-inducibfe members of HSP7O family; HSP70a has also been called HSP72 or 72K HSP (4), HSP70b has been called HSX70 (129); in the text both are referred to as HSP70.

expressed during normal growth and development in a wide variety of cell types, especially in secretory cells (11,IZ).

Regulation of the Expression of Heat Shock Protein

Genes

The heat-induced synthesis of HSPs in eucaryotic cells is mainly, but not soieiy, controlled at the transcriptional level (35, 36). The activation of HSP genes by heat is Regulation of Heat Shock Response dependent on a positive regulatory DNA motif, the heat shock element (HSE), present in the 5' flanking region of Heat shock response is characterized by a rapid and all HSP genes (37-41). HSE is a contiguous array of at specific synthesis of heat shock proteins, and a least two 5 basepair sequences (NGAAN] arranged in reduction in the synthesis of other proteins when cells or alternating orientations. HSE serves as a binding site for whole organisms are abruptly exposed to supraoptimal a transcriptional activator, heat shock factor (HSF), present in inactive form in resulting cells (42,431. temperatures. It is the most highly conserved genetic Genes encoding HSFs from yeast (44, 45), tomato system known, existing in every organism in which it has been sought (for review see refs 2, 12). In addition to the (46), fruit fly (47), human (48, 49) and mouse (50) have induction of a new synthesis of HSPs, heat treatment recently been cloned. Since the binding sequence in the also alters cellular distribution and phosphorylation of DNA is conserved, it is not surprising that HSFs from constitutively expressed HSPs (13-1 5). various species share large sequence homologies in the areas important for DNA-binding. They also share canserved leucine-zipper motifs and several heptad repeats Inducers of Hear Shock Response of hydrophobic residues, while the rest of the sequences When it was found that various forms of cellular stress, are high!y divergent. Also, the regulation of HSF activity other than heat, can induce the synthesis of HSPs, the differs between species. Yeast HSF is constitutively term stress proteins was introduced. Viral infection (16), bound to the DNA and activated by phospllorylation ethanol (?7), gfucose depletion (18),calcium ionophores upon heat shock (44). In fruit fly, human and mouse, heat (la), heavy metal ions (1 Q), steroid hormones (201, shock is required to convert the pre-existing HSF into an hydroxen peroxide (21 1, 1,25-dihydroxyvitamin 0, (21), active form, capable of binding to HSE and stimulating interleukin 2 (22),DNA damaging agents (23).anoxia (24, transcriptional initiation (51-54). Recent evidence 251,recovery from anoxia (24),and phagocytosis (26) are suggests that the activation of fruit fly HSF involves among the numerous reported inducers of HSPs. oligomerization and subsequent chromosomal relocaliSeveral of the known inducers of the heat shock zation of HSF (43). Interestingly, the active multimers do not only bind to the known heat shock puff sites, but also response cause changes in the intracellular pH or to 150 additional chromosomal sites including key conactivate the productions of free radicals. The role of these changes in signal transduction is, however, trol loci for growth and development. This suggests that speculative. HSF could function as a repressor of normal gene The expression of some members of HSP27, HSP70 activity and shut down developmental programmes and HSPSO families is also induced at certain stages of during unfavourableconditions (43). development (27-31). It is also important to note that In addition to HSE, many heat shack promoters conseveral members of HSPs are constitutively expressed tain additional regulatory sequences allowing complex and serve important functions in unstressed cells (12, patterns of expression (19, 55-57). Growing evidence 32-34]. suggests that different inducers may activate distinct

25 1

Heat Shock Proteins binding proteins, interacting with at least three different binding domains in HSP promotors (19,57, 58). This may

A.

explain variations in stress responses fotlowing diverse treatments. Moreover, post-transcriptional mechanisms including alternations in the stability and translationability of mRNA and the half-life of the proleins may account for the complicated pattern of expression (35, 36). While our knowledge of different transcription factors and their activation has grown enormously during .the last few years, there is still a gap to be filled in understanding what triggers the activation, i.e. How do cells sense changes in their environment?

