The cell surface-localized heat shock protein 70 epitope TKD induces migration and cytolytic activity selectively in human NK cells

Profiling of surface-bound proteins uncovers a tumor-selective heat shock protein 70 (Hsp70) membrane expression that provides a target structure for human NK cells. Hsp70 peptide TKD (TKDNNLLGRFELSG; aa 450-463) was found to enhance the cytolytic activity of NK cells. In this study, we demonstrate that TKD-activated CD3-CD56+CD94+ NK cells are selectively attracted by Hsp70 membrane-positive tumor cells, and supernatants derived thereof. Hsp70 membrane-negative tumors failed to attract these NK cells. The capacity to migrate was associated with a substantial lytic activity against Hsp70-positive tumor cells. Because NK cell migration was independent of cell-to-cell contact, the involvement of a soluble factor was assumed. Interestingly, synthetic Hsp70 protein and Hsp70 peptide TKD, mimicking surface-bound Hsp70, initiates migration of NK cells in a concentration-dependent (1-5 microg/ml), highly selective, and chemokine-independent manner. In summary, our results indicate that Hsp70 peptide TKD not only stimulates cytolysis but also chemotaxis in CD3-CD56+CD94+ NK cells.     

Heat shock protein 70-reactivity is associated with increased cell surface density of CD94/CD56 on primary natural killer cells

Previously we described an involvement of the C-type lectin receptor CD94 and the neuronal adhesion molecule CD56 in the interaction of natural killer (NK) cells with Hsp70-protein and Hsp70-peptide TKD. Therefore, differences in the cell surface density of these NK cell-specific markers were investigated comparatively in CD94-sorted, primary NK cells and in established NK cell lines NK-92, NKL, and YT after TKD stimulation. Initially, all NK cell types were positive for CD94; the CD56 expression varied. After stimulation with TKD, the mean fluorescence intensity (mfi) of CD94 and CD56 was upregulated selectively in primary NK cells but not in NK cell lines. Other cell surface markers including natural cytotoxicity receptors remained unaffected in all cell types. CD3-enriched T cells neither expressing CD94 nor CD56 served as a negative control. High receptor densities of CD94/CD56 were associated with an increased cytolytic response against Hsp70 membrane-positive tumor target cells. The major histocompatibility complex (MHC) class I-negative, Hsp70-positive target cell line K562 was efficiently lysed by primary NK cells and to a lower extent by NK lines NK-92 and NKL. YT and CD3-positive T cells were unable to kill K562 cells. MHC class-I and Hsp70-positive, Cx + tumor target cells were efficiently lysed only by CD94-sorted, TKD-stimulated NK cells with high CD94/CD56 mfi values. Hsp70-specificity was demonstrated by antibody blocking assays, comparative phenotyping of the tumor target cells, and by correlating the amount of membrane-bound Hsp70 with the sensitivity to lysis. Remarkably, a 14-mer peptide (LKD), exhibiting only 1 amino acid exchange at position 1 (T to L), neither stimulated Hsp70-reactivity nor resulted in an upregulated CD94 expression on primary NK cells. Taken together our findings indicate that an MHC class I-independent, Hsp70 reactivity could be associated with elevated cell surface densities of CD94 and CD56 after TKD stimulation.     

Influence of Hsp70 and HLA-E on the killing of leukemic blasts by cytokine/Hsp70 peptide-activated human natural killer (NK) cells

