RESUMEN
Generation of an effective immune response requires that antigens be processed and presented to T lymphocytes by antigen-presenting cells, the most efficient of which are dendritic cells (DC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected to result in a broad impairment of immune function, not unlike that observed in astronauts during or after space flight. In the study reported here, we investigated whether DC generation and function are altered in a culture environment that models microgravity, i.e., the rotary-cell culture system (RCCS). We observed that RCCS supported the generation of DC identified by morphology, phenotype (HLA-DR+ and lacking lineage-associated markers), and function (high allostimulatory activity). However, the yield of DC from RCCS was significantly lower than that from static cultures. RCCS-generated DC were less able to phagocytose Aspergillus fumigatus conidia and expressed a lower density of surface HLA-DR. The proportion of DC expressing CD80 was also significantly reduced in RCCS compared to static cultures. When exposed to fungal antigens, RCCS-generated DC produced lower levels of interleukin-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. These data suggest that DC generation, and some functions needed to mount an effective immune response to pathogens, may be disturbed in the microgravity environment of space.
Asunto(s)
Células Dendríticas/citología , Ingravidez , Antígenos CD34/inmunología , Técnicas de Cultivo de Célula , División Celular , Células Dendríticas/inmunología , Humanos , Interleucina-12/biosíntesis , Receptores de Lipopolisacáridos/inmunología , Prueba de Cultivo Mixto de Linfocitos , FagocitosisRESUMEN
We studied: (a) the adverse effects of tumour necrosis factor-alpha (TNF) given during whole body hyperthermia (WBH) on mean arterial pressure (MAP) and gut mucosa in anaesthetized rats; (b) the potential protective effect of NG-monomethyl-L-arginine (L-NMA), an inhibitor of nitric oxide synthase; and (c) the influence of L-NMA on the antitumour effect of the trimodality therapy, WBH + TNF + Carboplatin (CBDCA). In normothermic rats, TNF alone (10(5) or 10(6) U/kg) did not cause hypotension, but increased MAP (p < 0.05). L-NMA alone (5, 10 and 20 mg/kg) increased MAP moderately and dose-dependently (p < 0.05). WBH (41.5 degrees C for 2 h) increased MAP markedly (from 103 +/- 4 to 161 +/- 4 mm Hg). This increase in MAP was sustained throughout the hyperthermia, but was followed by a transient relative hypotension (MAP = 80 +/- mm Hg) on cessation of WBH and an eventual return to near baseline at 30 min post-WBH (MAP = 94 +/- 5 mm Hg). WBH + TNF (10(5) or 10(6) U/kg) initially increased MAP similarly to WBH alone. During the second hour of WBH, however, MAP decreased towards pre-treatment levels, and cessation of WBH was followed by sustained hypotension. This late hypotensive state was associated with a mortality during the early (first 2 h) post-WBH period of 17 and 100% at TNF dose of 10(5) and 10(6) U/kg TNF, respectively. L-NMA given to rats receiving WBH + TNF (10(6) U/kg) maintained MAP at levels similar to WBH alone during WBH treatment. L-NMA prevented the post-WBH hypotension, and extended the survival beyond the early (first 2 h) post-WBH period. No rat, however, receiving high dose TNF (10(6) U/kg) survived more than 12 h even with L-NMA (totally 40 mg/kg). WBH + TNF (10(5) and 10(6) U/kg) also produced marked histopathological injury to the gut mucosa at 2 h post-treatment. L-NMA substantially protected the gut from this injury. In rats bearing a transplantable fibrosarcoma, L-NMA did not decrease the antitumour effect consisting of WBH + TNF (10(5) U/kg) + CBDCA, while it decreased (p < 0.05) the general toxicity (weight loss, diarrhea and foot oedema) of this combination. We conclude that L-NMA may prevent or ameliorate the early toxicity but not the late lethal effects of WBH + high dose TNF (10(6) U/kg). Additionally, L-NMA reduces some of the toxicity of WBH + TNF (10(5) U/kg) + CBDCA without decreasing the antitumour effect of this trimodality therapy. Inhibitors of nitric oxide synthase such as L-NMA may provide a novel approach to overcoming the toxicity of TNF in combination with WBH.
