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Molecular and Cellular Biochemistry 196: 117–123, 1999.© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

Stress genes and species survival

Prasanta K. RayDepartment of Immunotechnology and Environmental Sciences, Bose Institute, Calcutta, India

Abstract

Stress genes can be ascribed to have been generated by the organism for their intrinsic urge to survive against the changingenvironmental odds, during the evolutionary process. This concept has been supported by a large number of reports describingindividual types of phenomena. These have been reconciled and globalised in terms of their relevance in this article. Supportingevidences have been drawn from the literature which indicated that by using different types of inducer one can express heatshock proteins. Similarly, several types of stress inducers, such as calorie restriction, LPS stimulation and StaphylococcalProtein-A stimulation, it was possible to induce a wide array of biological, biochemical and immunological reactions. Suchbiological reactions rendered protection against toxic, carcinogenic, metabolic, as well as biological stresses induced bymicroorganisms. Heat shock proteins have been implicated as having a role in providing resistance to the host against differenttypes of stressors. In this article, some mechanistic schemes have been proposed as possible pathways globalising suchphenomena. A minute amount of stress inducers has been observed to have helped expression of stress resistance genes, providingincreased capability to the host to protect itself against myriads of both biotic and abiotic stressors. More understanding aboutsuch phenomena would help in keeping our physiological systems vigilant and our bodies healthy, fighting out the stress-relatedevents effectively. (Mol Cell Biochem 196: 117–123, 1999)

Key words: stress, resistance, protection, stress gene superfamily, protein A, lipopolysaccharide, heat shock protein, calorierestriction, metabolic stress, carcinogenic stress, chemical stress

Abbreviations: DNA – deoxyribonucleic acid; SGS – stress gene superfamily; BCG – bacillus calmette guerin; LPS –lipopolysaccharide; HSP – heat shock proteins; IL – interleukin; TNF – tumor necrosis factor; NK – natural killer; ADCC –antibody dependent cell-mediated cytotoxicity; LAK – lymphokine activated killer; Cyt – cytochrome.

multicellular organisms – invertebrates – fishes – terrestrialplants – birds – mammals . . . to Man. In the evolutionarypathway, more than 99% of all species of living organisms,which existed at one time or the other, are now extinct. Theyhave no present day descendants. They could not survive thepotentially damaging environmental pressure and wereeliminated. Throughout the entire process of biologicalevolution, the surviving organisms were better equipped withthe ability to transform into non-lethal mutants as a result ofchemically and physically induced changes in the genomicstructure. Thus, it was possible to select the best suited tosurvive the contemporary environmental conditions of life[1]. It was because of the evolution of ‘Stress ResponseGenes’ (SRG), which appear to be one of the most highlyconserved and abundant genomic sequences found in nature.

Introduction

Evolutionary dogma revisited

Some three thousand million years ago, when the first livingcell evolved on the surface of the earth, it had to survive inthe face of seemingly insurmountable environmental odds.It had to traverse a long and arduous path on its way tobecome a more organised form suitable to cope with adversecircumstances. A series of biochemical manipulations ofgenomic organization were required to adapt to the changingcircumstances. The result was the evolution of more or-ganised life forms → from photosynthetic bacteria → bluegreen algae → aerobic bacteria → eucaryotic cells →

Address for offprints: P.K. Ray, Director, Bose Institute and Head, Immunotechnology and Environmental Sciences Section, P-1/12, C.I.T. Scheme, VII-M,Calcutta – 700 054, India

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Evolutionary Gains

A large number of reports are available in the literature whichsuggest that there were unique genomic sequences whichimparted high efficiency for survival of the organism. Theyappear to have been ‘conserved’ in the genome, even acrossspecies barriers [2–6]. When required, the organisms may usethem by allowing them to be expressed freely. These genesappear to be activated to cope with certain stressful events,such as heat shock [5–7], metabolic stress [8], exposure tomicrogravity [9], hydration [10], nicotine, ethanol or heatstress [11], genotoxic stress [12], oxidative stress [13],osmotic stress [14], radiation stress [15], carcinogenic stress[16, 17] etc. It is very interesting that even though the natureof stress inducers are different in terms of their both physicaland chemical nature, the biological reactions they induce maybe similar. They all induce Heat-Shock Proteins (HSPs). Thatis to say that those SRGs can be induced by altogetherdifferent and varied nature of stress inducers or stressors, asthey will be termed throughout this article.