This study compared the effects of the human 70-kDa stress protein (Hsp70) peptide, TKDNNLLGRFELSG (TKD), proinflammatory cytokines, or a combination of both on the repertoire of receptors expressed by human natural killer (NK) cells and their capacity to kill human CX colon carcinoma cells, K562 erythroleukemic cells, and leukemic blasts from two patients with acute myelogenous leukemia. Low-dose interleukin (IL) 2/IL-15 and TKD increase the expression density of activatory (NKG2D, NKp30, NKp44, NKp46, CD94/NKG2C) and inhibitory (CD94/NKG2A) receptors on NK cells. Concomitantly, IL-2/TKD treatment enhances the cytotoxicity of NK cells (as reflected by their secretion of granzyme B) against Hsp70 membrane-positive and human leukocyte antigen (HLA)-E membrane-negative (Hsp70⁺/HLA-E⁻) CX⁺ and K562 cells. However, it had no effect on the responsiveness to Hsp70⁻/HLA-E⁻ CX⁻ cells over that induced by IL-2 alone. The cytotoxicity of IL-2/TKD-activated, purified NK cells and peripheral blood mononuclear cells against Hsp70⁺/HLA-E⁺ leukemic blasts was weaker than that against Hsp70⁺/HLA-E⁻ K562 cells. Hsp70-blocking and HLA-E transfection experiments confirmed membrane-bound Hsp70 as being a recognition/activatory ligand for NK cells, as cytotoxicity was reduced by the presence of the anti-Hsp70 monoclonal antibody cmHsp70.2 and by inhibiting Hsp70 synthesis using short interference ribonucleic acid. HLA-E was confirmed as an inhibitory ligand, as the extent of NK cell-mediated lysis of K562 cell populations that had been transfected with HLA-E^R or HLA-E^G alleles was dependent on the proportion of HLA-E-expressing cells. These findings indicate that Hsp70 (as an activatory molecule) and HLA-E (as an inhibitory ligand) expression influence the susceptibility of leukemic cells to the cytolytic activities of cytokine/TKD-activated NK cells.     

Inhibition of tumor growth in mice with severe combined immunodeficiency is mediated by heat shock protein 70 (Hsp70)-peptide-activated,CD94 positive natural killer cells

Previously, we reported that the major stress-inducible heat shock protein 70 (Hsp70) acts as a recognition structure for natural killer (NK) cells, if localized on the cell surface of tumor cells. Incubation of purified NK cells with low-dose interleukin (IL)-2 (100 IU/mL) plus recombinant Hsp70-protein or the immunogenic 14-mer Hsp70-peptide TKDNNLLGRFELSG450-463, termed TKD (2 microg/mL), enhances the cytolytic activity against Hsp70 membrane-positive (CX+) but not against Hsp70-negative (CX-) tumor cells. Here, we show that the cytolytic activity against Hsp70-positive tumor cells is inducible by incubation of unseparated peripheral blood mononuclear cells (PBMNC) with low-dose IL-2 plus TKD. Cell sorting experiments revealed that within the PBMNC population CD94(+)/CD3(-) NK cells, and not CD94(-)/CD3(+) T cells, mediate the cytotoxic activity against Hsp70-positive tumor cells. The antitumoral effect of PBMNC stimulated either with IL-2 plus TKD or with IL-2 alone was assessed in tumor-bearing severe combined immunodeficiency/beige mice. A single intravenous (iv) injection of 40 x 10(6) IL-2 plus TKD-stimulated PBMNC (containing 5.2 x 10(6) NK cells) on day 4 results in a 60% reduction in tumor size, from 3.89 g to 1.56 g. In contrast, the adoptive transfer of the identical amount PBMNC stimulated with low-dose IL-2 only (containing 4.4 x 10(8) NK cells) reduces the tumor size only less than 10% (3.64 g). A phenotypic characterization of the excised tumors revealed that predominantly Hsp70-positive tumor cells were eliminated by TKD-activated PBMNC. Kinetic studies demonstrate that the in vivo cytolytic capacity of TKD-stimulated PBMNC is dependent on the effector to target cell ratio. An iv injection of effector cells on day 1 or 2 after tumor cell inoculation results in significantly smaller tumors (0.77 g or 0.89 g) on day 21 as compared with mice that were immunoreconstituted on day 4 or 8 (1.39 g or 2.23 g). The tumor size of nonimmunoreconstituted control animals was 3.55 g.     

Interaction of heat shock protein 70 peptide with NK cells involves the NK receptor CD94

Full-length Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) both stimulate the cytolytic activity of naive natural killer (NK) cells against Hsp70-positive tumor target cells. Here, we describe the characterization of Hsp70-NK cell interaction with binding studies using the human NK cell line YT. Binding of recombinant Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) to YT cells is demonstrated by immunofluorescence studies. A phenotypic characterization revealed that none of the recently described HSP-receptors (alpha2-macroglobulin receptor CD91, Toll-like receptors 2, 4, 9, CD14) are expressed on YT cells. Only the C-type lectin receptor CD94 is commonly expressed by YT cells and Hsp70 reactive NK cells. A correlation of the cell density-dependent, variable CD94 expression and the binding capacity of Hsp70 was detected. Furthermore, Hsp70 binding could be completely abrogated by preincubation of YT cells with a CD94-specific antibody. Competition assays using either unlabeled Hsp70 protein or an unrelated protein (GST) in 20-fold excess and binding studies with escalating doses of Hsp70 protein provide evidence for a specific and concentration-dependent interaction of Hsp70 with YT cells. In addition to Hsp70 and Hsp70C, a 14-mer Hsp70 peptide termed TKD is known to exhibit comparable stimulatory properties on NK cells. Similar to full-length Hsp70 protein (10 microg/ml-50 microg/ml), a specific binding of this peptide to YT cells was observed at 4 degrees C, at equivalent concentrations (2.0 microg/ml-8.0 microg/ml). Following a 30 min incubation period at 37 degrees C, membrane-bound Hsp70 protein and Hsp70 peptide TKD were completely taken up into the cytoplasm.     

