Tumor-derived inducible heat-shock protein 70 (HSP70) is an essential component of anti-tumor immunity.

The anti-apoptotic function and tumor-associated expression of heat-shock protein 70 (HSP70) is consistent with HSP70 functioning as a survival factor to promote tumorigenesis. However, its immunomodulatory activities to induce anti-tumor immunity predict the suppression of tumor growth. Using the Hsp70.1/3(-/-)(Hsp70(-/-)) mouse model, we observed that tumor-derived HSP70 was neither required for cellular transformation nor for in vivo tumor growth. Hsp70(-/-) murine embryonic fibroblasts (MEFs) were transformed by E1A/Ras and generated tumors in immunodeficient hosts as efficiently as wild-type (WT) transformants. Comparison of Bcr-Abl-mediated transformation of WT and Hsp70(-/-) bone marrow and progression of B-cell leukemogenesis in vivo revealed no differences in disease onset or survival rates, and Eµ-Myc-driven lymphoma in Hsp70(-/-) mice was phenotypically indistinguishable from that in WT Eµ-Myc mice. However, Hsp70(-/-) E1A/Ras MEFs generated significantly larger tumors than their WT counterparts in C57BL/6 J immune-competent hosts. Concurrent with this was a reduction in intra-tumoral infiltration of innate and adaptive immune cells, including macrophages and CD8(+) T cells. Evaluation of several potential mechanisms revealed an HSP70-chemokine-like activity to promote cellular migration. These observations support a role for tumor-derived HSP70 in facilitating anti-tumor immunity to limit tumor growth and highlight the potential consequences of anti-HSP70 therapy as an efficacious anti-cancer strategy.

Fas ligand gene expression is directly regulated by stress-inducible heat shock transcription factor-1.

Heat shock transcription factor-1 (HSF-1) is the primary stress responsive transcription factor that regulates expression of heat shock proteins (Hsps) in response to elevated temperature. We show that the transcriptional activity of HSF-1 can also directly mediate hyperthermia-induced Fas ligand (FasL) expression in activated T cells. We identify a conserved region within the human FasL promoter spanning from -276 to -236 upstream of the translational start site that contains two 15 bp non-identical adjacent HSF-1-binding sites or heat shock elements (HSEs) separated by 11 bp. Both the distal HSE (HSE1) (extending from -276 to -262) and the proximal HSE (HSE2) (spanning from -250 to -236) consist of two perfect and one imperfect nGAAn pentamers. We show the direct binding of HSF-1 to these elements and that mutation of these sites abrogates the ability of HSF-1 to bind and drive promoter activity. HSF-1 associates with these elements in a cooperative manner to mediate optimal promoter activity. We propose that the ability of HSF-1 to mediate stress-inducible expression of FasL extends its classical function as a regulator of Hsps to encompass a function for this transcription factor in the regulation of immune function and homeostasis.

Stressed to death: regulation of apoptotic signaling pathways by the heat shock proteins.

Cellular damage can engage two fundamental cellular responses: apoptosis, a precisely regulated form of cell death; and the heat shock protein (Hsp), or stress response, which functions to protect cells and to mediate an accelerated recovery following damage. The coordinated balance between these two opposing pathways governs the ultimate fate of the cell--whether it lives or dies. The self-destruction of a cell is mediated by one of many signaling pathways culminating in the activation of the caspase proteases. The Hsps regulate the activity of multiple intracellular signaling intermediates, many of which are intimately involved in the execution of the apoptotic signaling pathways. This review addresses whether the antiapoptotic activities of several Hsps, including Hsp70, Hsp90, and Hsp27, can be attributed to their collective ability to regulate the activities, expression, or both of apoptotic signaling molecules. In summary, the functional interface between the ancient heat shock or stress protein response and the highly conserved biochemical pathways leading to the activation of apoptosis governs the susceptibility of a cell to damaging stimuli.

Stress management - heat shock protein-70 and the regulation of apoptosis.