Usually, the urge of an organism is to adjust itself to theprevalent conditions to come back to a normal or steady state,conducive enough for perpetuation of life processes. Weknow that all organisms are continuously getting exposed toenvironmental, toxic or carcinogenic, physiological andmetabolic stressors. How are they alive then? It is becausethey possess the intrinsic capabilities to withstand divergenttypes of stressors. Unless they would have had such built-incapacity, they would not have existed thus far. Those inherentmechanisms of stress-resistance can be considered, therefore,as our evolutionary gains.

Stress gene superfamily (SGS)

It is only logical to assume that a large number of SRGs mayhave organised themselves together in a cluster in the formof a Stress Gene Superfamily (SGS). Such genes may act andfunction in harmony as per the needs of the organism. Foryears, the scientists have been trying to elucidate suchintrinsic mechanisms as are responsible for protecting the‘self’. A large number of such studies have been reported inthe literature from time to time, which looked at this problemfrom one point or the other, studying one type of stressor orthe other. It is pertinent therefore to take a note of a few ofsuch interesting observations to find out if there exists anycommon mechanism which so far escaped the attention of theresearchers.

First of all, it may be observed that although the nature ofthe stressors (inducers) used by various investigators aredifferent, the reactions they induce appear to be similar innature [5–7, 11]. On the other hand, a particular type ofstressor (inducer) was also observed to be able to induce a

large array of reactions, which are often required to protectthe host from the deleterious effects of different types ofphysical, chemical and biological stressors. These includealso the toxic or carcinogenic insults caused by a wide varietyof toxic or carcinogenic chemicals. These observations [9–34] provide us with the understanding that by using a specificstressor one may be able to activate a number of genesequences of the ‘SGS’. As a result of such activation a largenumber of biomolecules may be expressed, in a cascade typeof reactions to abrogate the harmful effects of differentstressors. This appears to be true in case of both biologicaland chemical stressors [19, 20–23].

DNA – still a puzzle

If we have to understand how such stress resistance mech-anisms function, we have to look at the genomic organization.It is known that significant stretches of the genomic se-quences still remain a great puzzle to us and their functionsare yet to be elucidated. Because of our inability to understandthe functional property of most parts of such sequences, theyhave wrongfully been labelled as ‘Junk’. Interestingly, naturehas not created anything without a meaning. There have beenscores of examples in scientific literature that some of thesame ‘Junk’ portion of the genome is made operative at times.Normally they remain dormant as reserve force and are usedwhen there is a need. It is quite understandable that theorganism keeps them on reserve, only to deal with unusualand abnormal (stressful) circumstances to safeguard the ‘self’survival. SGS appears to be still a part of this hithertounknown region of cellular DNA. It is also not knownwhether or not all cells process all the stretches of SRG or ifthey are distributed in different cell types. It is logical toassume that the latter may be the case. Further research willdelineate the actual mechanism governing the expression ofSGS.

Stress response genes versus stress resistance genes

When attempting to withstand the onslaughts of the abnormalstressful conditions the organisms may activate ‘genes’ toproduce specific proteins, each responsible for counteractingspecific or non-specific stress induced abnormalities [17–38].Such proteins may have a variety of functions: as enzymesto catalyze biotransformation and detoxification reactions(Phase I and Phase II reactions), as hormones to potentiatecell proliferation and differentiation, as structural proteins tosupply the building blocks to repair structural damages, asantibodies to nullify antigenic bacteria, fungus, virus, foreigncells and tissues etc., as cytokines to potentiate cell pro-liferation, differentiation and production of growth factors,

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carrier or transport proteins (such as chaperones, heat shockproteins), as signal proteins transmitting molecular messagesfrom membrane to the genomic sequences in the nucleus forexpressions of gene products, and also as DNA bindingproteins, transcription factors, transcription activating factors,transactivating factors etc., for proper activation and ex-pression of gene products.

