AREA DE ESTUDIOS

When cells of S. cerevisiae are shifted from a low growth temperature (2Q0C) to a lethal temperature (48oC) the cells are rapidly killed. However, if cells are first pre-adapted by incubation at a higher non-lethal temperature (ca. 37-4Q0C for 30 minutes) the rate of killing upon upshift to a lethal temperature is dramatically decreased (M cAlister and Finkelstein, 1980). This phenomenon is exhibited by a variety of systems including bacteria. Drosophila, plant and mammalian cells (Hall, 1983 and references therein), and is referred to as acquired thermotolerance. A cquired therm otolerance has been extensively studied, especially in mammalian cells. This is largely because of the upsurge in interest in the use of heat as an adjunctive technique for the treatment of certain cancers in humans.

1.7.1 The role of hsps and other factors in acquired thermotolerance.

M any studies demonstrate that preferential synthesis of hsps occurs during the period when development of thermotolerance is at its maximal rate. The argument that hsps are involved is strengthened by the fact that other inducers of thermotolerance (apart from heat) also induce hsps. In addition, individual hsps have been im plicated in acquired thermotolerance, including hsp70 (Angelidis et at., 1991) and hsp 104 (Sanchez and Lindquist, 1990). However the importance of hsps in acquired thermotolerance may be overestimated. For instance, an S. cerevisiae strain carrying a deletion of the gene coding fo r hsp 104 may show defects in acquired therm otolerance, but they only m anifest themselves after five minutes at the lethal temperature (De Virgilio et al., 1991; Sanchez and Lindquist, 1990 and Parsell et al., 1991). Furtherm ore, the reduction in acquired thermotolerance exhibited by this mutant alm ost disappears when the time o f pre­ adaptation at the higher non-lethal temperature is increased from 30 to 60 minutes (De Virgilio et al., 1991). This may suggest a m inor role for individual hsps in acquired thermotolerance. A major role for a combination of hsps may be inferred from this, but

studies with inhibitors of protein synthesis make this doubtful.

Many groups have investigated the developm ent of therm otolerance by using inhibitors of protein synthesis to eliminate the participation of hsps. This has yielded mixed results. Some groups maintain that hsps do play a role (McAlister and Finkelstein, 1980; Li and Werb, 1982); others report that blocking hsp synthesis has no effect (Hall, 1983; W atson et al., 1984; W idelitz et al., 1986). Some of the latter reports may be dismissed because the effectiveness of the inhibitors had not been tested. However this does not apply in every case. Two of the more obvious explanations for such anomalies are; i) incorrect interpretation of thermotolerance data and ii) the selection of single time points at which to measure thermotolerance resulting in the participation of hsps being missed. For example, choosing early points (< 5 minutes) in the killing curves of S.

cerevisiae will not reveal the role of hsp 104 in acquired thermotolerance.

All available data lead to the conclusion that, though hsps may play a minor role in thermotolerance, the mechanisms that make the greatest contribution are poorly-understood hsp-independent events associated with heat shock. It is obvious that experiments designed to investigate such mechanisms (involving inhibitors of protein synthesis or transcription) have their drawbacks. For instance, the widely employed transcriptional inhibitor 1,10- phenanthroline enhances the abundance of certain heat-shock genes (Adams and Gross, 1991). These problems can be overcome by using the recently-discovered hsf 1 -m3 mutation which prevents activation of heat-shock transcription factor; and, even though it causes a general block in heat inducible transcription, has hardly any effect on inducible (acquired) thermotolerance (Smith and Yaffe, 1991).

The question arises as to the nature of these hsp independent mechanisms that confer thermotolerance to the cell. A useful perspective from which to study them is to determine the cellular changes that occur on heat shock (these are discussed fully in sections 1.8-1.8.5). One of the most dramatic consequences of heat shock is the increase in levels of the non-reducing disaccharide trehalose. This has been observed in S.

cerevisiae, S. pombe and Neurosporra crassa (Hottiger et al., 1987; De Virgilio et al.,

1990 and Neves et al., 1991, respectively). Moreover, thermotolerance increases in parallel to trehalose accumulation and decreases in parallel to the trehalose levels when cells are shifted back to normal tem peratures, irrespective o f the presence or absence of cycloheximide (De Virgilio et al., 1990). A ccum ulation of trehalose also occurs on exposure to other inducers of thermotolerance, such as ethanol and hydrogen peroxide (Attfield, 1987).

It is well established that physiological state has a dramatic effect on both basal and acquired thermotolerance. The level of cAMRdepende nt protein kinase (protein kinase A) plays a major role here. Yeast in rapid fermentative growth has high levels of cAMP and low basal thermotolerance. In contrast, quiescent cells have low levels of cAMP and high levels of basal thermotolerance. Moreover, studies employing mutants of the yeast cAMP

system show that low protein kinase A activity correlates with high thermotolerance (lida, 1988), whereas high protein kinase A activity results in low thermotolerance (Tanaka et al.,

1989).

Acquired thermotolerance in rapidly growing cells is also depend/mt on the activity o f this kinase. Compared to its isogenic wild type, a m ild heat shock will not induce acquired thermotolerance in a strain with constitutively high levels o f protein kinase A activity (Shin et al., 1987). Conversely, cells with constitutively low levels o f cAMP are constitutively thermotolerant i.e cells in exponential phase of growth behave as if they have already experienced a sub-lethal, pre-adaptive heat shock (Shin et al., 1987).

It has been found that, like stationary phase cells, wild type S. cerevisiae is transiently arrested at the pre-replicative phase of the cell cycle by a sub-lethal heat shock (230-36^0 (Johnson and Singer, 1980). Paradoxically, the implied depression in cAMP level due to heat shock does not occur; levels o f this metabolite rise instead (see section 1.6).

In addition to heat itself many agents can induce thermotolerance. In S. cerevisiae^

for example,these agents include ethanol (Plesset etal., 1982), osmotic stress (Trollmo et al., 1988) and hydrogen peroxide (C ollinson and D awes, 1992). H ow ever the physiological overlap between some o f these agents and heat shock is unidirectional i.e. heat conditioning does not confer tolerance to osmotic shock (Trollmo et al., 1988) or oxidative stress (Collinson and Dawes, 1992). These phenomena are poorly understood, and understanding stress tolerance remains a future research goal.