Apoptosis, molecularly regulated cell death, can be induced by a range of environmental, physical or chemical stresses, and is characterized by a sequence of precisely regulated events that culminate in the self-destruction of a cell. Fascinating biochemical and genetic parallels exist between the cell death pathways of different animal species. However, an even more highly conserved and evolutionarily ancient cellular response can be engaged as a consequence of stress, which functions to maintain cellular survival. This response is mediated by the heat-shock or stress proteins. This article discusses the functional interactions between the stress response and the apoptotic cell death pathway and how these might impact on determining cellular survival.

A 'non-canonical' DNA-binding element mediates the response of the Fas-ligand promoter to c-Myc.

Cell number is regulated by maintaining a balance between cell proliferation and cell death through apoptosis. Key regulators of this balance include the oncogene product c-Myc, which promotes either entry into the cell cycle or apoptosis [1]. Although the mechanism of c-Myc-induced apoptosis remains unclear, it is susceptible to regulation by survival factors [2,3] and can proceed through the interaction of Fas ligand (FasL) with its receptor, Fas [4]. Activated T lymphocytes are eliminated by an apoptotic process known as activation-induced cell death (AICD), which requires the transcriptional induction of FasL expression [5-7] and sustained levels of c-Myc [8]. The FasL promoter can be driven by c-Myc overexpression, and functional inhibitors of Myc and its binding partner, Max, inhibit the transcriptional activity of the FasL promoter [9,10]. We identified a non-canonical binding site (ATTCTCT) for c-Myc-Max heterodimers in the FasL promoter, which, when mutated, abolished activity in response to c-Myc. Exchange of the canonical c-Myc responsive elements (CACGTG) in the ornithine decarboxylase (ODC) promoter [11] with the non-canonical sequence in the FasL promoter generated an ODC-FasL promoter that was significantly more responsive to c-Myc than the wild-type ODC promoter. Our findings identify a precise physiological role for c-Myc in the induction of apoptosis as a transcriptional regulator of the FasL gene.

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

Bcl-xL does not inhibit the function of Apaf-1.

Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1-5 microM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9.

Selective inhibition of topoisomerase II by ICRF-193 does not support a role for topoisomerase II activity in the fragmentation of chromatin during apoptosis of human leukemia cells.

Specific inhibitors of topoisomerase II (e.g., ICRF-193, an inhibitor of the catalytic activity of topoisomerase II and etoposide that stabilizes enzyme/DNA cleavable complexes) have been used to probe the role of topoisomerase II in the fragmentation of DNA during drug-induced apoptosis of human HL-60 leukemia cells. Topoisomerase II plays a role in the attachment of 50-kilobase domains of DNA to the nuclear matrix; fragments of this size are cleaved during apoptosis. Apoptosis was induced by 50 microM etoposide or 300 mM N-methylformamide (NMF), a nongenotoxic agent. Treatment with etoposide or NMF induced the morphology of apoptosis within 4 hr. Analysis of DNA integrity by electrophoresis showed coincident fragmentation from 50 kb and to integers of 200 bp. Transient protein-associated DNA strand breaks, characteristic of etoposide-induced damage, were visualized as DNA fragments of > 600 kb. Preincubation with ICRF-193 (100 microM) reduced the number of etoposide-induced DNA strand breaks by 50% and delayed the appearance of DNA fragmentation by approximately 18 hr. However, ICRF-193 had no effect on either NMF- or camptothecin-induced DNA fragmentation. The induction of apoptosis by both etoposide and NMF was associated with a reduction in the cellular levels of topoisomerases II alpha and II beta. ICRF-193 inhibited proteolytic cleavage of topoisomerase II induced by etoposide but not by NMF. The data suggest that the activity of topoisomerase II is not required for the cleavage of DNA to 50-kb fragments but that proteolysis of topoisomerase II represents a conserved event of apoptosis.