Asunto(s)
Fiebre , Hipotensión/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , omega-N-Metilarginina/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Carboplatino/toxicidad , Inhibidores Enzimáticos/farmacología , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Histocitoquímica , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/terapia , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Endogámicas F344RESUMEN
The response of tumour necrosis factor (TNF)-sensitive murine L929 cells to TNF was enhanced approximately 1000-fold after step-down heating (SDH) for 30 min at a sensitizing temperature (ST) of 43 degrees C and a subsequent 24 h incubation at a test temperature (TT) of 40.5 degrees C, compared to continuous treatment at 37 degrees C. The TNF-resistant phenotype of murine EMT-6 mammary adenocarcinoma cells could be overcome by 24 h heating at a TT of 40.5 degrees C, and their sensitivity to TNF could be further increased by preheating at the ST for up to 60 min. The response of TNF-sensitive HCT-15 human colon adenocarcinoma cells was somewhat similar to that of L929 cells except that there was u approximately 2.5 log increase in TNF-sensitivity due solely to heating at 40.5 degrees C. The response of TNF-resistant DLD-1 human colon adenocarcinoma cells was similar to that of EMT-6 cells. In contrast, three normal cell lines demonstrated greater resistance to any TNF/SDH treatment examined. Our results suggest that SDH may overcome the resistance or enhance the response of tumour cells to TNF while minimizing cytotoxic effects on normal cells.
Asunto(s)
Hipertermia Inducida/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Terapia Combinada , Resistencia a Medicamentos , Proteínas de Choque Térmico/biosíntesis , Humanos , Ratones , Neoplasias/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/terapia , Fenotipo , Temperatura , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
Hyperthermia sensitizes tumor cells to killing by tumor necrosis factor-alpha (TNF). Sensitization is greater in cells exposed to TNF before heating begins than with the reverse sequence, and heat-shock proteins (hsp) have been suggested to protect cells from TNF cytotoxicity. Here we examined the role of Hsp27 in TNF resistance. Murine L929 cells were stably transfected with the vector pRc/CMV constitutively to express an inserted human hsp27 complementary DNA (cDNA) sequence. Parental cells produced no detectable murine homolog to human hsp27. Hsp27-sense clones expressed hsp27 messenger RNA (mRNA) and protein at 37 degrees C. Cells transfected with the cDNA in the anti-sense orientation produced anti-sense mRNA but no protein, and cells transfected with the vector alone produced neither product. Expression of hsp27 conferred significant resistance to TNF cytotoxicity in both neutral red cytotoxicity and clonogenic survival assays. Vector along and hsp27 anti-sense transfectants had a TNF response similar to that of parental L929 cells. Kinetic studies in L929 cells showed that hsp27-expressing clones exhibited resistance relative to parental cells beginning 6 h after TNF exposure, and this differential response increased by 12 and 24 h. Addition of actinomycin D to the TNF cytotoxicity assays accelerated the cytotoxicity development in parental and transfected cells, but the hsp27-sense clones were still more resistant. Hsp27-sense clones of L929 cells were also resistant to oxidative stress induced by menadione and released less arachidonic acid in response to TNF induction. These results show that hsp27 can negatively regulate the TNF cytotoxic mechanism.
Asunto(s)
Citotoxicidad Inmunológica/efectos de los fármacos , ADN Complementario/genética , Proteínas de Choque Térmico/genética , Transfección/inmunología , Factor de Necrosis Tumoral alfa/toxicidad , Animales , Ácido Araquidónico/metabolismo , ADN Complementario/inmunología , Proteínas de Choque Térmico/biosíntesis , Humanos , Células L , Ratones , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/inmunologíaRESUMEN
We evaluated the in vitro cytotoxic effects of combined human tumour necrosis factor alpha (TNF), human interferon gamma (IFN-gamma), melphalan (L-PAM) and hyperthermia (HTX) on human melanoma cell lines using the crystal violet assay. HTX (40 degrees C, 1 h) alone had no effect. The responses of the cell lines to TNF were in the rank order of 939 cells > 987 > 284 > C8161 > 852 > A2058 approximately 0, and all displayed shallow dose-response curves; no significant thermal enhancement of TNF cytotoxicity was apparent with this heat dose. All cell lines were sensitive to L-PAM, with 284 cells being the most sensitive; HTX caused only slightly increased sensitization to L-PAM. The combination of TNF and L-PAM resulted in generally subadditive or additive cytotoxicity, with or without HTX. The response to IFN-gamma alone was heterogeneous; the 939, 284 and 852 cell lines were sensitive to a dose as low as 20 ng/ml, whereas the 987 line was resistant to 2.0 micrograms/ml, even with HTX. IFN-gamma enhanced the response to TNF only of the TNF-resistant A2058 cell line, but there was no enhancement of the response to L-PAM for any line. Thus, this tetramodality combination achieved generally subadditive or additive cytoxicity in vitro.
