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Cell Growth & Differentiation Vol. 10, 759-767, November 1999
© 1999 American Association for Cancer Research


Articles

Proteolysis of Heat Shock Transcription Factor Is Associated with Apoptosis in Rat Nb2 Lymphoma Cells1

Mingyu Zhang, Michael J. Blake, Peter W. Gout, Donna J. Buckley and Arthur R. Buckley2

College of Pharmacy [M. Z., D. J. B., A. R. B.] and Department of Molecular and Cellular Physiology [A. R. B.], College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0004; Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202 [M. J. B.]; and Department of Cancer Endocrinology, British Columbia Cancer Agency, Vancouver, British Columbia, V5Z 4E6 Canada [P. W. G.]

Abstract

Previously, we reported that prolactin (PRL)-dependent Nb2 lymphoma cells exhibit an aberrant heat shock response because of cysteine protease-mediated fragmentation of the heat shock transcription factor (HSF). Moreover, exposure of the cells to PRL abrogated heat-induced HSF proteolysis. The present study was conducted to investigate whether HSF proteolysis is a component of the apoptotic process in this model. Initially, the effect of heat stress (41°C for 1 h) on apoptosis, determined by agarose gel electrophoresis and flow cytometric analysis, was evaluated in PRL-dependent Nb2-11 cells and in an autonomous subline (Nb2-SFJCD1). Heat was found to induce HSF proteolysis concomitant with activation of apoptosis in each cell line; treatment with PRL blocked these effects. To determine whether HSF proteolysis occurred as a generalized phenomenon associated with apoptosis, the effects of other activators of this process were evaluated. Vinblastine, cycloheximide, and thapsigargin stimulated fragmentation of HSF and hydrolysis of DNA in each cell line. The addition of PRL blocked the effects of vinblastine but was ineffective in cells treated with either cycloheximide or thapsigargin. Iodoacetamide, a cysteine protease inhibitor that blocks HSF fragmentation, also inhibited apoptosis. In addition, Z-VAD, a general caspase antagonist, blocked vinblastine-induced fragmentation of HSF and DNA, suggesting that the enzyme responsible for proteolysis of the transcription factor was likely a caspase family member. The results suggest that proteolysis of HSF reflects the action of one or more caspases activated as a consequence of stimulation of cell death. It is concluded that HSF may represent a previously unrecognized substrate for caspases or other cysteine proteases activated during apoptosis.

Introduction

Programmed cell death, or apoptosis, plays an important role in a variety of physiological processes, including ontogenesis, tissue homeostasis, and immune regulation (1, 2, 3, 4) . It can occur spontaneously in cancer and as such has therapeutic implications for the treatment of the disease (1 , 2) . Apoptosis can be triggered by a wide range of stimuli, including neoplastic agents, physical treatments (heat and radiation) and hormone or growth factor ablation (1 , 2) . Furthermore, interaction of Fas, a member of the tumor necrosis factor receptor family, with its ligand or with an agonistic anti-Fas antibody can enhance apoptosis in lymphoid cell lines (1 , 5) . Evidence has been obtained that apoptosis can be regulated by proto-oncogenes (1) .

The mechanisms by which apoptosis is initiated vary and are dependent upon the nature of its activation and the specific cell or tissue type involved (2 , 5) . However, most cells undergoing apoptosis display similar morphological and biochemical changes that are considered characteristic of the phenomenon. For example, the fragmentation of cellular DNA, resulting from increased endonuclease activity, is well accepted as a hallmark of apoptosis (1 , 6) . Current evidence suggests that activation of specific proteases occurs concomitant with increased endonuclease activity and is a crucial event in the execution of this process (7 , 8) .

Several classes of proteases have been shown to regulate apoptosis, including serine and cysteine proteases (8) . ICE3 and ICE-like proteases are the founding members of the caspase family of cysteine proteases showing high cleavage specificity for certain amino acid motifs containing an aspartic acid (8) . ICE (caspase-1), originally identified as responsible for cleavage of pro-interleukin-1ß into the active cytokine, has been implicated in apoptosis induced by the Fas-ligand and by growth factor deprivation (9, 10, 11) . Inhibitors of ICE, such as the cowpox virus protein Crm A, can suppress apoptosis induced by various stimuli (11 , 12) . In addition to caspase-1, other caspases, including caspase-2 (Ich-1/Nedd2), caspase-3 (CPP32/Yama), caspase-4 (TX/Ich-2), and others, have been implicated in the regulation of apoptosis (13, 14, 15, 16) . For example, overexpression of caspase-3 and related proteases can cause apoptosis, and specific inhibitors of these proteases can block apoptotic cell death (8 , 14 , 17) . To date, at least 10 caspases have been implicated as mediators of apoptosis (18) .