Retinoid- and sodium-butyrate-induced decrease in heat shock protein 70 membrane-positive tumor cells is associated with reduced sensitivity to natural killer cell lysis, growth delay, and altered growth morphology

Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX+/CX-) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 microM, 1 microM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX+ tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX+ and ML-1 but not in CX- tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX+ and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology.     

Plasmodium falciparum-infected erythrocytes induce granzyme B by NK cells through expression of host-Hsp70

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo.     

The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress‐inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain‐related molecule A (MICA) in a natural killer group 2 member D (NKG2D‐) dependent manner. The HSP70‐derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full‐length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress‐inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.     

Control of metastasized pancreatic carcinomas in SCID/beige mice with human IL-2/TKD-activated NK cells

Pancreatic carcinoma, the fifth leading cause of cancer-related mortality, frequently presents the stress-inducible heat shock protein 70 (Hsp70) on the cell membrane. Therefore, we explored an immunological approach exploiting the efficacy of NK cells activated either with low dose IL-2 plus Hsp70-peptide TKDNNLLGRFELSG (TKD; IL-2/TKD) or with IL-2 alone in a xenograft pancreatic carcinoma model. An orthotopic injection of either 2.5 x 10(6) or 1 x 10(6) Colo357 cells in SCID/beige mice resulted in rapidly growing primary tumors and the development of hepatic metastases on days 5 and 10, respectively. In line with results of in vitro migration assays, these NK cells also had the capacity to infiltrate pancreatic tumors and liver metastases in tumor-bearing mice. In vitro, a combined treatment of NK cells with IL-2/TKD but neither of the two treatments alone causes a profound increase in the lytic capacity against Hsp70 membrane-positive Colo357 cells. In vivo, a single i.v. injection of these NK cells on day 15 post-tumor inoculation resulted in a significant reduction in tumor weights, a delayed onset of hepatic metastases, and a prolonged life expectancy. In contrast, identically treated T cells and NK cells treated with IL-2 alone were significantly less efficient in controlling pancreatic tumors and metastases. Most importantly, four repeated i.v. infusions of IL-2/TKD-activated NK cells eradicated primary tumors and prevented hepatic metastases. In summary, our mouse data have implicated that NK cells preactivated with IL-2/TKD might provide a novel therapeutic tool for the treatment of aggressive, Hsp70-positive pancreatic carcinoma.     

A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity

Compared with normal cells, tumor cell lines exhibit an unusual plasma membrane localization of heat shock protein 70 (Hsp70). This tumor-selective Hsp70 membrane expression has been found to correlate with an increased sensitivity to lysis mediated by human natural killer (NK) cells that transiently adhere to plastic following cytokine stimulation. A human Hsp70-specific monoclonal antibody (mAb) detects membrane-bound Hsp70 on viable tumor cells and blocks the immune response of NK cells against Hsp70-expressing tumor cells. By peptide scanning (pep-scan) analysis, the epitope of this mAb was mapped as the C-terminal-localized 8-mer NLLGRFEL (NLL, amino acids [aa] 454-461). Most interestingly, similar to full-length Hsp70 protein, the N-terminal-extended 14-mer peptide TKDNNLLGRFELSG (TKD, aa 450-463) was able to stimulate the cytolytic and proliferative activity of NK cells at concentrations equivalent to full-length Hsp70 protein. Blocking studies revealed that an excess of the 14-mer peptide TKDNNLLGRFELSG inhibits the cytolytic activity of NK cells similar to that of Hsp70 protein. In comparison, other TKD-related peptides, including the 8-mer antibody epitope NLLGRFEL (aa 454-461), the 12-mer TKDNNLLGRFEL (aa 450-461), the 13-mer C-terminal-extended peptide NLLGRFELSGIPP (aa 454-466), the 14-mer TKD-equivalent sequences of Hsp70hom TKDNNLLGRFELTG (aa 450-463), Hsc70 TKDNNLLGKFELTG (aa 450-463), and DnaK AADNKSLGQFNLDG (aa 447-460) failed to activate NK activity.     

