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Cancer extracellular vesicles (EVs), or exosomes, promote tumor progression through enhancing tumor growth, initiating epithelial-to-mesenchymal transition, remodeling the tumor microenvironment, and preparing metastatic niches. Three-dimensionally (3D) cultured tumoroids / spheroids aim to reproduce some aspects of tumor behavior in vitro and show increased cancer stem cell properties. These properties are transferred to their EVs that promote tumor growth. Moreover, recent tumoroid models can be furnished with aspects of the tumor microenvironment, such as vasculature, hypoxia, and extracellular matrix. This review summarizes tumor tissue culture and engineering platforms compatible with EV research. For example, the combination experiments of 3D-tumoroids and EVs have revealed multifunctional proteins loaded in EVs, such as metalloproteinases and heat shock proteins. EVs or exosomes are able to transfer their cargo molecules to recipient cells, whose fates are often largely altered. In addition, the review summarizes approaches to EV labeling technology using fluorescence and luciferase, useful for studies on EV-mediated intercellular communication, biodistribution, and metastatic niche formation.

Genetic amplification, overexpression, and increased signaling from the epidermal growth factor receptor (EGFR) are often found in oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed effects of cetuximab in control of EGF-driven malignant traits of OSCC cells. EGF stimulation promoted progression level of mesenchymal traits in OSCC cells, which were attenuated by cetuximab but incompletely. We pursued a potential mechanism underlying such incomplete attenuation of OSCC malignant traits. Cetuximab promoted secretion of EGFR-EVs by OSCC cells and failed to inhibit EGF-driven secretion of EGFR-EVs. Cetuximab was also found to be robustly secreted with the EGFR-EVs by the OSCC cells. Thus, EGF promotes the level of mesenchymal traits of OSCC cells and secretion of EGFR-EVs, which involve cetuximab resistance.

RNA polymerase II (Pol II) dependent transcription in stimulus-inducible genes requires topoisomerase IIβ-mediated DNA strand break and the activation of DNA damage response signaling. Here we report a novel function of DNA repair proteins, involving a breast cancer 1 (BRCA1)-BRCA1 associated RING domain 1 (BARD1) complex, in transcriptional activation of stimulus-inducible genes in humans. We have found that BRCA1 is phosphorylated at S1524 by the kinases ATM and ATR during gene activation and that this phosphorylation event is essential for productive transcription. The major TOPIIβ binding site in the EGR1 TSS was mapped in the promoter between −132 and −14. We showed that BARD1 interacts with and could ubiquitinate TOPIIβ, and that BRCA1 regulates this interaction. Together, our findings indicate that the BRCA1-BARD1 complex controls TOPIIβ stability and gene association during Pol II transcription.

SREC-I is a class F scavenger receptor with key role in the immune response, particularly in antigen presenting cell (APC) such as macrophages and dendritic cells (DC). This receptor is able to mediate engulfment of dead cells as well as endocytosis of heat shock protein (HSP)-antigen complexes. SREC-I could thus potentially mediate the tolerizing influence of apoptotic cells or the immunostimulatory effects of HSP-peptide complexes, depending on context. This receptor was able to mediate presentation of external antigens, bound to HSPs through both the class II pathway as well as cross presentation via MHC class I complexes. In addition to its recently established role in adaptive immunity, emerging studies are indicating a broad role in innate immunity and regulation of cell signaling through Toll Like Receptors (TLR). SREC-I may thus play a key role in APC function by coordinating immune responses to internal and external antigens in APC.

Prenatal protein malnutrition (PPM) in rats causes enduring changes in brain and behavior including increased cognitive rigidity and decreased inhibitory control. A preliminary gene microarray screen of PPM rat prefrontal cortex (PFC) identified alterations in KCNJ3 (GIRK1/Kir3.1), a gene important for regulating neuronal excitability. Follow-up with polymerase chain reaction and Western blot showed decreased KCNJ3 expression in the PFC, but not hippocampus or brainstem. To verify localization of the effect to the PFC, baseline regional brain activity was assessed with 14C-2-deoxyglucose. Results showed decreased activation in the PFC but not hippocampus. Together these findings point to the unique vulnerability of the PFC to the nutritional insult during early brain development, with enduring effects in adulthood on KCNJ3 expression and baseline metabolic activity.