Asunto(s)
Interferón gamma/uso terapéutico , Melanoma/terapia , Melfalán/uso terapéutico , Neoplasias Cutáneas/terapia , Factor de Necrosis Tumoral alfa/uso terapéutico , Supervivencia Celular , Terapia Combinada , Relación Dosis-Respuesta a Droga , Humanos , Hipertermia Inducida , Interferón gamma/administración & dosificación , Melanoma/tratamiento farmacológico , Melanoma/patología , Melfalán/administración & dosificación , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/administración & dosificaciónRESUMEN
The responses of DLD-1 and HCT-15 human colon adenocarcinoma cells to hyperthermia, 5-fluorouracil (5-FU)/leucovorin, carboplatin and tumor necrosis factor-alpha, singly and in multiple combinations, were evaluated in clonogenic assays. The combination of hyperthermia with the lower dose combination resulted in a survival fraction of about 0.005 to 0.001 for both cell types, whereas estimated additive interactions alone would have resulted in a survival fraction of about 0.5 (DLD-1) or 0.05 (HCT-15). A survival fraction of 0.00001 or greater was observed when the higher dose levels were combined with hyperthermia, whereas additive interactions alone would have achieved a decrease of only 0.001 or 0.0001 in the surviving fraction. The combination of the three other modalities at either dose level under conditions of hyperthermia or normothermia achieved statistically significant apparently supra-additive losses of clonogenicity in HCT-15 cells; similar results were obtained with the lower dose level in DLD-1 cells. Our results suggest that human colon tumor cells are markedly sensitive to this combination of modalities when used at clinically achievable dose levels.
Asunto(s)
Carboplatino/toxicidad , Fluorouracilo/toxicidad , Hipertermia Inducida , Leucovorina/toxicidad , Factor de Necrosis Tumoral alfa/toxicidad , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Neoplasias del Colon , Relación Dosis-Respuesta a Droga , Humanos , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
Hyperthermia has been shown to potentiate the cytotoxicity of exogenously added tumor necrosis factor (TNF) against tumor cell targets. The mechanism for that interaction is not known, but among the possibilities are that heat enhances internalization of ligand-bound TNF or enhances processing of internalized TNF. In this study, we found that NIH 3T3 cells transfected with an expression vector containing the full-length human pro-TNF secreted TNF and that hyperthermic treatment of chromium-labeled L929 target cells at 43 degrees C for 1 h potentiated the cytotoxicity of these transfectants against the L929 cells in clonogenic survival and chromium-release assays. On the other hand, transfectants expressing a transmembrane, nonsecretable pro-TNF mutant that kills L929 cells by cell-to-cell contact without internalization also exhibited enhanced cytotoxicity against heated L929 cell targets. Thus, potentiation of the cytotoxicity of TNF by hyperthermia is not strictly dependent on enhanced internalization of ligand-bound TNF or enhanced processing of internalized TNF.