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6 = HSPTO

Heat Shock Proteins Function as Molecular Chaperones The term morecular chaperone is used for proteins which prevent incorrect interactions and assist the assembly of other proteins, without being part of the final protein structure (59).Strong evidence is accumulating that HSPs of HSPGO and HSP70 families participate in folding and unfolding of cellular proteins. The interactions of HSPSO with steroid receptors, actin, tubulin and severat protein kinases, the association of HSP47 with ptocoltagen and the tendency of HSP27 to form aggregates and to associate with oestrogen receptor suggest that other HSPs may function also as chaperones (Fig. 1 ; for recent reviews see refs 2,601. The best characterized chaperone HSP is the bacterial heat shock protein, GroE. GroE consist of two subunits, GroEL and GroES, the former being analogous to the mammalian HSPGO (7). GroEL binds unfolded proteins tightly and feleases them in a folded form, either assembled or able to become so. This event requires hydrolysis of ATP, and interaction of GroES. The function of GroES could be to prevent the bound protein from dissociating from GroEL, until it has completed folding. The folded protein dissociates from GroES upon ATP hydrolysis (61).In mammalian cells HSPGO is located in mitochondria (62-64). Growing evidence suggests that mammalian HSPGO plays a crucial role in the proper folding and assembly of imported proteins, including enzyme complexes in the mitochondria1 matrix (63,64). Studies of bovine mitochondria have also revealed the presence of a 9 kD protein involved in the assembly, probably the mammalian homolog of bacterial GroES (65). HSP6O has also been shown to associate with p21 oncoprotein (66).The significance of this interesting association remains unclear, but it has been suggested that HSP6O could help p21ras to associate with its activators or effectors (66). The bacterial homolog to HSP70, DnaK, functions also as a chaperone. DnaK participates in an ordered assembly and partial disassembly of the initiation cornplex essential for bacteriophage 1 DNA replication (for review see ref. 67). It has been suggested that members of mammalian HSP7O family, especially constitutively expressed HSC70 and heat-inducible HSPIO, bind to hydrophobic protein sequences exposed during normal cellular processes, such as translocation across mem-

6. Nucleus

a.

-- Steroid receptor

Mitochondrion

Q = HSPZT

0 = HSPBO 17 = HSPSO L

e - .ATP+ .. .

ADP+ Pi.

Figure 1. Schematic presentation 01 possibte chaperone functions of heat shock proteins. (A) HSC70 binds to newly synthesized unfolded proteins to assist their proper folding and assembly (a) and lo transport them across membranes into various cellular compartmenls (b). Wrongly folded proteins are transported to lysosomes for degradation (d). Heat and other environmental stresses denaturale cellular proteins and induce synthesis of HSP70. Newly synthesized HSP70 binds to denalurated proteins to keep them soluble and probably assists tfleir renaturation after stress (d}. ( 8 ) HSP27 associates with itself and with oestrogen receptors (a). HSPBO binds 10 steroid receptors keeping them from binding to DNA in the absence of steroid stimulation (b). Mitochondria1 HSPGO takes part in the folding and assembly of newly imported milochondtial proteins (Cl.

branes and translation of new polypeptides or after partial denaturation during heat shock (68).Proteins can then be released from HSC70/HSP70 with the aid of ATP hydrolysis. The transient binding of HSC7WHSP70 to exposed sequences could lhus prevent the wrong protein-protein interactions, aggregation of denatured proteins and premature folding of nascent polypeptides. This model is supported by the structural studies of HSC70 and HSP70 showing that they have both an ATPbinding domain and a peptide-binding domain, and by in vitro binding studies of unfolded proteins to HSC70 in the presence and absence of ATP (69-71).Chiang and

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Jaattela

co-workers have suggested that the binding of HSC70 to denatured proteins leads to the transportation of these proteins into lysosomes and subsequent degradation (72). Thus, HSPs seem to serve many vital housekeeping functions in normal cells.