Once activated, caspases usually cleave structural or catalytic nuclear protein substrates including fodrin (19) , protein lamin B1 (20) , a component of the U1 small nuclear ribonucleoprotein (21) , and PARP (22) . Proteolytic degradation of PARP disrupts DNA repair and facilitates apoptosis and has been used as an early biochemical marker of apoptosis (23 , 24) . Current evidence indicates a clear association between caspase activity and the progression of apoptotic processes (9 , 21 , 24 , 25) .

In previous studies, we examined the effect of heat stress (1 h at 41°C) on the expression of heat shock proteins in PRL-dependent rat Nb2-11 lymphoma cells that had been rendered quiescent via PRL starvation (26) . The unusual lack of increased heat shock protein expression following the heat stress appeared to be attributable to heat-induced, proteolytic cleavage of the HSF. In subsequent studies, evidence was obtained indicating that the proteolytic cleavage of the HSF was mediated by cysteine proteases, because the proteolysis of the HSF could be blocked by incubation with IOD, an inhibitor of these enzymes (27) . In the present study, we investigated whether an intimate relationship existed between HSF fragmentation and apoptosis induced by heat or chemical agents. The results indicate that, in Nb2 lymphoma cells, HSF may serve as a substrate for caspase-mediated proteolysis initiated by diverse apoptotic stimuli.

Results

Concomitant HSF Proteolysis and DNA Fragmentation Resulting from Heat Shock Treatment of Proliferating Nb2-11 and Nb2-SFJCD1 Cells.
We have shown previously that heat shock (1 h at 41°C) of quiescent, PRL-starved Nb2-11 cells led to cysteine protease-mediated fragmentation of HSF (27) . To investigate whether heat-induced proteolysis also occurred in proliferating PRL-dependent Nb2-11 or autonomous Nb2-SFJCD1 cells, exponentially growing cultures of each cell line were similarly subjected to heat stress (1 h at 41°C) and then returned to 37°C for further incubation. In either case, fragmentation of HSF was consistently observed as a progressive, time-dependent decline in the intensity of the Mr 90,000 HSF band, with occasional accumulation of smaller protein fragments. The onset of HSF fragmentation in the proliferating cell lines was delayed compared with that obtained previously in quiescent Nb2-11 cells, which followed immediately after the heat shock procedure (26) . As shown in Fig. 1ACitation , HSF fragmentation in the Nb2-SFJCD1 cells was first detected after 1.5 h, whereas in the Nb2-11 cells, fragmentation of HSF appeared after 2 h. Thus, proliferating Nb2-11 and Nb2-SFJCD1 cells appear more resistant to heat shock-induced proteolysis of HSF than quiescent Nb2-11 cells that, as a result of PRL starvation, are on the verge of apoptosis.



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Fig. 1. Heat shock causes HSF and DNA fragmentation in Nb2 lymphoma cells. Cells from exponentially growing Nb2-11 and Nb2-SFJCD1 cultures were subjected to heat shock (1 h at 41°C), returned to 37°C for further incubation, and then harvested at intervals. The time points represent the hour of incubation after the heat shock treatment. Control cells (C) were not subjected to heat shock. A, HSF fragmentation determined by immunoblotting. B, DNA fragmentation determined by gel electrophoresis. MW, molecular weight.

 
Heat treatment is a well-recognized stimulus for induction of apoptosis (28, 29, 30) . To investigate whether heat stress (1 h at 41°C) induced apoptosis in addition to HSF proteolysis and whether there was a correlation between the two phenomena in proliferating Nb2-11 and Nb2-SFJCD1 cells, exponentially growing cultures of each cell line were evaluated for heat-provoked DNA fragmentation. As shown in Fig. 1BCitation , the heat stress led to substantial DNA fragmentation in both cell lines. Taken together, the results presented in Fig. 1Citation suggest that heat stress, applied to both lymphoma cell lines in the proliferative phase, causes apoptosis coincident with HSF proteolysis.