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells,B Cells and NKT Cells

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide.     

Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background

We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner.

Methodology/Principal Findings

Optical imaging of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes.

Conclusions/Significance

These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors.     

A hypoxia-induced decrease of either MICA/B or Hsp70 on the membrane of tumor cells mediates immune escape from NK cells

Recent findings suggest that hypoxia of the tumor microenvironment contributes to immune escape from natural killer (NK) cell-mediated cytotoxicity. Heat shock protein 70 (Hsp70) and the stress-regulated major histocompatibility class I chain-related protein A and B (MICA/B) both serve as ligands for activated NK cells when expressed on the cell surface of tumor cells. Herein, we studied the effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) on the membrane expression of these NK cell ligands in H1339 with high and MDA-MB-231 tumor cells with low basal HIF-1α levels and its consequences on NK cell-mediated cytotoxicity. We could show that a hypoxia-induced decrease in the membrane expression of MICA/B and Hsp70 on H1339 and MDA-MB-231 cells, respectively, is associated with a reduced sensitivity to NK cell-mediated lysis. A knockdown of HIF-1α revealed that the decreased surface expression of MICA/B under hypoxia is dependent on HIF-1α in H1339 cells with high basal HIF-1α levels. Hypoxia and HIF-1α did not affect the MICA/B expression in MDA-MB-231 cells but reduced the Hsp70 membrane expression which in turn also impaired NK cell recognition. Furthermore, we could show that the hypoxia-induced decrease in membrane Hsp70 is independent of HIF-1α in MDA-MB-231. Our data indicate that hypoxia-induced downregulation of both NK cell ligands MICA/B and Hsp70 impairs NK cell-mediated cytotoxicity, whereby only MICA/B appears to be regulated by HIF-1α.     

Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3

Background

Human tumors differ from normal tissues in their capacity to present Hsp70, the major stress-inducible member of the HSP70 family, on their plasma membrane. Membrane Hsp70 has been found to serve as a prognostic indicator of overall patient survival in leukemia, lower rectal and non small cell lung carcinomas. Why tumors, but not normal cells, present Hsp70 on their cell surface and the impact of membrane Hsp70 on cancer progression remains to be elucidated.

Methodology/Principal Findings

Although Hsp70 has been reported to be associated with cholesterol rich microdomains (CRMs), the partner in the plasma membrane with which Hsp70 interacts has yet to be identified. Herein, global lipid profiling demonstrates that Hsp70 membrane-positive tumors differ from their membrane-negative counterparts by containing significantly higher amounts of globotriaoslyceramide (Gb3), but not of other lipids such as lactosylceramide (LacCer), dodecasaccharideceramide (DoCer), galactosylceramide (GalCer), ceramide (Cer), or the ganglioside GM1. Apart from germinal center B cells, normal tissues are Gb3 membrane-negative. Co-localization of Hsp70 and Gb3 was selectively determined in Gb3 membrane-positive tumor cells, and these cells were also shown to bind soluble Hsp70-FITC protein from outside in a concentration-dependent manner. Given that the latter interaction can be blocked by a Gb3-specific antibody, and that the depletion of globotriaosides from tumors reduces the amount of membrane-bound Hsp70, we propose that Gb3 is a binding partner for Hsp70. The in vitro finding that Hsp70 predominantly binds to artificial liposomes containing Gb3 (PC/SM/Chol/Gb3, 17/45/33/5) confirms that Gb3 is an interaction partner for Hsp70.

Conclusions/Significance

These data indicate that the presence of Gb3 enables anchorage of Hsp70 in the plasma membrane of tumors and thus they might explain tumor-specific membrane localization of Hsp70.     