Heat shock protein 70 (Hsp70) is released from cells of many types and plays a significant signaling role, particularly in the inflammatory and immune responses. However, Hsp70 does not contain a consensus secretory signal and thus cannot traverse the plasma membrane by conventional mechanisms. However, Hsp70 can be released from cells by active mechanism that are independent of de novo Hsp70 synthesis or cell death. This pathway is similar to one utilized by the leaderless protein interleukin 1β. Hsp70 release involves transit through an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of Hsp70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of Hsp70 into this secretory compartment appears to involve the ABC-family transporter proteins. While the cell signals involved in triggering Hsp70 release through this lysosomal pathway are largely unknown, recent data suggest a regulatory role for extracellular ATP. These mechanisms are also shared by interleukin 1β secretion. Following release it has been shown that Hsp70 binds to adjacent cells, suggesting that the secreted protein participates in paracrine or autocrine interactions with adjacent cell surfaces. Thus an outline is beginning to of the mechanisms of Hsp70 secretion. Much further study will be required to fully elucidate mechanisms involved in targeting Hsp70 towards the non-canonical secretion pathways and its regulation.

The response of breast cancer patients to endocrine therapy is guided by the expression of two steroid hormone receptors (HR): estrogen receptor alpha (ERalpha) and/or progesterone receptors (PR). In most laboratories the expression of these predictive markers is studied by immunohistochemistry (IHC) in the breast cancer biopsy samples. Another molecular marker that is being increasingly examined in breast cancer is the oncoprotein Her-2/neu, whose expression/amplification predicts the response to anti-Her-2/neu immunotherapy. The co-expression of HR with that of Her-2/neu is infrequent (most reports agree on this), however, there are some conflicting reports about the clinical implications in term of response to endocrine therapy in the patients that co-express HR and Her-2/neu. We have examined these molecular markers for a number of years in our tumor bank, in this dissertation we will present the method and cut-off to study these markers, the correlations between their expression, and the follow-up of the patients that received tamoxifen-based endocrine therapy, alone or following chemotherapy. We confirmed that the co-expression of HR with Her-2/neu is infrequent, and that these patients presented both a shorter disease free survival and overall survival. Our results will be compared with others related recently published. For example, the aromatase inhibitor anastrozole appears to be an effective endocrine treatment in HR+ patients, irrespective of the Her-2/neu status. We will present data on the molecular mechanisms that could explain the relatively poor outcome of these patients. Heregulin has been found to be a potent inducer of heat shock factor 1 (HSF1) activity and of heat shock protein (Hsp) synthesis in breast cancer cells and HSF1 activation plays a role in the tumorigenic changes induced by heregulin, heregulin exerts its tumorigenic changes through the cell surface tyrosine kinase receptors c-erbB-3 and c-erbB-4 which are able to form dimers with the "ligandless" Her-2/neu. We found that HSF1 associates with metastasis associated protein 1 (MTA1) on the promoters of genes as well as other molecules involved in gene repression (HDAC1, HDAC2) in a manner that is enhanced by either heregulin exposure or heat shock. ERs, although promoting the growth of breast cancer cells are less associated with invasion/metastasis and ER-induced gene expression is involve in this effect. Heregulin can overcome the protective effects of ER and at least a component of this appears to be due to MTA1 repression of ERE dependent transcription, HSF1 and MTA1 cooperate in gene repression. The co-expression of HSF1 and MTA1 was confirmed by IHC in human breast cancer biopsy samples.