Effects of IOD and PRL on Heat-induced Fragmentation of HSF and DNA.
We demonstrated previously that heat-induced HSF fragmentation in quiescent, PRL-starved Nb2-11 cells could be inhibited by treatment with IOD, a cysteine protease antagonist, or with PRL (27) . In the present study, it was examined whether these agents could similarly inhibit heat-induced HSF proteolysis in proliferating Nb2-11 or Nb2-SFJCD1 cells, as well as heat-induced DNA fragmentation and apoptosis. The results in Fig. 2ACitation indicate that incubation with IOD (0.05 mM), beginning 1 h before heat stress application, completely inhibited heat-induced HSF fragmentation in both cell lines. Similarly, HSF fragmentation in Nb2-SFJCD1 cells was inhibited by incubation with PRL (20 ng/ml), starting 2 h before initiation of the heat stress. In contrast, PRL had no effect on HSF fragmentation in Nb2-11 cells. This lack of effect was expected because the Nb2-11 cells were already exposed to excessive amounts of lactogenic hormone present in the fetal bovine serum component of the maintenance medium. The results indicate that there is a significant difference between the two cell lines with regard to their response to the induced stress and the role of PRL in this process.



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Fig. 2. Effect of PRL and IOD on heat-induced HSF fragmentation, DNA cleavage, and percentage of apoptotic cells. Exponentially growing Nb2-11 and Nb2-SFJCD1 cultures were incubated with PRL (20 ng/ml) for 2 h or IOD (0.05 mM) for 1 h. Cells were then subjected to heat shock (1 h at 41°C; HS) and further incubated for 3 h at 37°C, after which they were harvested for analysis. Control cells (C) received neither drug nor heat shock treatment. A, HSF fragmentation determined by immunoblotting. B, DNA fragmentation determined by gel electrophoresis. C, percentage of apoptotic cells determined by flow cytometric analysis. *, those sample means that were significantly different from means of control cultures (P < 0.01); **, those sample means that were significantly different from means of heat-shocked cultures. Bars, SE.

 
As shown in Fig. 2BCitation , heat-induced DNA fragmentation was blocked by treatment with IOD in both Nb2-11 and Nb2-SFJCD1 cells but was inhibited by PRL only in the Nb2-SFJCD1 cultures. To quantitate the effects of heat shock on fragmentation of DNA in growing cells, identically treated cultures of the two cell lines were processed for flow cytometric analysis of propidium iodide-stained DNA. As shown in Fig. 2CCitation , heat stress consistently induced a high percentage of apoptotic cells in both cell lines. Whereas IOD significantly reduced the percentage of heat-induced apoptotic cells in each line, PRL inhibited heat-induced apoptosis only in Nb2-SFJCD1 cells.

Taken together, the results of Fig. 2Citation indicate that heat shock induces HSF proteolysis and apoptosis with similar kinetic patterns. Moreover, treatment with either IOD or PRL identically opposes either heat-provoked phenomenon, at least in the Nb2-SFJCD1 cells. It appears therefore that heat-induced proteolysis of HSF may be a common feature of apoptosis in Nb2 lymphoma cells.

Vinblastine-induced HSF Fragmentation.
To investigate whether the observed association between HSF proteolysis and apoptosis was specific for heat shock stimulation, the antimitotic agent vinblastine was evaluated as an alternative stimulus of programmed cell death (31) . HSF fragmentation was determined in quiescent Nb2-11 cells treated for 3–12 h with vinblastine (100 ng/ml). As shown in Fig. 3ACitation , proteolysis of HSF occurred within 3 h with maximal fragmentation apparent by 6 h. Concentration-response experiments revealed that about 100 ng/ml of vinblastine were required to induce HSF fragmentation within 6 h (Fig. 3B)Citation . Importantly, control cultures (no vinblastine) showed detectable HSF fragmentation during the same time period, consistent with PRL starvation-induced apoptosis (Fig. 3ACitation ; Ref. 32 ).