Fusion proteins of Hsp70 with tumor-associated antigen acting as a potent tumor vaccine and the C-terminal peptide-binding domain of Hsp70 being essential in inducing antigen-independent anti-tumor response in vivo

Hsp70s are a family of ATP-dependent chaperones of relative molecular mass around 70 kDa. Immunization of mice with Hsp70 isolated from tumor tissues has been proved to elicit specific protective immunity against the original tumor challenge. In this work, we investigated whether Hsp70 can be used as vehicle to elicit immune response to its covalence-accompanying antigen. A recombinant protein expression vector was constructed that permitted the production of recombinant protein fusing tumor-associated antigen (eg, Mela) to the C terminus of Hsp70. We found that the Hsp70-Mela fusion protein can elicit strong cellular immune responses against murine tumor B16, which expresses protein Mela. The Hsp70 peptide-binding domain deletion mutant of the fusion protein was sufficient for inducing Mela-specific cytotoxic T lymphocyte but was not sufficient for engendering potent anti-tumor immunity against B16. We also found that host natural killer (NK) cells were stimulated in vivo by C-terminal domain of Hsp70. We thus presume that Hsp70 fusion proteins suppress tumor growth via at least 2 distinct pathways: one is covalence-accompanying antigen dependent; another is antigen independent. The C-terminal domain of Hsp70 seemed to be the crucial part in eliciting antigen-independent responses, including NK cell stimulation, against tumor challenges. Furthermore, we found that immunization with multiple Hsp70 fusion proteins resulted in a better anti-tumor effect.     

Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells

CX+/CX- and Colo+/Colo- tumor sublines with stable heat shock protein 70 (Hsp70) high and low membrane expression were generated by fluorescence activated cell sorting of the parental human colon (CX2) and pancreas (Colo357) carcinoma cell lines, using an Hsp70-specific antibody. Two-parameter flow cytometry revealed that Hsp70 colocalizes with Bag-4, also termed silencer of death domain, not only in the cytosol but also on the plasma membrane. After nonlethal gamma-irradiation, the percentage of membrane-positive cells and the protein density of Hsp70 and Bag-4 were found to be strongly upregulated in carcinoma sublines with initially low expression levels (CX-, Colo-). Membrane expression of Hsp70 was also elevated in Bag-4 overexpressing HeLa cervix carcinoma cells when compared to neo-transfected cells. In response to gamma-irradiation, neo-transfected HeLa cells behaved like Hsp70/Bag-4 low-expressing CX- and Colo-, and Bag-4-transfected HeLa cells like Hsp70/Bag-4 high-expressing carcinoma sublines CX+ and Colo+. Immunoprecipitation studies further confirmed colocalization of Hsp70 and Bag-4 but also point to an association of Hsp70 and Hsp40 on the plasma membrane of CX+ and Colo+ cells; on CX- and Colo- tumor sublines, Hsp40 was detectable in the absence of Hsp70 and Bag-4. Other co-chaperones including Hsp60 and Hsp90 were neither found on the cell surface of CX+/CX-, Colo+/Colo- nor on HeLa neo-/HeLa Bag-4-transfected tumor cells. Functionally, Hsp70/Bag-4 and Hsp70/Hsp40 membrane-positive tumor cells appeared to be better protected against radiation-induced effects, including G2/M arrest and growth inhibition, on the one hand. On the other hand, membrane-bound Hsp70, but neither Bag-4 nor Hsp40, served as a recognition site for the cytolytic attack mediated by natural killer cells.     

Effects of antineoplastic agents on cytoplasmic and membrane-bound heat shock protein 70 (Hsp70) levels

Here we report on the study of the effects of different antineoplastic agents, including cytarabine, 4-hydroperoxyifosfamide, the activated form of ifosfamide, vincristine, and paclitaxel, with regard to their capacity to modulate the amount of cytoplasmic and membrane-bound heat shock protein 70 (Hsp70). Hsp70 levels were measured in the myelogenous leukemic cell line K562, in the human colon carcinoma cell line CX2, and in peripheral blood lymphocytes (PBL) under physiological conditions (37 degrees C), and following non-lethal heat shock at 41.8 degrees C. A concentration of 1 microM and an incubation period of 2 h were determined as non-lethal, since none of the different antineoplastic agents induced necrosis or apoptosis in untreated or heat-shocked cells under these conditions. Our results show that tubulin-interacting agents, including vincristine and paclitaxel, but not DNA-interacting agents, including cytarabine and ifosfamide, selectively increase the amount of cytoplasmic Hsp70 in tumor and normal cells, as measured by semi-quantitative Western blot analysis. Mechanistically, a vincristine- and paclitaxel-induced tubulin assembly, as demonstrated by immunofluorescence microscopy, might be responsible for the elevated cytoplasmic Hsp70 levels. Interestingly, an increased membrane expression of Hsp70 following treatment with vincristine or paclitaxel was selectively observed on tumor cells, but not on normal cells.     