Iodide-dependent regulation of thyroid follicular cell proliferation: a mediating role of autocrine insulin-like growth factor-I.

An inhibitory action of intracellular iodide on the autocrine production of insulin-like growth factor-I (IGF-I) by thyroid follicular cells (TFCs) in vitro has been investigated as a possible mechanism underlying the iodide-dependent control of TFC proliferation. IGF-I release from primary monolayer cultures of porcine TFCs increased 5-fold between 24 and 168 h of incubation. Confirmation of a mediating role of IGF-I in TFC proliferation was obtained by exposing TFCs to an immunoadsorbing IGF-I antiserum, which led to a significant (P < 0.05) decline in [methyl-3H]thymidine incorporation, relative to TFCs exposed to preimmune serum. Exposure of TFCs to sodium iodide (NaI; 0.1-100 mumol/l) led to an attenuation of the IGF-I content of the cell-conditioned medium. This was accompanied by a reduction in [methyl-3H]thymidine incorporation that was affected by IGF-I immunoneutralization. The inhibitory effect of NaI on IGF-I production and [methyl-3H]thymidine incorporation were reversed by the thionamide compound methimazole (MMI; 1 mmol/l), exposure to which also led to significant (P < 0.001) increases above control values. However, a residual suppressive effect of NaI on [methyl-3H]thymidine incorporation suggested that certain of the TFC growth-attenuating effects of iodide may not be dependent upon organification. While providing evidence, therefore, for a direct relationship between iodide exposure, suppression of autocrine IGF-I production and a regulation of TFC proliferation, the present studies also suggest that suppression of TFC proliferation by iodide may be partially mediated by MMI-insensitive events.

Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drug resistant and drug sensitive leukaemic cells.

Apoptosis, originally defined by specific morphological changes, is characterised biochemically by non-random cleavage of DNA. Depending on cell type, this DNA cleavage proceeds from 300 and 50kbp fragments prior to, concomitantly with, or in the absence of 180bp integer fragmentation. Incorporation into fragmented DNA of biotin-labelled nucleotides by terminal deoxynucleotidyl transferase (TdT) has recently become a standard flow cytometric assay for the identification and quantitation of apoptosis. Nucleotide incorporation is visualized using avidin-tagged fluorescein isothiocyanate (FITC) (Gorczyca et al.: Cancer Res 53:1945-1951, 1993; Jonker et al.: Cytometry (Suppl 13):Abstr 99A, 1993). Here, we characterise this assay further in three different haemopoietic cell lines. Drug-induced DNA damage is not identified by the TdT assay unless it is coupled to the apoptotic response. This was demonstrated using cells in which activation of the oncogenic Abelson-encoded protein tyrosine kinase suppressed drug-induced apoptosis, but did not inhibit drug-induced DNA damage (by melphalan, hydroxyurea, or etoposide). Furthermore, the TdT assay identifies DNA fragments formed during apoptosis induced by etoposide and N-methylformamide in HL60 and MOLT-4 cells, including those high molecular weight DNA fragments formed in MOLT-4 cells which were not further cleaved to 180-200bp integer fragments. Our results support the use of flow cytometry and the TdT assay to reliably measure apoptotic cells in heterogeneous cell samples.

Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis.