Heat shock transcription factor 1 (HSF1) monitors the structural integrity of intracellular proteins and its regulation is essential for the health and longevity of eukaryotic organisms. HSF1 also plays a role in the acute inflammatory response in the negative regulation of cytokine gene transcription. Here we show, for the first time, that HSF1 is regulated by the proinflammatory protein kinase MAPKAP kinase 2 (MK2). We have shown that MK2 directly phosphorylates HSF1 and inhibits activity by decreasing its ability to bind the heat shock elements (HSE) found in the promoters of target genes encoding the HSP molecular chaperones and cytokine genes. We show that activation of HSF1 to bind HSE in hsp promoters is inhibited through the phosphorylation of a specific residue, serine 121 by MK2. A potential mechanism for MK2-induced HSF1 inactivation is suggested by the findings that phosphorylation of serine 121 enhances HSF1 binding to HSP90, a major repressor of HSF1. Dephosphorylation of serine 121 in cells exposed to non-steroidal anti-inflammatory drugs leads to HSP90 dissociation from HSF1, which then forms active DNA binding trimers. These experiments indicate a novel mechanism for the regulation of HSF1 by proinflammatory signaling and may permit HSF1 to respond rapidly to extracellular events, permitting optimal physiological regulation.

Heat shock protein (Hsp) 90 is a ubiquitously expressed chaperone that stabilizes expression of multiple signaling kinases involved in growth regulation, including ErbB2, Raf-1, and Akt. The chaperone activity of Hsp90 requires ATP, which binds with approximately 10-fold lower affinity than ADP. This suggests that Hsp90 may be a physiological ATP sensor, regulating the stability of growth signaling cascades in relation to cellular energy charge. Here we show that lowering ATP concentration by inhibiting glycolysis or mitochondrial respiration in isolated myocytes triggers rapid dissociation of Hsp90 from ErbB2 and degradation of ErbB2 along with other client proteins. The effect of disrupting Hsp90 chaperone activity by ATP depletion was similar to the effect of the pharmacological Hsp90 inhibitor geldanamycin. ATP depletion-induced disruption of Hsp90 chaperone activity was associated with cellular resistance to growth factor activation of intracellular signaling. ErbB2 degradation was also induced by the physiological stress of beta-adrenergic receptor stimulation in electrically stimulated cells. These results support a role for Hsp90 as an ATP sensor that modulates tissue growth factor responsiveness under metabolically stressed conditions and provide a novel mechanism by which cellular responsiveness to growth factor stimulation is modulated by cellular energy charge.

The stress protein response involves the immediate reprogramming of gene expression in cells exposed to proteomic insult leading to massive synthesis of heat shock proteins (HSP). We have examined the outcome when cells are induced to activate two other gene expression programs--the acute inflammatory response and entry of quiescent cells into the cell cycle--and then exposed to protein stress. We find that these responses are mutually antagonistic with, on the one hand, heat shock factor 1 (HSF1) inhibition through the phosphorylation of inhibitory serine residues after inflammatory or mitogenic stimulus and, on the other hand, after stress, HSF1 directly repressing the promoters of genes that mediate acute inflammation and mitogenesis. The expression of the stress protein response during periods of acute protein damage was shown to lead to efficient activation of HSF1 and HSP expression accompanied by repression of other gene expression programs.