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Fig. 3. Vinblastine-induced HSF fragmentation in Nb2 lymphoma cells. A, time course of the effects of vinblastine on Nb2-11 cells. Quiescent, PRL-starved Nb2-11 cells were incubated with vinblastine (100 ng/ml) for 3, 6, 9, and 12 h or without the alkaloid (C). B, dose-effect relationship of vinblastine in cultures of quiescent Nb2-11 cells during a 6-h incubation period. C, combination of time course (3, 6, and 9 h) and dose (0.1 and 1 µg/ml) of vinblastine in Nb2-SFJCD1 cultures.

 
The effects of vinblastine on HSF fragmentation were also investigated in autonomous Nb2-SFJCD1 cells. Of the drug concentrations evaluated, only 1 µg/ml caused detectable HSF fragmentation after 3–6 h of incubation (Fig. 3C)Citation . Cells from exponentially growing Nb2-11 cultures exhibited a similar time-dependent concentration-response to vinblastine (data not shown). The results indicate that proliferating Nb2-11 and Nb2-SFJCD1 cells are equally resistant to vinblastine-induced HSF fragmentation and more so than quiescent, PRL-starved Nb2-11 cells.

Effects of IOD and PRL on Vinblastine-induced HSF Proteolysis and Apoptosis in Nb2-SFJCD1 Cells.
To further examine vinblastine-induced HSF fragmentation, Nb2-SFJCD1 cells were incubated with IOD or PRL, beginning 1 and 2 h before a 6-h exposure to vinblastine (1 µg/ml), respectively. As shown in Fig. 4ACitation , HSF proteolysis was markedly inhibited or reduced by IOD and PRL, respectively. The results obtained are very similar to the inhibitory effects of the two agents on heat stress-induced HSF fragmentation and suggest that treatment with vinblastine also leads to activation of cysteine proteases.



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Fig. 4. Effect of IOD and PRL on vinblastine-induced HSF fragmentation, DNA cleavage, and appearance of apoptotic cells in Nb2-SFJCD1 cell cultures. Nb2-SFJCD1 cells were incubated with vinblastine (1 µg/ml) for 6 h in the absence and presence of PRL (20 ng/ml) or IOD (0.05 mM), added to the cultures 2 or 1 h prior to vinblastine addition, respectively. Control cultures (C) received no drug treatment. A, HSF fragmentation determined by immunoblotting. B, DNA fragmentation determined by gel electrophoresis. C, percentage of apoptotic cells determined by flow cytometric analysis. *, those sample means that were significantly different from means of control cultures (P < 0.01); **, those sample means that were significantly different from means of cultures treated with only vinblastine (Vin). Bars, SE.

 
To determine whether treatment with vinblastine also led to apoptosis, the Nb2-SFJCD1 cultures were evaluated for DNA fragmentation, using agarose gel electrophoresis and flow cytometry. As shown in Fig. 4BCitation , vinblastine caused apoptosis as characterized by DNA fragmentation. The latter was markedly inhibited by IOD or PRL, added to the cultures prior to treatment with the antimitotic agent (Fig. 4B)Citation . Quantitation of the apoptotic response also indicated that vinblastine-induced apoptosis is significantly reduced by IOD and PRL (Fig. 4C)Citation .

Vinblastine-induced apoptosis, and blocking of this process by IOD or PRL, was also examined using proliferating Nb2-11 cells. The results indicate that vinblastine (1 µg/ml, 6 h) induced apoptosis and that IOD blocked both HSF fragmentation and apoptosis similar to its demonstrated effects in Nb2-SFJCD1 cells. However, as expected, PRL did not inhibit either HSF fragmentation or apoptosis in the Nb2-11 cultures (data not shown).