Studies carried out on heat-shock proteins [5–7, 11], andvarious other stress induced proteins [8, 11–40] have yieldeda wealth of information in recent years. Genes dealing withthe resistance mechanisms in microorganisms against theimmune attack of the host, in addition to those which induceimmunity against them in the host [18–20, 25–28, 34], addedsome important information regarding the existence of StressResponse Genes in microbes. Abrogating such mechanismswould help in controlling their growth and proliferation.Nature has evolved such unique gene sequences as to provideadequate resistance mechanisms against environmental oddsof physical, chemical and biological nature in every species.The evolutionary dogma of ‘survival of the fittest’, therefore,lies in the ability of the organisms to perpetuate the StressResponse Genes vis-a-vis the Stress Resistance Genes.

Stress induction and cross tolerance phenomena

It is very intriguing to note that several immunomodulatorswhen used in very small dose, which induces small stress,such as BCG vaccine, S. aureus, S. faecalis, C. parvum,Coley’s toxin, various types of viruses, bacterial lipopoly-saccharides, lipid A, staphylococcal Protein A etc., not onlypotentiate non-specific immune responses against a varietyof bacteria and viruses, but also have been observed to causeregression of various tumors [41]. As is well understood, thetumor is a major stress inducer so far as the host is concerned.One of the major outcomes of the progressive growth of atumor is its profound suppressive effect on the immunesystem, causing lymphocytotoxicity, inhibition of phagocyticresponse, induction of humoral and cellular immunosup-pressive factors etc. Thus, it gives rise to total anergy [41] inadvanced cases of malignancy. It has been well establishedthat sensitising the host with small amounts of various typesof stressors, as are mentioned above, helps in activating thehost resistance mechanism to fight out tumor growth [41].

The various compartments of the host immune system havebeen observed to be activated by such immunomodulators,resulting in the proliferation of the lymphoid cell populations,potentiation of the natural killer cell and lymphokine acti-vated killer cell activity. It also stimulates the antibodydependent cell mediated cytotoxicity, phagocytic phenomena,increased antibody production, and an increased elicitationof growth factors and cytokines. These observed facts canonly be explained if it is accepted that the activation of any

one of the gene(s) of the SGS may help in coactivation andcoexpression of multiple gene products, perhaps in a cascadetype of reaction. It is not known, however, what are thosesequences of reactions and also how many gene products areliberated at a time, and also whether different set of SRGsare located in different cell types. Future research will throwmore light in this direction to help us in developing strategyas to when and how to induce such SRGs for sufficientprotection.

Stress-induced preconditioning

During the past decades, a tremendous volume of literaturehad indicated that ‘Calorie Restriction’ was able to providethe host with an increased ability to fight out major toxic orcarcinogenic stresses [35]. Caloric restriction definitelycauses various forms of physiological stresses in the body.

LPS-induced induction of minor oxidative stress has beenreported to stimulate the anti-oxidant mechanisms of thebody, such that the reperfusion-induced cardiac damagescould be lessened [23].

It is now well established that the resistance of the myo-cardium to ischemia can be substantially enhanced both bypreconditioning of the host and by upregulating the cyto-protective proteins, particularly heat shock proteins (HSPs).

Currie et al. [36] described the association between the heatshock proteins and myocardial protection. They showed thatby raising the body temperature in rats, both cardiac HSPsand catalase activity were increased, and at this point heartsbecame resistant to ischemia/reperfusion injury.

