Biomedical subjects
Publications and source records attributed to Mathias Gehrmann.
The apoptosis stimulator STA-4783 acts by inducing expression of heat shock protein (Hsp)70 on tumor cell surfaces and disrupting the cytoskeletal network. Currently in development by Synta Pharmaceuticals for the potential treatment of solid tumors, phase II clinical trials in non-small-cell lung cancer, melanoma and sarcoma have been initiated.
Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.
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.
PURPOSE: Modulation of the heat shock protein (HSP) response affects sensitivity to therapeutic agents in cancer. Here, drugs with anti-inflammatory potential (cyclooxygenase 1/2 inhibitors) and peroxidase proliferator-activated receptor-gamma agonists were analyzed for their capacity to affect Hsp70 expression in human cancer cells with a divergent Hsp70 membrane expression pattern. EXPERIMENTAL DESIGN: In dose kinetics, the nonlethal concentration of acetyl-salicyl acid, celecoxib, rofecoxib, and the insulin-sensitizer pioglitazone was identified for the human adenocarcinoma cell line CX-. With the exception of CLX, which was diluted in DMSO, all reagents were dissolved in water. After treatment with the different compounds at nontoxic concentrations for 6 h, followed by a 1-h recovery period, the cytosolic Hsp70 levels were measured in CX-2 and CX- tumor cells by Western blot analysis. Fold increase was calculated in relation to the housekeeping protein tubulin. Membrane-bound Hsp70 was analyzed by flow cytometry using a FITC-labeled Hsp70-specific monoclonal antibody. Untreated cells and cells incubated with equivalent amounts of the diluting agents served as controls. The immunological function was tested in granzyme B apoptosis assays, standard (51)Cr release assays, and antibody blocking studies. RESULTS: Compared with aqua dest, the cytoplasmic amount of Hsp70 was equally enhanced in CX-2 and CX- cells by all compounds. An increase in membrane-bound Hsp70, detected selectively in CX- cells, corresponded to an enhanced sensitivity to granzyme B- and natural killer cell-mediated kill that was blockable by using a Hsp70-specific antibody. CONCLUSIONS: Although increase in cytosolic Hsp70 levels conferred resistance to further stress, membrane-bound Hsp70 rendered tumor cells more sensitive to the immunological attack mediated by granzyme B and natural killer cells. Our data provide a biological rational for combining anti-inflammatory drugs with immunotherapy in cancer therapy.
PURPOSE: Heat shock protein 70 (Hsp70) was detected on the cell membrane of human tumor cell lines, but not on normal cells. Here we studied Hsp70 membrane expression as a target for natural killer (NK) cells on tumor material and control tissues of head-and-neck cancer patients. METHODS AND MATERIALS: Membrane-bound Hsp70 was determined by flow cytometry on single-cell suspensions of tumors and the corresponding normal tissues of head-and-neck cancer patients. The cytolytic activity of NK cells against Hsp70-positive tumor cells was measured in a standard cytotoxicity assay. RESULTS: In total, 54 of 74 primary tumors were found to be Hsp70 membrane-positive (73%); tongue/mouth, 21 of 24 (88%); oropharynx, 13 of 20 (65%); hypopharynx, 3 of 6 (50%); larynx, 8 of 11 (73%); trachea 1 of 2 (50%); esophagus, 4 of 5 (80%); lymph node metastases, 4 of 6 (67%). The corresponding control tissue was negative for membrane-bound Hsp70. Biopsies (6 of 6) of patients after in vivo gamma-irradiation (fractionated 5 x 2 Gy) were strongly Hsp70 membrane-positive. Irradiated, Hsp70-positive tumor cells are targets for Hsp70-peptide stimulated NK cells. CONCLUSION: An irradiation-inducible, tumor-selective Hsp70 membrane localization provides a target structure for Hsp70-peptide stimulated human NK cells.
Cell membrane localization of the 72 kDa heat shock protein 70 (Hsp70) has been found on different tumour cell lines, on biopsy material from solid tumours and metastases and on leukaemic blasts from acute myelogenous leukaemia patients, but not on the corresponding normal tissues, as determined by flow cytometry using the Hsp70-specific monoclonal antibody C92F3B1. In the present study Hsp70 membrane expression was studied on primary malignant melanomas, melanoma metastases, melanocytes, human skin fibroblasts and peripheral blood lymphocytes, together with expression of the melanoma-associated markers Mel-1, Mel-2 and Mel-5, major histocompatibility complex class I and the fibroblast-specific marker ASO2. As previously shown, fibroblasts and peripheral blood lymphocytes from healthy human volunteers were found to be negative for Hsp70 and for the melanoma-associated markers Mel-1, Mel-2 and Mel-5. Human melanocytes from healthy human donors were also negative for Hsp70, but were positive for Mel-1 and Mel-5. Independent of the Clark's level, all the malignant melanomas (n = 9) and metastases (n = 11) exhibited were positive for both Mel-1 and Mel-2. The primary melanomas could be divided into two groups according to their Hsp70 and Mel-5 expression pattern: those with an Hsp70-negative and a Mel-5-positive phenotype (-/+) (five out of nine), and those with an Hsp70-positive and a Mel-5-negative phenotype (+/-) (four out of nine). All the melanoma metastases (n = 11) had an Hsp70-positive, Mel-5-negative phenotype (+/-). These data provide the first hint that the marker combination Hsp70 positive/Mel-5 negative might be useful in estimating the metastatic potential of a melanoma. Investigations on changes in the marker combination Hsp70/Mel-5 during onset of melanoma disease and progression will clarify its potential as a prognostic risk factor.
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BACKGROUND: Chronic kidney disease is characterized by increased synthesis and inhibited destruction of collagenous and non-collagenous matrix proteins. Elevation of collagen fragments has been demonstrated in the serum and urine of patients with renal disease, but the dynamics of renal matrix deposition remain difficult to determine. METHODS: To obtain a further insight into renal matrix metabolism we have assessed whether serum and urine concentrations of the non-collagenous protein, tenascin, and of the tissue inhibitor of metalloproteinases 1 (TIMP-1) are altered in association with renal disease. Serum and urine concentrations of both proteins were determined using a newly developed magnetic particle enzyme immunoassay and were compared with levels of N-terminal procollagen III-peptide (PIIINP) and related to the degree of renal failure and proteinuria. RESULTS: Circulating levels of tenascin and TIMP-1 were moderately, but significantly, higher in patients with chronic renal disease (n=54; mean creatinine clearance, 62 ml/min) than in healthy controls (n=176). Urine concentrations per mg creatinine of tenascin and TIMP-1 were significantly lower than serum levels, but were on average six- and 18-fold higher, respectively, in patients with renal disease than in controls. Urinary concentrations increased with progressive reduction in renal function, but were unrelated to proteinuria. TIMP-1 concentrations in urine correlated with tenascin, which is compatible with the impact of TIMP-1 on the accumulation of matrix proteins. The concentrations of proteins measured did not differ depending on the aetiology of renal disease. CONCLUSION: Urinary concentrations of tenascin and TIMP-1 are elevated in association with renal disease and may reflect specific aspects of renal fibrosis.
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.
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.