HSF Proteolysis, a Common Feature Associated with Apoptosis in Nb2 Lymphoma Cells.
To investigate whether proteolysis of HSF was a general, apoptosis-associated event in Nb2 lymphoma cells, experiments were initiated using additional inducers of apoptosis including TG and CHX. TG is a specific inhibitor of an endoplastic reticulum-associated Ca2+-ATPase, the inhibition of which has been reported to lead to apoptosis (33) . Nb2-SFJCD1 cells were incubated with TG (100 nM) for 6 h, and its effects on HSF proteolysis and percentage of cells undergoing apoptosis were determined (Fig. 5, A and B)Citation . As shown in Fig. 5ACitation , TG provoked HSF proteolysis; IOD inhibited this effect and also blocked TG-induced apoptosis (Fig. 5B)Citation . However, PRL did not suppress the HSF proteolysis or apoptosis induced by treatment of the Nb2-SFJCD1 cells with TG (Fig. 5, A and C)Citation , in contrast to its inhibitory effects on these phenomena when induced by treatment with heat shock or vinblastine (see Figs. 2Citation and 4Citation ). The degree of DNA fragmentation induced by TG was found to be consistent with its effect on HSF proteolysis with regard to time course and concentration-response (data not shown). The response to TG of proliferating Nb2-11 cells was similar to that of the Nb2-SFJCD1 cells (data not shown).



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Fig. 5. Effect of IOD and PRL on HSF fragmentation induced in Nb2-SFJCD1 cells by TG and CHX. Nb2-SFJCD1 cultures were incubated with PRL (20 ng/ml) for 2 h or IOD (0.05 mM) for 1 h. Following this, CHX (10 µg/ml) or TG (100 nM) was added for an additional 6-h incubation, after which cells were harvested for HSF immunoblotting and DNA flow cytometric analysis. A control group (C) received no treatment, and some cultures received either TG or CHX alone. A and B, HSF fragmentation. C, percentage of apoptotic cells. *, those sample means that were significantly different from means of control cultures (P < 0.01); **, those sample means that were significantly different from means of cultures treated with only TG or only CHX. Bars, SE.

 
Inhibition of protein synthesis by CHX has been reported to induce apoptosis in other systems (34) . Incubation of Nb2-SFJCD1 cells with CHX (10 µg/ml) caused HSF fragmentation, an effect that was blocked by IOD but not by PRL (Fig. 5B)Citation . Similarly, CHX induced apoptosis, as determined by DNA fragmentation (data not shown) and flow cytometric analysis (Fig. 5C)Citation , a process that was inhibited by IOD but not by PRL. Proliferating Nb2-11 cells had similar responses to treatment with CHX as the Nb2-SFJCD1 cells (data not shown).

Inhibition of HSF and DNA Fragmentation in Nb2-SFJCD1 Cells by Caspase Inhibitor Z-VAD-fmk.
Caspases are known to play a pivotal role in apoptosis (18) . Therefore, the possibility that the cysteine protease(s) responsible for the apoptosis-associated HSF fragmentation was a member(s) of the caspase family was investigated. Toward this end, a series of specific caspase inhibitors were evaluated for inhibition of HSF fragmentation including: Z-YVAD-fmk (caspase-1); Z-VDVAD-fmk (caspase-2); Z-DEVD-fmk (caspase-3); Z-VEID (caspase-6); Z-IETD-fmk (caspase-8); and Z-VAD-fmk (a general inhibitor). The use of caspase antagonists is a widely used experimental approach to assess involvement of this family of cysteine proteases in cellular responses (28 , 35, 36, 37, 38) . The inhibitors (100–300 µM) were added to Nb2-SFJCD1 cells 1 h before a 6-h treatment with vinblastine (1 µg/ml). As shown in Fig. 6ACitation , the general caspase inhibitor, Z-VAD-fmk, markedly inhibited vinblastine-induced HSF fragmentation, characterized by dissolution of HSF (Mr 90,000). In contrast, the caspase-3 inhibitor, Z-DEVD-fmk, did not significantly inhibit the vinblastine-induced HSF cleavage (Fig. 6A)Citation , nor did any of the other specific caspase antagonists (data not shown). The effects of Z-VAD-fmk on vinblastine-induced DNA fragmentation in the cultures were also assessed. Similar to its effect on HSF fragmentation, Z-VAD-fmk substantially inhibited vinblastine-induced DNA fragmentation (Fig. 6B)Citation . These results indicate that the cysteine protease(s) responsible for the vinblastine-induced cleavage of HSF is probably a caspase family member but is most likely not caspase 1, 2, 3, 6, or 8.