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Heat Shock Proteins Protect Cells from EnvironmentalStress It is widely assumed that HSPs protect organisms from toxic effects of heat and other forms of environmental stress, Thus, HSPs may give an explanation to the old belief of the protective effects of fever. This hypothesis was originally based on experiments, where a short pretreatment of cells at supraoptimal temperatures protected them from further exposure to higher, otherwise lethal, temperatures (73, 74). More compellingly, the acquired thermotolerance coincides with the synthesis and degradation of HSPs (74, 75). Some results show that treatments capable of inducing HSPs in normal temperatures also render cells more resistant to heat. This suggests that the effect is not due to other accommodations in cell physiology brought about by raising the temperature (13, 17, 75). Furthermore, an association between HSPs and thermotolerance has been found in studies showing that Chinese hamster fibroblasts, selected for their ability to survive at high temperatures, constitutively produce higher levels of HSP70 than the parental cells (76). Nevertheless, the role of HSPs in cellular resistance to heat has been widely discussed, because most results show only a correlative relationship between HSPs and thermotolerance. Moreover, numerous similar experiments to those listed above have failed to show an association between the levels of HSPs and heat resistance (77-81 ). However, more direct evidence supporting the role of HSP70 in thermotolerance comes from recent studies by Riabolow and co-workers showing that fibroblasts, microinjected with antibodies against HSP70, are significantly more vulnerable to heat shock than cells injected with control antibodies (82). Moreover, competitive inhibition of HSP70 gene expression and protein synthesis cause thermosensitivity in Chinese hamster ovary cells (83).Finally, Li and co-workers have succeeded in establishing stable transfected rat fibroblast cell lines constitutively expressing human HSP70 gene (84, 85). In these cell lines the thermotolerance correlates with the level of expression of HSP70. Most of the studies have concentrated on examining the correlation between HSP70 and thermotolerance, whereas the role of other HSPs has been overlooked. However, recent results provide good evidence that the synthesis of the low molecularweight heat shock protein, HSP27, is associated also with thermotolerance in mammalian cells. Heat-resistant variants of Chinese hamster cells obtained by chemical mutagenesis followed by a single heat treatment express high levels of HSP27 constitutively (86). More convincingly, transfection of Chinese hamster and mouse cells with human HSP27 gene confers permanent thermoresistant phenotypes

Wissing (87). However, microinjection of antibodies against HSP27 into fibroblasts do not render them sensitive to heat treatment (82) and heat-sensitive Chinese hamster cells not expressing HSP70, synthesis normal levels of HSP27 in response to heat (83). From the above it seems that acquired thermoresistance may be mediated by combined action of HSP27 and HSP70, although the role of other heat-inducible proteins cannot be excluded. Landry and co-workers have suggested that a rapid phosphorylation of HSP27, in response to heat treatment, sets up an immediate protective mechanism to limit heat-induced cascade reactions that lead to cellular damage (87). The role of other HSPs could then be to repair lesions and prevent further damage at a later point. This idea is supported by results showing that overexpresslon of HSP70 leads to an accelerated recovery of nucleolar structures after heat shock (88).Moreover, members of HSP70 family prevent inappropriate interactions of proteins that have not yet achieved their final state of assembly and abnormal proteins unable to fold properly (1 1, 68). Thus, binding of denatured proteins to HSP70 may maintain their solubility and thereby reduce the cellular damage brought about by heat shock. The role of various HSPs in cellular resistance against heat and other forms of stress, however, remains a matter of debate until more information on their actions in stressed cells is available.

Heat Shack Proteins in Immunology HSPs appear to serve a variety of important functions in immunity (for reviews see refs 89, 90). Recent evidence suggests that certain HSPs participate in synthesis of immunoglobulins and in antigen processing and presentation. HSPs are also among the major antigens recognized in immune response to a wide spectrum of pathogens. As HSPs are highly conserved in evolution, immune responses against pathogen HSPs are often cross-reactive and can lead to anti-self reactivity. In some cases, overstimulation of the immune response towards HSPs may lead to autoimmune disease.