Recent studies have implicated heat shock proteins (HSP) and heat shock transcription factor 1 (HSF1) in tumor progression. We have examined the role of HSF1 in the malignant phenotype of PC-3 prostate carcinoma cells. We have developed a dominant negative construct of HSF1 that antagonizes transcription from HSP promoters and results in the depletion of intracellular HSP 70. Our studies indicate that expression of DN-HSF1 dramatically alters the DNA content of PC-3 cells (derived from p53 null prostatic carcinoma) and inhibits aneuploidy in these cells. This effect is due to prolonged expression of DN-HSF1, and transient expression of the dominant negative factor from an inducible promoter failed to cause the effect. Inhibition of aneuploidy in p53 null PC-3 cells by DN-HSF1 expression was recapitulated by expression within the cells of wild type p53. Furthermore, cells expressing DN-HSF1 showed a profound inhibition in the development of aneuploidy when exposed to chemical agents that disrupt the mitotic spindle and prevent progression through metaphase. Inhibition of aneuploidy in PC-3 cells expressing DN-HSF1 was associated with delayed breakdown of cyclin B1 compared with controls, consistent with a role for wild type HSF1 in the regulation of cyclin B1 degradation, a key step in the control of mitosis. Our experiments therefore demonstrate that HSF1 plays a functional role in cancer cells under nonstress conditions and influences cell cycle behavior and progression through mitosis and promotes the development of the aneuploid state.

Heat shock factor 2 (HSF2) belongs to a family of structurally related transcription factors, which share the property of binding to heat shock elements in the promoters of hsp molecular chaperone genes. However, unlike HSF1, which is essential for hsp gene transcription, the cellular functions of HSF2 are not well known. Here we show that human HSF2, although an ineffective activator of the hsp70 promoter in vitro and in vivo in the absence of stress, participates in the activation of the hsp70 promoter by heat shock. HSF2 was not, however, activated by heat shock in cells deficient in functional HSF1, suggesting a requirement for HSF1 in HSF2-mediated transcriptional enhancement. In addition, HSF2 regulation involves differential activity of two isoforms, HSF2A and HSF2B, which arise from alternative splicing of a common hsf2 gene. Under basal conditions, both HSF2 isoforms are ineffective in activating the hsp70 transcription. However, heat shock differentially activates HSF2A in vivo. This phenomenon appears to be physiologically significant, as human myeloprogenitor cells differentiating along the erythroid lineage express HSF2A de novo and undergo a large increase in capacity to activate the hsp70 promoter. Our experiments further show that HSF1 is physically associated with HSF2 in the cell and that such binding is enhanced by heat shock. Our data suggest a mechanism involving the formation of heterocomplexes between HSF1 and HSF2 with enhanced activity to activate the hsp70 promoter when compared with HSF1 or HSF2 homotrimers.

Heat shock factor 1 (HSF1), in addition to its pivotal role as a regulator of the heat shock response, functions as a versatile gene repressor. We have investigated the structural domains involved in gene repression using mutational analysis of the hsf1 gene. Our studies indicate that HSF1 contains two adjacent sequences located within the N-terminal half of the protein that mediate the repression of c-fos and c-fms. One region (NF) appears to be involved in quenching transcriptional activation factors on target promoters and binds to the basic zipper transcription factor NF-IL6 required for activation of c-fms and IL-1beta. The NF domain encompasses the leucine zipper 1 and 2 sequences as well as the linker domain between the DNA binding and leucine zipper regions. The function of this domain in gene repression is highly specific for HSF1, and the homologous region from conserved family member HSF2 does not restore repressive function in HSF2/HSF1 chimeras. In addition, HSF2 is not capable of binding to NF-IL6. The NF domain, although necessary for repression, is not sufficient, and a second region (REP) occupying a portion of the regulatory domain is required for repression. Neither domain functions independently, and both are required for repression. Furthermore, we constructed dominant inhibitors of c-fos repression by HSF1, which also blocked the repression of c-fms and IL-1beta, suggesting a shared mechanism for repression of these genes by HSF1. Our studies suggest a complex mechanism for gene repression by HSF1 involving the binding to and quenching of activating factors on target promoters. Mapping the structural domains involved in this process should permit further characterization of molecular mechanisms that mediate repression.