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Fig. 6. Effect of caspase inhibitors on vinblastine-induced HSF and DNA fragmentation in Nb2-SFJCD1 cells. Nb2-SFJCD1 cultures were incubated with Z-VAD-fmk (100 µM) or Z-DEVD-fmk (300 µM) for 1 h, after which vinblastine (final concentration, 1 µg/ml) was added for an additional 6-h incubation and subsequent analysis of HSF fragmentation (immunoblotting; A) and DNA fragmentation (gel electrophoresis; B). A control group (C) received no treatment, and some cultures received only vinblastine (Vin).

 
Discussion

There is considerable evidence indicating that multiple intracellular proteases, most notably caspases, are central to apoptosis. In the present study, we have demonstrated that proteolytic cleavage of HSF, mediated by cysteine proteases, is associated with apoptosis in Nb2 lymphoma cells induced by a variety of disparate stimuli. Heat shock, vinblastine, TG, CHX, and deprivation of growth factors are known to cause apoptosis in other systems via diverse mechanisms (27 , 31 , 33 , 34) . Importantly, exposure of Nb2 lymphoma cells to these agents each induced fragmentation of HSF as well as apoptosis. In addition to exogenous activation of apoptosis, PRL deprivation of Nb2-11 cells also caused apoptosis (32) and provoked HSF proteolysis (Fig. 3A)Citation . Moreover, a cysteine protease inhibitor, IOD, concomitantly blocked proteolysis of HSF and apoptosis. These observations indicate that proteolysis of HSF stems from activation of a cysteine protease that is likely to be common to multiple signaling pathways leading to apoptosis in Nb2 lymphoma cells.

Activation of a cascade of related caspases has been implicated in apoptosis (13) , and several substrates of these proteases have been identified. They include nuclear proteins such as nuclear matrix protein lamin B1 (20) , the DNA repair enzyme PARP (22) , the Mr 70,000 protein component of the U1 small nuclear ribonucleoprotein (21) , and the catalytic subunit of DNA-dependent protein kinase (39) . A cycloplasmic protein, fodrin, has also been shown to be proteolytically cleaved during apoptosis (19) . Degradation of these proteins appears to proceed in a selective fashion. Although the exact role of the proteolytic cleavage in the execution phase of apoptosis remains to be established, it is noteworthy that many of the identified substrates have a role in DNA repair or are involved in maintaining the integrity of the nucleus or cytoskeleton. The disruption of these activities appears to facilitate the process of apoptosis. Identification of additional cleavage targets may be one approach to clarify regulatory mechanisms of apoptosis. Here we present results that suggest that the transcription factor, HSF, may represent a substrate for caspase-mediated degradation in the apoptotic process.

Accumulation of heat shock proteins tends to render cells more resistant to programmed cell death and increase cell survival (40 , 41) . Conversely, disruption of heat shock protein expression by antisense oligonucleotides enhances sensitivity of cells to apoptosis (42) . These observations suggest that expression of heat shock proteins forms an endogenous protective mechanism against apoptosis. In view of this, selective cleavage of HSF, a key factor in heat shock protein production at the transcription level (43) , could abolish this cytoprotection and hence facilitate apoptosis. The results of the present study, which suggest that selective cleavage of HSF may be a scheduled event in apoptosis, are consistent with this hypothesis.

The identity of the protease(s) responsible for the cleavage of HSF was not fully established in the present study. Because the general caspase antagonist Z-VAD-fmk blocked the degradation of the protein, the protease involved is most likely a caspase family member. However, none of the specific caspase inhibitors used blocked HSF proteolysis, indicating that caspases 1–3, 6, or 8 are not likely to have key roles in this process. Future studies in which the putative caspase cleavage site(s) of HSF can be identified, modified, and used in transfection experiments will provide important insight into the mechanism of transcription factor fragmentation and the protease(s) responsible for it.