Apoptosis is characterized by the nonrandom cleavage of DNA. After continuous treatment of MOLT-4 human T lymphoblastoid cells with the topoisomerase II inhibitor etoposide (50 microM) and the nongenotoxic agent N-methylformamide (300 mM), apoptosis was confirmed by electron microscopy. Analysis of DNA integrity by conventional gel electrophoresis failed to detect internucleosomal DNA cleavage. Resolution of DNA by field inversion gel electrophoresis showed fragments of 50 kilobases (kb). Etoposide induced the transient appearance of an additional DNA band of > 600 kb, which was temporally coincident with DNA-protein complex formation and was rapidly reversible upon drug removal. This DNA band was not observed after N-methylformamide treatment. In situ DNA end-labeling showed the incorporation of biotinylated dUTP into 50-kb DNA fragments but not etoposide-induced DNA fragments of > 600 kb. DNA end-labelling with terminal deoxynucleotidyltransferase was therefore not dependent upon intenucleosomal DNA cleavage, and fragments of approximately 50 kb were characterized by free 3'-OH termini that were not occluded by topoisomerase II protein. Although we considered that topoisomerase II potentially played an active role in the fragmentation of higher order chromatin during apoptosis, the results showed that DNA cleavage by topoisomerase II induced reversible, protein-associated fragments of > 600 kb and not irreversible cleavage to 50-kb fragments. The reversible cleavage of DNA to fragments of > 600 kb appears to be a signal for the engagement of apoptosis and is not an initial step in the sequential unwinding of chromatin.

Differentiation: a suitable strategy for cancer chemotherapy?

Differentiation therapy focuses on the development and use of specific agents designed to selectively engage the process of terminal differentiation, leading to the eventual elimination of tumorigenic cells and rebalance of normal cellular homeostasis. Extensive in vitro study of the molecular mechanism involved during drug-induced maturation has allowed the realization and application of a differentiation-based therapy to the clinic. Rationalization of this mode of therapy has included the combined use of differentiation agents with low-dose chemotherapy to lessen adverse cytotoxicity and to enhance the efficacy of differentiation agents, allowing some success in their application to conditions resistant to conventional therapy. This review discusses some biological principles that underlie the concept of a differentiation therapy and compares the in vitro and in vivo effectiveness of the two differentiation agents, in particular retinoic acid (RA) and hexamethylene bisacetamide (HMBA). It also evaluates the prospects for differentiation therapy as an effective strategy in the treatment and management of malignancy.

Investigations of mechanisms of drug-induced changes in gene expression: N-methylformamide-induced changes in synthesis of the M(r) 72,000 constitutive heat shock protein during commitment of HL-60 cells to granulocyte differentiation.

HL-60 cells were treated with the differentiating agent N-methylformamide and early changes in gene expression and protein content were investigated. Analysis of protein synthesis had previously shown an early (< 12 h) fall in the synthesis of M(r) 70,000 heat shock proteins (F. M. Richards, A. Watson, and J. A. Hickman. Cancer Res., 48: 6715-6720, 1988). The changes have now been characterized in detail and their kinetics compared to those of the expression of the c-myc protein. Immunoanalysis, using antibodies to either the stress-inducible heat shock protein hsp70 (4G4) or a pan-M(r) 70,000 heat shock protein antibody (3A3), showed that there was a striking reduction in the levels of the constitutive heat shock protein hsc70 when cells were incubated continuously with 170 mM N-methylformamide. A reduction in the level of hsc70 RNA was observed within 3 h and continued thereafter. In contrast, transcription of the hsc70 gene was induced within 1-2 h, after which the rate returned to basal level. There were no significant changes in the rate of transcription of the stress-inducible heat shock proteins hsp70 or hsp90. When N-methylformamide was removed from the cells, prior to commitment to differentiation, the levels of hsc70 were reestablished, whereas after 36 h of treatment there was no recovery. Western blotting with an antibody to the c-myc protein showed this to fall to virtually undetectable levels by 3 h under the same conditions. The results suggest that the loss of hsc70, which may perform a protein chaperoning role, was mediated at both transcriptional and posttranscriptional levels of regulation and was an early event closely associated with the commitment of HL-60 cells to differentiation. The fall in hsc70 was not associated with alterations in the cell cycle, nor were the kinetics of the change suggestive of a relationship with the decrease in content of c-myc protein.

Mechanisms of cytotoxicity caused by antitumour drugs.