Recent studies have initiated a paradigm shift in the understanding of the function of heat shock proteins (HSP). It is now clear that HSP can and do exit mammalian cells, interact with cells of the immune system, and exert immunoregulatory effects. We recently demonstrated that exogenously added HSP70 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca2+ flux, activate NF-κB, and up-regulate the expression of pro-inflammatory cytokines in human monocytes. Here for the first time, we report that HSP70-induced proinflammatory cytokine production is mediated via the MyD88/IRAK/NF-κB signal transduction pathway and that HSP70 utilizes both TLR2 (receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) to transduce its proinflammatory signal in a CD14-dependent fashion. These studies now pave the way for the development of highly effective pharmacological or molecular tools that will either up-regulate or suppress HSP70-induced functions in conditions where HSP70 effects are desirable (cancer) or disorders where HSP70 effects are undesirable (arthritis and arteriosclerosis).

Heat shock factor (HSF) 1 is the major heat shock transcription factor that regulates stress-inducible synthesis of heat shock proteins and is also essential in protection against endotoxic shock. Following our previous study, which demonstrated the transcriptional repression of the IL-1beta gene by HSF1 (Cahill, C. M., Waterman, W. R., Xie, Y., Auron, P. E., and Calderwood, S. K. (1996) J. Biol. Chem. 271, 24874-24879), we have examined the mechanisms of transcriptional repression. Our studies show that HSF1 represses the lipopolyliposaccharide-induced transcription of the IL-1beta promoter through direct interaction with the nuclear factor of interleukin 6 (NF-IL6, also known as CCAAT enhancer binding protein (C/EBPbeta), an essential regulator in IL-1beta transcription. We show for the first time that HSF1 binds directly to NF-IL6 in vivo and antagonizes its activity. The HSF1/NF-IL6 interaction involves a sequence of HSF1 containing the trimerization and regulatory domains and the bZip region of NF-IL6. HSF1 has little effect on IL-1beta promoter activity stimulated by the essential monocytic transcription factor Spi.1 but is strongly inhibitory to transcriptional activation by NF-IL6 and to the synergistic activation by NF-IL6 and Spi.1. Because of its ability to bind to specific C/EBP elements in the promoters of multiple genes and its ability to interact with other transcription factors, NF-IL6 is involved in transcriptional regulation of a wide range of genes. Interaction between HSF1 and NF-IL6 could thus be an important mechanism in HSF1 regulation of general gene transcription during endotoxin stress.

We have examined the functional antagonism between the regulator of the heat shock response, HSF1, and NF-IL6, which plays a major role in control of the acute phase response (APR). Agents that activate HSF1 such as heat shock and sodium salicylate inhibit NF-IL6 induced transcription while NF-IL6 activators such as lipopolysaccharide (LPS) and interleukin 6 (IL-6) repressed the stress responsive HSP70B promoter. In transfection studies, the inhibitory effects of HSF1 and NF-IL6 on the c-fms promoter were shown to be highly dose-dependent. Furthermore, heat shock is inhibitory to differentiation-linked expression of macrophage colony stimulating factor (M-CSF) receptor, product of the c-fms gene, which is transcriptionally activated by NF-IL6 but repressed by HSF1. Our studies suggest a strong mutual antagonism between the heat shock response and APR, which may influence the sensitivity and duration of inflammatory responses.

Tumor necrosis factor-alpha (TNFalpha) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5-40 degrees C) markedly attenuates TNFalpha expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNFalpha gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5'-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5'-UTR of the TNFalpha gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFalpha gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFalpha promoter (-85/+138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (-1080/+138) TNFalpha promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions -1080/-845, -533/-196, and -326/-39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal -85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimal TNFalpha promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5 degrees C. This suggests that HSF-1 might repress TNFalpha transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1.