Heat Shock Proteins in Antigen Processing and Presentation The finding of characteristic functions of HSPs, namely unfolding,translocation and disintegration of proteins, led to speculations that members of HSPs may participate in processing and presentation of antigens. Moreover, it seems more than coincidental that genes encoding HSP70 have been mapped in the human major histocompatibility complex (MHC) on chromosome 6 between the loci for complement components (C2, factor 6, and C4) and tumour necrosis factors alpha and beta (91). During the past few years, evidence has accumulated supporting the role of HSPs in antigen processing. In the search for a peptide-binding protein playing a role in antigen processing a 72-4 kD protein (PBP72/ 74), with specific binding to a known antigenic peptide of pigeon cytochrome c (Pc), was purified (92). Antibodies raised against PBP72/74 block effectively the process-

253

Heat Shock Proteins Table 2. Heat shock proteins as antigens in various infections. Pathogen ~~~

Eorellia burgdorferi Brugia malayi Chlamydia trachomatis Coxiella burnetii Legionella pneumophila Mycobacterium tuberculosis Mycobacterium leprae Plasmodium falciparium Schistosoma mansoni Treponema pallidurn Trypanosoma cruzi

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Disease

HSP family

Lyme disease

HSPGO HSPi'O HSPGO HSPGO HSPGO HSPGO, HSP70, small HSPs HSPGO,HSP70, small HSPs HSP70, HSPSO HSP7O HSPGO HSP70

~

~~

Filariasis Trachoma

Q-fever

Legionnaires' disease Tuberculosis Leprosy Malaria Schitsosomiasis Syphilis Chagas disease

ing and presentation of Pc to a Pc-specific T-cell hybrid and of ovalbumin to an ovalbumin-specific T-cell. More evidence supporting the role of PBP72/74 in antigen processing comes from studies showing that PBP72174 purified from antigen-processing cells which have processed radiolabelled antigen have radiolabelled processed antigen bound to it (93). PBP72/74 has been found in cell surfaces of B-cells, T-cells and fibroblasts, and it is recognized by antibodies against conserved epitopes in HSP70 family members (93). Moreover, PEP72174 shares another characteristic feature of HSP70 proteins, which is its ability to bind ATP. These findings indicate that a member of HSP70 family plays a role in processing antigens through the class II pathway. Whether other members of HSPs serve similar functions remains to be studied.

Immune Recognition of Pathogen Shock Proteins Variety of HSPs are among the major targets of the immune response to various pathogens. HSPs are abundant proteins in many pathogens and their expression is often increased during infection. Expression of pathogen HSPs can be induced by temperature shift during transmission from vector to host and in response to reactive oxygen metabolites released from host immune cells (89,90). Why HSPs serve as major immune targets is unclear. It could be simply becguse of their abundance or, probably, because they are suitable for antigen presentation. Also, the conservation of these proteins may contribute to immunogeneity through frequent stimulation of immune response by various pathogens with similar HSPs. Immune response against HSPs has been best characterized in mycobacterial infections. Mycobacterial HSPs, especially HSP60 and HSP70, are among the immunodominant targets of both antibody and T-cell (including T-cells with alp- and y/d-receptors) responses in mouse and humans (89). Antibodies and T-cells reactive to mycobacterial HSPGO do not recognize a single epitope, but a wide variety of epitopes along the entire protein (94). In addition to mycobacterial infections, HSPs are major targets for antibody-mediated and cellular responses in a large variety of bacterial and parasitic infections (Table 2; ref. 95 and references therein).