Although the nature of the interaction between a drug or toxin and its target is of critical importance in determining the fate of a cell, we have argued here that the biological outcome of that interaction will also be determined by the nature of cellular events "downstream" of the initial interactions. We suggest that some type of coupling must take place between the formation of a drug-target interaction (the stimulus?) and the response of the cell. That response will depend upon the phenotypically determined repertoire of response open to the cell as well as upon the quantitative and qualitative measures of the events that the drug induces (DNA or protein damage, inhibition of growth etc.). For example we have described how the HL-60 cell appears to respond to low levels of toxins by engaging a programme of terminal differentiation whilst at greater concentrations apoptosis becomes engaged. Consideration of the cellular response to a toxic insult may provide valuable insights into the selective toxicity of agents as well as providing avenues for the discovery of toxins which might be useful in the treatment of cancer.

Insulin-like growth factor-I production and action in porcine thyroid follicular cells in monolayer: regulation by transforming growth factor-beta.

The regulation of thyroid follicular cell growth in vitro involves autocrine or paracrine actions of insulin-like growth factor-I (IGF-I), which are partially suppressed by transforming growth factor-beta (TGF-beta). Using subconfluent monolayers of porcine thyroid follicular cells, the aims of this study were to establish whether the actions of TGF-beta involve changes in the synthesis of, or response to, IGF-I. We also investigated the extent to which inhibitory actions of iodide on IGF-I-dependent proliferation of thyroid follicular cells may be attributable to the production of TGF-beta by follicular cells, as opposed to iodide-mediated autoregulation events. Exposure of porcine thyroid follicular cells in subconfluent monolayer culture to TGF-beta over a 7-day period reduced both IGF-I release and the incorporation of [methyl-3H]thymidine into trichloroacetic acid-precipitable cellular material, while preincubation of cells with NaI (0.1 mmol/l) for 24 h prior to the addition of TSH reduced the stimulatory effect of the latter on IGF-I release over the following 7 days. Preincubation of cells with iodide also reduced basal (i.e. autonomous) [methyl-3H]thymidine incorporation. This effect was partially reversed when, following initial exposure to follicular cells, iodide-containing preincubation medium was immunoadsorbed with a neutralizing TGF-beta antiserum, and subsequently re-added to the cells. Furthermore, similar immunoadsorption of iodide-free preincubation medium resulted in an enhancement of the control level of [methyl-3H]thymidine incorporation when the treated medium was returned to the original cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Iodide autoregulation of functional and morphological differentiation events in the FRTL-5 rat thyroid cell strain.

The role of cyclic AMP (cAMP) attenuation in mediating the autoregulatory actions of iodide on thyroid cell iodide uptake and surface morphological responses to TSH was investigated in the rat thyroid cell strain FRTL-5. Preincubation of cells for 6 h with up to 1 mmol sodium iodide/l led to a progressive reduction in both accumulation of cAMP and iodide uptake responses to TSH. Forskolin-mediated accumulation of cAMP and iodide uptake responses were similarly reduced after preincubation with iodide, whilst the iodide accumulation response to dibutyryl cAMP (dbcAMP) was unaffected. The inhibitory effects of iodide on TSH or forskolin-responsive iodide accumulation were not seen if preincubation was limited to 3 h, and were also abolished by the thionamide drug methimazole (1 mmol/l). Medium containing 1 mumol iodide/l prevented the appearance of the surface microvilli and pseudopodia normally observed after re-addition of TSH or forskolin, although cytoplasmic retraction was still apparent under such conditions. In contrast, iodide was without effect on the ability of dbcAMP (1 mmol/l) to induce cytoplasmic retraction and the formation of microvilli and pseudopodia. Inclusion of 1 mmol sodium perchlorate/l together with iodide during preincubation failed to prevent or reduce the suppression by iodide of either iodide uptake or surface morphological differentiation, suggesting that both aspects of autoregulation may involve surface actions of organified iodide. These observations indicate that in FRTL-5 cells, autoregulation by iodide of both the functional and surface morphological actions of TSH principally reflects the attenuating activities of organified iodide on intracellular cAMP generation.