Heat shock protein 70 is able to prevent heat shock-induced resistance of target cells to CTL

Heat shock or transfection with heat shock protein 70 (Hsp70) genes has been shown to protect tumor cell lines against immune mechanisms of cytotoxicity. We have reported previously that heat shock confers resistance to CTL in the rat myeloma cell line Y3 that is Hsp70 defective. Evidence is now presented that Hsp70 is able to prevent the induction of the resistant phenotype. In Con A-stimulated lymphocytes and in lymphocyte x Y3 somatic cell hybrid clones a severe, non-Hsp70-inducing heat shock elicits resistance to CTL in contrast to a heat shock that results in Hsp70 expression. Thus, Hsp70 expression appears to be negatively associated with the development of resistance. Furthermore, loading of Y3 cells with recombinant Hsp70 protein before heat shock is able to prevent resistance. Because apoptosis induced in Y3 cells by heat shock is not affected, Hsp70 appears to interfere selectively with the CTL-induced lethal pathway that is found to be calcium but not caspase dependent. It is suggested that after heat shock Hsp70 enhances the CTL-induced apoptotic pathway by chaperoning certain proteins in the target cell that are involved in the execution of cell death. Thus, although shown to confer protection against many cytotoxic mechanisms, Hsp70 does not appear to be generally cytoprotective. This observation could also be of relevance when interpreting the effectiveness of tumor immunity.     

Anticancer drugs up-regulate HspBP1 and thereby antagonize the prosurvival function of Hsp70 in tumor cells

The 70-kDa heat shock protein (Hsp70) is up-regulated in a wide variety of tumor cell types and contributes to the resistance of these cells to the induction of cell death by anticancer drugs. Hsp70 binding protein 1 (HspBP1) modulates the activity of Hsp70 but its biological significance has remained unclear. We have now examined whether HspBP1 might interfere with the prosurvival function of Hsp70, which is mediated, at least in part, by inhibition of the death-associated permeabilization of lysosomal membranes. HspBP1 was found to be expressed at a higher level than Hsp70 in all normal and tumor cell types examined. Tumor cells with a high HspBP1/Hsp70 molar ratio were more susceptible to anticancer drugs than were those with a low ratio. Ectopic expression of HspBP1 enhanced this effect of anticancer drugs in a manner that was both dependent on the ability of HspBP1 to bind to Hsp70 and sensitive to the induction of Hsp70 by mild heat shock. Furthermore, anticancer drugs up-regulated HspBP1 expression, whereas prevention of such up-regulation by RNA interference reduced the susceptibility of tumor cells to anticancer drugs. Overexpression of HspBP1 promoted the permeabilization of lysosomal membranes, the release of cathepsins from lysosomes into the cytosol, and the activation of caspase-3 induced by anticancer drugs. These results suggest that HspBP1, by antagonizing the prosurvival activity of Hsp70, sensitizes tumor cells to cathepsin-mediated cell death.     

The role of Hsp70 chaperone in the reaction of human leukemic cells to anticancer drugs

Most of anti-tumor factors are designed to kill selectively cancer cells; in most cases this action is related to the ability of the above substances to induce apoptosis. One of potent anti-apoptotic mechanisms is based on Hsp70 protein. Since the level of this protein is often higher in malignant tumors than in normal tissues, the aim of this study was to establish whether the elevated Hsp70 content may influence the process of apoptosis induced by anti-tumor drugs in cancer cells population. The increase of intracellular content of Hsp70 in human leukemia U-937 cells was attained by a mild heat stress or by transfection of cells with the human hsp70 gene. The elevation of Hsp70 quantity, irrespective of the way it was performed, leads to the inhibition of apoptosis in cells treated with two substances, etoposide or adriamycin. The inhibition of apoptosis was accompanied with the reducing of the share of cells with fragmented nuclei and with the delay in caspase activation. It is suggested that in addition to the previously discovered targets, whose activity is suppressed by the Hsp70 chaperone, this protein can inhibit the activity of caspase-3 and -7; this delays the onset of apoptosis in part of a cancer cell population.