We previously reported that expression of tumor necrosis factor-α (TNFα) was attenuated in macrophages exposed to febrile range temperatures. In this study, we analyzed the influence of temperature on TNFα transcription in the Raw 264.7 macrophage cell line during incubation at 37 and 39.5 °C. The initial activation of TNFα transcription in response to endotoxin (LPS) was comparable in the 37 and 39.5 °C cell cultures, peaking within 10 min of LPS stimulation. However, the duration of transcriptional activation was markedly reduced in the 39.5 °C cells (30–60 min) compared with the 37 °C cells (2–4 h). Deletion mapping of the TNFα gene revealed that the proximal 85-nucleotide promoter sequence and the 5′-untranslated region were sufficient for temperature sensitivity. This sequence contains six heat shock response element (HRE) half-sites but no complete HREs. Electrophoretic mobility shift and immunoblot assays demonstrated that nuclear transclocation of heat shock factor (HSF) and its activation to a DNA-binding form occurred in the 39.5 °C cells in the absence of heat shock protein-70 gene activation. The proximal TNFα promoter/5′-untranslated region sequence competed for HSF binding to a classic HRE. Overexpression of HSF-1 reduced activity of the TNFα promoter. These data suggest that partial activation of HSF-1 during exposure to febrile, sub-heat shock temperatures may block TNFα transcription by binding to its proximal promoter or 5′-untranslated region.

Recent studies have shown that the non-steroidal anti-inflammatory drugs (NSAIDs) activate heat shock transcription factor (HSF1) from a latent cytoplasmic form to a nuclear, DNA binding state. As HSF1 can function as both an activator of heat shock genes and a repressor of non-heat shock genes such as IL1B and c- fos, we have examined the potential role of HSF1 in the effects of NSAIDs on gene expression in a human monocytic cell line THP-1. We found that two members of the NSAIDs, sodium salicylate and sulindac repress the IL1B promoter to similar degree to heat shock or HSF1 overexpression. In addition, sodium salicylate and additional NSAIDs used at concentrations that activate HSF1 also inhibited the expression of other monocytic genes (TNF-alpha, IL-1beta, IL-6, IL-8, IL-10, ICAM-1) activated by exposure to a pro-inflammatory stimulus (lipopolysaccharide, LPS). At least in the case of the IL1B promoter, repression did not seem to involve another factor whose activity is affected by the NSAIDs, NFkappaB as the IL1B promoter fragment used in our studies is not NFkappaB responsive and binds specifically to HSF1. Exposure to NSAIDs had a complex effect on HSP gene expression and while sulindac activated the stress responsive HSP70B promoter, sodium salicylate did not. In addition, only a subset of the NSAIDs induced HSP70 mRNA species. These findings reflect the properties of HSF1 which can be activated to at least two DNA binding forms only one of which activates heat shock promoters and suggest that individual NSAID family members may differentially induce one or other of these forms. Overall therefore, exposure to NSAIDs leads to a profound switch in gene expression in monocytic cells, with suppression of genes involved in macrophage activation and induction of stress genes and HSF1 appears to play a regulatory role in these effects.

Cells of the LA7 rat mammary tumor line stimulate proliferation of normal mouse mammary epithelial cells in culture when in direct physical contact with them. We examined junctional connections between these LA7 feeders and the recipient mouse mammary epithelial cells in order to study the role these junctions may play in growth signaling. Tight junctions and desmosomes between LA7 feeders and mouse mammary cells were detected by immunocytochemistry. These junctions connected every cell of either type with each of its neighbors. Adherens junctions, although evident between mouse mammary cells, could not be detected between LA7 and mouse mammary cells or between LA7 cells themselves. However, E-cadherin, the transmembrane protein of adherens junctions, was present in LA7 cell lysates. beta-catenin, which normally binds cadherins, was detected at the borders of LA7 cells. Presence of gap junctions between LA7 and mouse mammary cells was determined by traverse of lucifer yellow from an injected LA7 cell to surrounding mouse mammary epithelial cells. The experiments thus indicate that three types of intercellular junctions occur between cells active in direct cell-cell-stimulated proliferation signaling.