The mechanisms involved in the induction of HSF fragmentation by heat shock and other apoptotic stimuli are also not understood. In this report, several apoptotic stimuli, each acting via a different mechanism, induced both HSF proteolysis and apoptosis in Nb2 lymphoma cells. It is possible that individual molecular pathways, triggered by the different stimuli, converge to activate a particular protease, thereby setting in motion the process of programmed cell death. Alternatively, a common mediator may be activated by the diverse stimuli which, in turn, activates one or more proteases. In this context, it is notable that Ca2+ apparently has a key role in certain apoptotic events that can be triggered by a variety of stimuli (44 , 45) ; Ca2+-independent mechanisms, however, also exist (46 , 47) . TG, an inhibitor of sarcoplasmic and endoplasmic reticular Ca2+-ATPases, was found to induce HSF proteolysis and apoptosis in Nb2 cells (Fig. 5)Citation , suggesting that Ca2+ is involved in the triggering of protease activation and apoptosis in this lymphoma model. However, depletion of extracellular Ca2+ with EGTA did not affect HSF fragmentation induced by heat shock or vinblastine treatment (data not shown). Moreover, 1,2-bis (2-amino phenoxy) ethane-N,N,N1,N1-tetraacetic acid acetoxy-methylester, a chelator of intracellular Ca2+, did not prevent HSF fragmentation (data not shown). It appears, therefore, that Ca2+ is very likely not a common signaling component involved in protease activation and induction of apoptosis in Nb2 lymphoma cells.

Commitment to apoptotic cell death is thought to be determined by a balance in cells between negative and positive regulations that are executed by groups of gene products (48) . The observation that quiescent, PRL-starved Nb2-11 cells are more sensitive to apoptotic stimuli than proliferating Nb2-11 and Nb2-SFJCD1 cells can be explained by a lack of expression of antiapoptosis genes in the quiescent Nb2-11 cells that, as a result of PRL deprivation, are on the verge of apoptosis. This suggestion is supported by the observation that the expressions of bcl-2 and pim-1 genes in quiescent Nb2-11 cells are very low, in contrast to those in PRL-stimulated Nb2-11 cells and in Nb2-SFJCD1 cells, in which they are constitutive but nevertheless enhanced by PRL (49 , 50) . Therefore, augmentation of antiapoptosis gene expression by PRL in Nb2-SFJCD1 cells may contribute to the protective effect of the hormone against HSF cleavage and apoptosis. This idea is supported by the observation that inhibition of protein or RNA synthesis abrogated the inhibitory effects of PRL (data not shown). It is possible that gene products that inhibit activation of caspases or other proteases may be located upstream in the apoptotic signaling pathway. This could explain the observation that PRL can inhibit HSF fragmentation and apoptosis induced in Nb2-SFJCD1 cells by heat shock or treatment with vinblastine but not the HSF cleavage induced by CHX.

In summary, HSF was found to be a substrate for apoptosis-associated proteolysis in Nb2 lymphoma cells. Further studies of the mechanism(s) by which PRL can prevent HSF fragmentation and accompanying apoptosis and identification of the enzyme(s) mediating the proteolysis could lead to a better understanding of the molecular pathways leading to apoptotic cell death.

MATERIALS AND METHODS

Materials.
Ovine PRL (NIDDK oPRL-20 and AFP 10677 C) was obtained through the NIH Pituitary Hormone and Antisera Program (Bethesda, MD). Rat monoclonal antibodies that recognize HSF1 or HSF2 were generously provided by Dr. R. I. Morimoto (Chicago, IL). Caspase inhibitors were obtained from Enzyme Systems Products (Livermore, CA). Unless otherwise specified, all other reagents were of molecular biology grade and obtained from Sigma Chemical Co (St. Louis, MO).

Cell Cultures.
The PRL-dependent, cloned rat pre-T lymphoma cell line, Nb2-11, and the PRL-independent subline, Nb2-SFJCD1, developed from the parental Nb2 line by lactogen starvation and cloning of surviving cells, were used (26 , 27) . Nb2-11 cell cultures were maintained at 37°C in Fischer’s medium containing 10% fetal bovine serum (BioWhittaker, Walkersville, MD) as a source of lactogen, 10% horse serum (BioWhittaker), 2-ME (10-4 M), penicillin (50 units/ml), and streptomycin (50 mg/ml; maintenance medium), as originally described for the parental Nb2 lymphoma line (51) . The Nb2-SFJCD1 subline was cultured in maintenance medium from which fetal bovine serum had been omitted. In most experiments, log phase Nb2-SFJCD1 and Nb2-11 cells were used. When required, Nb2-11 cells were first rendered quiescent via PRL starvation by a preincubation for 18–24 h in PRL (lactogen)-free medium, i.e., Fischer’s medium supplemented with 2-ME, antibiotics, and 10% nonmitogenic gelding serum (ICN, Irvine, CA). To ensure proper comparison, Nb2-SFJCD1 cells were similarly treated. Under these conditions, Nb2-11 cells were growth arrested in the early G1 phase of the cell cycle, whereas Nb2-SFJCD1 cells continued to cycle.