Heat Shock Proteins in Autoimmune Disorders Several lines of evidence indicate a role for HSPs in autoimmunity (ref. 96 and references therein). The general theme of such observations is that T-cells and antibodies specific for HSPs of pathogenic organisms cross-react with HSPs of the host. Because infection induces stress response both in pathogens and host, increased synthesis of highly similar sets of autologous and foreign molecules occur at the time of active immune response, thereby allowing activation of specific immunity against conserved regions of the hosts' own HSPs (26,96). It has also been suggested that the immune system of healthy individuals has the capacity to react against its own HSPs in a manner that could help eliminate its own cells if they are transformed, infected, or otherwise stressed. The role of HSPs in autoimmunity is supported by data showing that: (i) microbial HSPs induce a formation of autoantibodies in the host (97); (ii) patients with various autoimmune disorders, including systemic lupus erythematosus and rheumatoid arthritis, have autoantibodies to HSPs (98, 99); (iii) mycobacterial HSPGO stimulates the growth of synovial fluid T-cells from patients with rheumatoid arthritis (100); (iv) T-cells from diabetogenic mice recognize an epitope in human HSPGO which can be used to treat insulin-dependent autoimmune diabetes in mice (101); and (v) high levels of human HSPGO can be found in the cartilage-pannus junction and in rheumatoid nodules of patients with rheumatoid arthritis, but not in normal tissues or other types of chronic inflammation (102). Moreover, several animal models strongly support the role of HSPs as triggering antigens in arthritis in genetically predisposed individuals and their central role in the etiology of arthritis appears likely (96). Intensive studies on the role of HSPs in other autoimmune disorders are expected to shed light on the basic mechanisms underlying autoimmunity.

Heat Shock Proteins in Tumours HSP70 Protects Tumour Cells from Lysis by Turnour Necrosis Factors Turnour necrosis factor (TNF) is a multifunctional cytokine capable of causing haemorrhagic necrosis in

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tumours in vivo and lysis of various tumour cell lines in vitro (103). The mechanism of action by which TNF kills tumour cells is not fully understood. The production of free radicals is, however, essential for its cytoxicity and various antioxidants and free radical scavengers protect cells from TNF-mediated lysis (ref, 104 and references therein). Interestingly, a short heat treatment followed by the synthesis of heat-inducible proteins effectively protects sensitive tumour cells from subsequent killing by TNF (105, 106). Transfection studies employing cDNAs encoding for HSP27 and HSP70 suggest that the major heat-inducible heat shock protein, HSP70, plays an important role in this protection (107). In concordance with the data showing that HSP70 protects cells from heat stress (84, 85),these results support the idea that HSPs play a role in protection of cells from various types of stress associated with increase in cellular free radical content. Whereas growth of most tumours is probably due to activation of oncogenes promoting cell proliferation and inactivation of tumour suppressor genes, tumour growth in vivo may also be enhanced by activation of the genes whose products allow cells to escape from immunological surveillance. Thus, the data showing that heat shock and HSP70 protect tumour cells from TNFmediated cytotoxicity, suggests that environmental stress/HSP70 may increase the oncogenic potential of some tumour cells by providing them with an escape mechanism from immunological surveillance.

Association of Heat Shock Proteins with Oncoproteins and Tumour Suppressors Mutations in the p53 tumour suppressor gene are the most common genetic alternations in human malignancies (ref. 108 and references therein). Recently, it has become evident that the wild-type p53 negatively regulates cell growth and that loss of function mutations are needed for its oncogenicity (109, 110). The tumourigenic mutants of p53 are often expressed at elevated levels in human tumours and various transformed cell lines (111, 112). Accumulation of mutated p53 is partly due to its considerably longer half-life compared to the wild-type p53. A characteristic of some mutant forms of p53 is that they bind to the constitutive member of HSP70 family, HSC70 (113-1 16). This binding could contribute to the longer half-life and higher expression of mutated p53 in some tumour cell lines. The significance of the binding of p53 and HSC70 to the turnourigenicity of mutated p53, however, remains, elusive. It has been suggested that in cells with a single copy of mutated p53 gene, aggregates of HSC70 and mutated p53 protein also bind wild-type p53 and thereby inactivate it. Another interesting property of HSP70 in tumour cells is its colocalization with c-myc and v-myc oncoproteins (1 17). In both heat-shocked and untreated cells overexpressing either c-myc or v-myc protein, these proteins accumulate in. amorphous, phase-dense nuclear structures in codistribution with HSP70. This colocalization suggests that HSP7O may regulate the activity of highly expressed oncogenes by accumulating them into insoluble, and thereby inactive, structures. Moreover,