Heat shock factor 1, the critical molecular regulator of the stress response is conserved throughout eukaryotic organisms and activates the transcription of heat shock genes. We now show that heat shock factor 1 inhibits the expression of c-fos, an immediate early gene that controls responses to extracellular stimuli for growth and differentiation. Heat shock factor 1 inhibits the transcription of the c-fos gene and antagonizes the activating effects of the signal transducing protein Ras on the c-fos promoter and on the promoter of another Ras responsive gene uPA. This property was specific for heat shock factor 1; c-fos repression was not seen with the structurally related protein heat shock factor 2. Repression involved different molecular mechanisms compared with those involved in transcriptional activation by heat shock factor 1 and specifically did not require binding to the c-fos promoter. Thus, in addition to its known role as a transcriptional activator of the cellular heat shock response, heat shock factor 1 also antagonizes the expression of Fos, a key component of the ubiquitous AP-1 transcription factor complex and as such could influence multiple aspects of cell regulation.

In an exciting recent paper Richard Vile and coworkers describe a novel approach to tumor immunotherapy based on the killing of a population of normal cells within an inflammatory environment. This approach builds on recent studies showing that the molecular chaperone Hsp70 can act at multiple stages during tumor antigen presentation to enhance the generation of CD8(+) T lymphocyte-mediated immunity and lead to regression of primary and metastatic tumors.

Mammalian heat shock genes are regulated at the transcriptional level by heat shock factor-1 (HSF-1), a sequence-specific transcription factor. We have examined the role of serine phosphorylation of HSF-1 in the regulation of heat shock gene transcription. Our experiments show that mitogen-activated protein kinases (MAPKs) of the ERK-1 family phosphorylate HSF-1 on serine residues and repress the transcriptional activation of the heat shock protein 70B (HSP70B) promoter by HSF-1 in vivo. These effects of MAPK are transmitted through a specific serine residue (Ser-303) located in a proline-rich sequence within the transcriptional regulatory domain of human HSF-1. However, despite the importance of Ser-303 in transmitting the signal from the MAPK cascade to HSP70 transcription, there was no evidence that Ser-303 could be phosphorylated by MAPK in vitro, although an adjacent residue (Ser-307) was avidly phosphorylated by MAPK. Further studies revealed that Ser-303 is phosphorylated by glycogen synthase kinase 3 (GSK3) through a mechanism dependent on primary phosphorylation of Ser-307 by MAPK. Secondary phosphorylation of Ser-303 by GSK3 may thus repress the activity of HSF-1, and its requirement for priming by MAPK phosphorylation of Ser-307 provides a potential link between the MAPK cascade and HSF-1. Our experiments thus indicate that MAPK is a potent inhibitor of HSF-1 function and may be involved in repressing the heat shock response during normal growth and development and deactivating the heat shock response during recovery from stress.

Heat shock factor 1 activates the promoters of heat shock genes at elevated temperatures through its interaction with heat shock elements. We have examined a new role for heat shock factor 1 in the repression of the prointerleukin 1β gene in human monocytes responding to stimulation with lipopolysaccharide. Both exposure to elevated temperatures and heat-independent heat shock factor 1 expression repressed the transcription of the prointerleukin 1β gene, and repression was strictly dependent on an intact consensus heat shock element in the prointerleukin 1β promoter to which heat shock factor 1 bound. This is the first demonstration of heat shock factor 1 as a transcriptional repressor and suggests a role for the factor in the counter-regulation of cytokine gene transcription.

Interaction of heat shock transcription factor-1 (HSF-1) with the seventy kilodalton heat shock cognate protein (HSC70) was examined in NIH 3T3 cells. HSF-1 was found in the cytoplasm of non-stressed cells associated with HSC70 in large (Mr 400-500,000) complexes. After heat shock, HSF-1 became concentrated in the nucleus in smaller, more stable complexes that did not contain HSC70, an indication of significant rearrangement within the complexes. These experiments show a profound effect of heat shock on the structure and stability of HSF-1 complexes during nuclear localization and support the hypothesis that HSC70 binding may control HSF-1 function.