Of greater pathological relevance was the observation thatischaemia itself could induce evolution of HSPs and involu-tion of cardiac stress [37], by simultaneously increasing theconcentration of myocardial antioxidant enzyme, superoxidedismutase [37, 38].

Thus, it is quite evident that a very minute amount ofstress-induced shock may help potentiating the intrinsicability of the host to absorb more shock as it might have toface later. Heat shock proteins appear to play a major role inthis stress-induced resistance mechanism.

Diverse functions of Protein A of S. aureus

The above proposition is strengthened by our observationsspanned across the past two decades. We observed thatalthough Protein A of S. aureus Cowan-I was originallydiscovered for its Fc-binding affinity of IgG, and that suchproperty was utilized for complexing both IgG and immunecomplexes; the same Protein A had been observed to possessmultifarious properties, such as anti-toxic [18–23, 29–34, 28–36], anti-carcinogenic [21, 22] and anti-tumor [39–40]

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properties. It renders protection to the host against a widevariety of stressors, such as cyclophosphamide, carbontetrachloride, benzene, dimethyl benz-anthracene, Sal-monella endotoxin, Aflatoxin, various tumors, and alsointracellular parasites [18–23, 28–36, 39, 40]. Significantprotection against the toxic, carcinogenic and immuno-suppressive effects of those compounds were observed withprior sensitization of the host with a very small amount ofProtein A [18, 23, 29, 34]. Further, Protein A has been foundto cause increased phagocytic response, activate respiratoryburst phenomena, increase production of IL1, IL2, Gamma-interferon, TNF-alpha, CFU-E and CFU-MG, erythropoietin,and also effect an increase in NK cell, ADCC and LAK cellactivities. Our recent observations have indicated that ProteinA can induce selective apoptosis to tumor cells as againstspleen cells of the same tumor hosts (P.K. Ray et al., un-published observation). In general, Protein A has been shownto activate Th1 response and depress Th2 response. Thesecytoprotective and immunostimulatory properties of ProteinA, including its ability to elevate the Cyt-P450 dependenthepatic microsomal mixed function monooxygenases, Gluta-thione S-transferases Glutathione Content etc., simul-

taneously activating the bioelimination processes by activatingthe cells of monocytes and PMN lineages, have been impli-cated to be responsible for its anti-toxic, anti-carcinogenic,immunostimulatory, cytoprotective and cross-toleranceinducing properties.

The above observations provide supporting evidences toour contention that minute amount of stress induction by astressor–inducer molecule might offer increased resistanceto either the same or other stressors. These observations areindicative of the existence of SGS, expression of which wouldoffer protection to the host against various environmentalagents. One may be tempted to speculate that activation ofSGS should be able to provide cross tolerance and protectionagainst a wide variety of stressors [42]. Role of stress proteinsin myocardial protection [43] also supports our hypothesis.

Autoregulation of stress

The phenomenon of induced enzyme synthesis has been welldocumented over the years. This happens due to activationof an otherwise dormant gene sequence, which is induced by

Fig. 1. Possible mechanism of expression/coexpression of stress resistance superfamily genes by varied nature of inducers (I/II/III). The mechanism showsonly three different possibilities as an example. There could be many others. The direction of activation/coactivation is purely empirical and requiresvalidation. Obviously, the sequences of events cannot be predicted, and may depend on the nature of inducers (stressors) on the one hand, and regulatorymechanisms of the host on the other.

,

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Fig. 2. A hypothesis regarding the mechanism of small stress induction in rendering greater capability to withstand larger dosages of stressors.

the substrate itself. Also, the mechanisms of induced-drugresistance in microbes, parasites and tumour cells have beenwell established. Interestingly, there exists striking re-semblances among such inductive processes. The evolutionof heat shock proteins in plants, bacteria and mammalian cellsis yet another unique phenomena. The HSPs are liberated asa part of the host resistance phenomena. Feedback mechanismto inhibit the production of unwanted excesses of a productis a well known control mechanism in biological systems. Anelaborate control mechanism also exists to detoxify harmfulchemicals in various species, which again are mostly in-ducible systems.