Immunoblotting.
Cells were homogenized in ice-cold buffer containing 20 mM HEPES (pH 7.5), 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM DTT, 0.4 M NaCl, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM leupeptin at 4°C. Insoluble material was removed by microcentrifugation. Total protein in the supernatant was quantitated using a modified Lowry method (Bio-Rad). Protein samples (20 µg/lane) were resolved using 12% SDS-polyacrylamide gels and transferred to Immuno-Lite Blotting Membrane (Bio-Rad). Membranes were blocked in 5% nonfat dry milk, and immunoblotting was performed using rat monoclonal antibodies that recognize HSF1 or HSF2. A goat anti-rat IgG conjugated to alkaline phosphatase was used as a secondary antibody. Protein bands were visualized using a chemiluminescent reaction (Immun-Lite assay kit; Bio-Rad) followed by exposure to X-ray film.

DNA Fragmentation Analysis.
Fragmentation of DNA was used as a measure of apoptosis. DNA was isolated from cells essentially as described previously (52) . Briefly, cells (1–3 x 106 cells/treatment) were harvested, washed, resuspended in 1 ml PBS and fixed in 70% ice-cold ethanol. The fixed cells were centrifuged at 800 x g and resuspended in a buffer containing 194 mM Na2HPO4 and 4 mM citric acid and then were centrifuged at 1000 x g. The resulting supernatants were diluted with 0.25% NP40 and then incubated first with RNase A (1 mg/ml), followed by a second incubation in the presence of proteinase K. The samples were mixed with a buffer containing 0.25% bromphenol blue, 0.25% xylene cyanol FF, and 30% glycerol, resolved on 1.8% agarose gels, and following ethidium bromide staining, visualized using UV light.

Flow Cytometric Analysis.
Apoptosis in cells was quantitated using the method described by Nicoletti et al. (53) . Aliquots containing 1–2 x 106 cells were centrifuged at 200 x g and fixed in cold (-20°C) 70% ethanol. Samples were stored at 4°C for 60 min. The cells were subsequently washed twice in PBS, resuspended in RNase (1 mg/ml; type 1-A), and incubated at 37°C for 60 min. Cells were again washed in PBS and then resuspended in propidium iodide (50 mg/ml in PBS) solution for flow cytometric analysis. To this end, an Elite flow cytometer (Coulter Electronics, Hialeah, FL) was used with the 488-nm line of an enterprise laser (Coherent, Palo Alto, CA). Red fluorescence of the propidium iodide-stained cells was monitored through a 600-nm dichroic and a 610 LP filter. Forward scatter and side scatter were measured simultaneously. Time-of-flight measurement was used to exclude cellular debris and clumps. All samples were assessed under identical instrument settings.

Data Analysis.
Data are presented from experiments replicated three times unless otherwise specified. Where applicable, data are presented as the mean ± SE. Statistically significant differences among treatment groups were evaluated by ANOVA, followed by the student Newman Kuel’s post-test for multiple comparisons.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grants DK53452 and NS30493 from the NIH, Grant 95B086 from the American Institute for Cancer Research, and grants from the Ohio Cancer Research Associates and the British Columbia Cancer Agency. Back

2 To whom requests for reprints should be addressed, at College of Pharmacy and Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, 3223 Eden Avenue, Cincinnati, OH 45267. Phone: (513) 558-2575; Fax: (513) 558-0978; E-mail: Arthur.Buckley{at}uc.edu Back

3 The abbreviations used are: ICE, interleukin-1ß-converting enzyme; PARP, poly(ADP-ribose) polymerase; PRL, prolactin; HSF, heat shock transcription factor; IOD, iodoacetamide; TG, thapsigargin; CHX, cycloheximide; 2-ME, 2-mercaptoethanol. Back

Received for publication 6/ 3/99. Revision received 9/17/99. Accepted for publication 9/20/99.

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