Products of a number of mutant p53 genes bind with high affinity to members of the hsp70 family of chaperonin proteins, whereas wild type p53 lacks this type of association. Examination of the sequences of p53 genes from five different species enabled us to predict domains on p53 which may be involved in the association with hsp70 family members. A synthetic polypeptide (Pro-17-Gly) corresponding to the candidate hsp70 binding domain bound to in vitro translated hsp70 as determined by affinity chromatography and nondenaturing gel mobility shift assays. In addition, the Pro-17-Gly peptide competitively inhibited association between hsp70 and p53, an activity which was determined by immunoprecipitation with anti-p53 monoclonal antibody PAb240. The data indicate that p53 contains a hsp70 binding domain, which is located in a highly conserved region at the amino terminus of the protein, and may participate in the cellular function of wild-type p53 or in the transforming capacity of p53 mutants.

A heat-inducible member of the mouse hsp70 gene family has been isolated and characterized. The gene, hsp70.1 , has a primary transcript of 2.8 kb before and 3.1–3.5 kb after polyadenylation. It includes a 645-nucleotide (nt) 3′-nontranslated terminus and a 225-nt 5′ leader. The leader sequence is unusual in that it contains a 33-nt triple tandem repeat element proximal to the ATG codon. As expected, a single open reading frame encodes a 642-amino acid (aa) 70-kDa protein with 95% aa homology and 91% nt homology to a human hsp70 gene. While there is no homology through the 5′- and 3′-nontranslated regions, the human and mouse hsp70 gene promoters can be aligned to give 65% homology. Of three mouse cell lines studied, two expressed the 3.1- to 3.5-kb mRNA during heat shock. One line also expressed an additional, smaller, 2.6-kb hsp70 mRNA transcribed from a separate gene(s). Mouse erythroleukemia cells were unresponsive to heat shock, with neither hsp70 RNA being induced by heat shock. These results suggest that cell lineage has a major influence on the cellular response to heat shock and that the response is not universal in all cell types.

Binding of the polycation DEAE-dextran to the cell surface of HA-1 CHO cells caused a marked increase in 45Ca2+ exchange influx. The effect was fairly selective for Ca2+, undirectional (efflux was not increased) and was rapidly reversed by treatment with polyanion dextran sulfate. 45Ca2+ influx could not be stimulated by treatment with multivalent lectins or fibronectin. In addition to stimulating 45Ca2+ flux, DEAE-dextran inhibited the capping of concanavalin-A acceptor proteins. Inhibition of capping occurred over the same DEAE-dextran concentration range (20-200 micrograms/ml) which stimulated 45Ca2+ uptake, possibly implicating increased cellular [Ca2+] in the inhibition of concanavalin A acceptor protein capping in this cell type. The profound effect of DEAE-dextran on cellular Ca2+ uptake and the rapid reversal of the effect by dextran sulfate might make the polycation a useful agent for the induction of transient increases in cellular [Ca2+].

Membrane proteins may be key targets in thermal cell killing. In the present study, insulin binding to cellular receptors has been used as a probe for membrane protein behavior after heat shock (42-45 degrees C). Heating and binding studies were carried out on monolayers of HA-1 Chinese hamster ovary cells. Binding was unaffected by temperatures below 43 degrees C, but above this temperature (43-45 degrees C) it was inhibited in a time-temperature dependent manner. The kinetics of inhibition of insulin binding were similar to those for cell inactivation. Scatchard analysis indicated a decrease in receptor number rather than affinity for insulin in heated cells. In cells made resistant to further heat treatment (thermotolerant cells) by a mild pretreatment dose of hyperthermia (45 degrees C/10 min) insulin binding was also made heat resistant. Heating appeared to act directly on the insulin receptor rather than indirectly on subsequent energy dependent processes such as internalization. The data thus indicate that the fate of at least one membrane protein is closely tied up with the ultimate destiny of the cell per se after heating.