binding of HSC70 to the transforming protein, medium T antigen of polyoma transformation-defective mutants, but not to T antigen of wild-type transformationcompetent virus, suggest that HSC70 may protect cells from transformation by this mutated from of T antigen (118). Furthermore, HSP70 is expressed in the synthetic phase of a cell cycle and it interacts with a number of as yet unidentified proteins in a cell cycle-specific manner (34, 11 9).Thus, it may play a role in the regulation of cell growth and contribute to the oncogenicity of tumour cells. HSPSO binds to a well-characterized oncoprotein Rous sarcoma virus transforming protein, pp60src (120). Several other transforming tyrosine kinases, yes, fps, fes and fgr, also form stable complexes with a 90 kD protein. In some cases this 90 kD protein has been identified as HSPSO (ref. 2 and references therein). As tyrosine kinases bound to HSPSO are underphosphorylated and incapable of autophosphorylation, it has been suggested that HSPSO may regulate their turnover and activity by keeping them soluble and inactive during transportation to the proper location in the plasma membrane (121). In addition to protein tyrosine kinasas, HSPSO associates also with other cellular kinases, heme-regulated elF2-a kinase and casein kinase II (122). In contrast to tyrosine kinases, HSPSO seems to activate these kinases leading to autophosphorylation of the kinase or phosphorylation of HSPSO (2, 122). Also HSPGO binds to at least one oncoprotein, ~21'"" (66). It has been suggested that HSPGO could help p2lraS to associate with its activators or effectors (66). The interactions between products of oncogenes and tumour suppressor genes with various HSPs are intriguing but, at present, of uncertain importance. As all HSPs seem to bind more effectively to mutated than wild-type forms of oncoproteins and tumour suppressors, these mutated proteins may just have a more hydrophobic surface than the corresponding wild-type proteins and, therefore have a higher affinity for HSPs.

Correlation of HSP27 Expression and Turnourigenicity Small HSPs have been shown to have properties typical for anti-oncogenes in some systems and those typical for oncogenes in others. An inverse relationship between HSP27 expression and oncogenicity has been observed in adenovirus-transformed rat cells (123). Adenovirustransformed cells unable to form tumours in immunocompetent mice were found to express similar levels of HSP27 as non-transformed cells, whereas oncogenic cells lacked both HSP27 mRNA and protein even after heat shock. HSP27 is frequently expressed in human breast cancer (124) and its expression has been used as a marker for hormone-responsive breast tumours (125). A correlation of tumours expressing HSP27, and tumours responding to endocrine therapy has been shown (126) and recently it has become evident that HSP27 is an oestrogen receptor binding protein expressed in several breast and endometrial malignancies (127). A study of

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Heat Shock Proteins

300 breast cancer patients, with median follow-up at 8 years, shows a significant correlation between HSP27 overexpression and oestrogen receptor content, nodal metastases, tumour size, lymphaticlvascular invasion, and a shorter disease-free survival period, but not shorter overall survival, for the study population as a whote. The HSP27 overexpression and correlation with more aggressive tumours, suggest a biological role for HSP27 in oestrogen-responsjve carcinomas (128).

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Concluding Remarks As HSPs are highly conserved in evolution, it is not entirely surprising that they play crucial rules in a wide variety of cellular processes. The general theme throughout this review is the involvement of HSPs in protein-protein interactions. By binding to other proteins HSPs may regulate their activity, help them to fold properly and form multimers, inhibit incorrect interactions between highly adhesive hydrophobic protein surfaces and transport them to various cellular compartments. These are all functions essential for normal growth of cells. It is widely assumed that heat-induced HSPs protect Celts from various stresses. The protection may be brought about by binding of HSPs to partly denatured proteins or probably heat-inducible HSPs carry out some special as yet unknown functions in stressed cells. Interesting hypotheses suggesting a role for HSPs in tumour growth, antigen processing and autoimmunity are as yet mostly based on in vifro studies. Future results from the wide group of scientists working in the field are expected to shed light on the molecular mechanisms underfying these events and to determine the role of HSPs in various cellular processes. The work done in our laboratory was supported by granfs from the Danish Cancer Society and the Novo Foundation.

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