Further, it has been observed that when naturally occurringinnate immune resistance fails, the induced immunopro-tective mechanisms prevail. All these phenomena appear tobe genomically linked. Normally they remain dormant or lessfunctional under the normal conditions. Any stress inducer(physical, chemical and biological) which we encounter in

our everyday life from food, water, air or by deliberateingestion of drugs may effect activation of such stress genes.Thus, such dormant genes are made operative to keep ourphysiological system vigilant to fight out the odds. Con-tinuous search in this area might provide more insight intoour understanding about what they are, and how they are,especially how they are regulated during both normal andabnormal circumstances. In the event of availability ofsuitable inducers it might be possible to boost the ability ofour ‘physiological system to render greater protection againsttoxic, carcinogenic, immunosuppressive and infectiousagents than we would have otherwise experienced [42].

Our hypothesis

It appears more and more evident from the above that a largenumber of genes may have clustered together because of the

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survival pressure in the form of a superfamily. Any one ofthem, following activation, may help triggering coexpressionof the others in the form of a cascade type of reaction. Apossible but hypothetical scheme is presented in Fig. 1.

It would have been a self-limiting mechanism if more thanone such gene were not to be coexpressed. Since to repairvarious damages caused by any environmental agent, amultitude of biochemical reactions are necessary, suchrequirement would necessitate the expression of multiplegenes. Thus, it indicates the existence of SGS and thenecessity of their co-expression. The gene products mayconstitute repair enzymes, biotransformation and detoxi-fication enzymes, cytokines, growth factors and hormones,antioxidants, heat-shock proteins, antibody molecules etc. Itis not known, however, whether all such requirements are metwith by one type of cell or if it is the collective efforts of alarge number of different types of cells, located in differenttissues and organs. It is logical to assume that the latter is thepossibility. In such a case, the mechanisms of ‘cross-talk’is required to be elucidated to have more understandingabout it.

It seems likely that deliberate introduction of minutestamount of stress may sensitize the host, alerting it to switchon its SGS, thus rendering protection against secondaryexposures to larger amount of the same and/or differentstressors (Fig. 2).

This phenomenon appears to be quite similar to thedevelopment of immunity by vaccinating the host with verysmall amount of a sensitizing substance – an antigen orimmunogen. In the immune system, the infectious substancesare generally dealt with. The concept of repair and re-construction is virtually lacking in the overall concept of thedevelopment of immunity.

It is known that to provide protection against toxic chemi-cals, mutagens, carcinogens, radiation etc., other physio-logical systems, such as Phase I and II biotransformation anddetoxification mechanisms, together with the immuno-neuro-endocrine axis, antioxidant mechanisms, repair mechanisms,cytokine-growth factors, hormone mediated growth promo-tion and proliferation phenomena, the phenomena of heatshock protein chaperoning peptide/proteins etc., appear to beinvolved in rendering the overall protection to the self. Allor some of such systems may have to function in harmonywith one another to cope with the multitude of both biotic andabiotic stress-inducers to safeguard the ‘self’.

Acknowledgements

The hard work of all my students and co-workers is thankfullyacknowledged. Fruitful discussions and suggestions of Dr.Ronald W. Hart of National Center for Toxicological Re-

search, Arkansas, Prof. D.K. Das of Cardiovascular Division,University of Connecticut, Prof. P.K. Srivastava of the CancerCenter of University of Connecticut Health Sciences Center,USA, Prof. J.J. Ghosh of Calcutta University and Prof. R.K.Mandal of Bose Institute are gratefully acknowledged. Theauthor is thankful to Mr. Arup Sengupta and Mr. ShankarSinha for their secretarial assistance and to Parthasarathi Rayand Amartya K. Ray for their editorial assistance.

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