Asunto(s)
Terapia Genética , Calor , Factor de Necrosis Tumoral alfa/genética , Células 3T3 , Animales , Supervivencia Celular/efectos de los fármacos , Células Clonales , Hipertermia Inducida , Ratones , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The relationship of DNA fragmentation to the greatly enhanced cytotoxicity seen in vitro against tumour cells when recombinant human tumour necrosis factor-alpha (TNF-alpha) is combined with hyperthermia was investigated. The TNF-alpha-sensitive L929 and -resistant EMT6 cells were treated with 8.8 and 16 ng of TNF-alpha per ml, respectively, and then heated at 40.5 degrees C for 24 h (L929) or at 43 degrees C for 1 h (L929) or 1.5 h (EMT-6) beginning 1 h later. For both cell lines at both temperatures, the addition of heating to the TNF-alpha treatment significantly decreased viability and increased DNA fragmentation at earlier time points than seen with either TNF-alpha or heat alone. DNA fragmentation was further studied using agarose gel electrophoresis to examine the size distribution of the DNA fragments and the ability of intracellular calcium buffering agents BAPTA and quin-2 to inhibit fragmentation. At 4.5 h after L929 cells were treated with TNF-alpha at 43 degrees C, the size distribution of DNA fragments more closely resembled the oligonucleosome sized apoptotic DNA fragmentation, as seen in irradiated rat thymocytes, than the spectrum of DNA fragments seen in necrotic fragmentation. However, while BAPTA and quin-2 inhibited the calcium-dependent apoptotic fragmentation seen in thymocytes they did not inhibit the DNA fragmentation in L929 cells. In addition, the loss of membrane integrity in both L929 and EMT-6 cells preceded or approximated the appearance of DNA fragmentation, whereas loss of membrane integrity usually follows DNA fragmentation in apoptosis. However, morphological studies showed that apoptotic bodies were present in L929 cell cultures treated with TNF-alpha and heat, and were distinguishable from necrosing cells. We conclude that both types of DNA fragmentation are operant in some cell lines exhibiting a cytotoxic response to TNF-alpha and heat treatments, and that increased fragmentation reflects the greatly enhanced cytotoxic interactions seen with combination treatments in those cells.
Asunto(s)
Daño del ADN , Calor , Factor de Necrosis Tumoral alfa/farmacología , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We have investigated the cytotoxic responses in vitro of three human colon tumor cell lines with epithelial-like morphology, DLD-1, HCT-15, and HT-29, to thermochemoimmunotherapy with hyperthermia (42 degrees C for 2 h), carboplatin, and recombinant human tumor necrosis factor (TNF). Dose ranges of carboplatin and recombinant human TNF were administered essentially simultaneously and were followed 1 h later by hyperthermia. A two-tiered approach was used to evaluate cytotoxicity. In the first tier, a 5-day microcytotoxicity assay using vital dye staining was done; the effect on surviving fraction of simultaneously varying carboplatin and recombinant human TNF doses was evaluated by response surface methodology. From this analysis doses were selected for use in the second-tier clonogenic survival assays. A similar treatment protocol was used in clonogenic assays. Both assays revealed significant interline treatment response heterogeneity. Only the HCT-15 cells were sensitive to TNF alone; carboplatin activity against all three tumor cell lines was enhanced by TNF. Hyperthermia had minimal effect as a sole agent but enhanced the effects of carboplatin and TNF in DLD-1 and HCT-15 cells. Triple modality treatment resulted in 3-4-log decreased survival and could reduce cytotoxic resistance expressed against single- or dual-modality treatments by some of these cells.
Asunto(s)
Adenocarcinoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Cisplatino/farmacología , Neoplasias del Colon/terapia , Hipertermia Inducida , Factor de Necrosis Tumoral alfa/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/microbiología , Cisplatino/administración & dosificación , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/microbiología , Terapia Combinada , Humanos , Factores Inmunológicos/farmacología , Inmunoterapia/métodos , Rojo Neutro/farmacocinética , Fenotipo , Sensibilidad y Especificidad , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/administración & dosificación , Ensayo de Tumor de Célula MadreRESUMEN
This study examined the effect of a trimodality therapy of the combination of recombinant human tumor necrosis factor alpha (TNF), whole-body hypertheria (WBH), and cis-diamminedichloroplatinum(II) (CDDP) or cis-diammine-1,1-cyclobutane dicarboxylate platinum(II) (CBDCA) on a fibrosarcoma and normal tissue in F344 rats. TNF (1 x 10(5) units/kg) increased the antitumor effect of both CDDP (1.5 mg/kg) + WBH (2 h at 41.5 degrees C) and CBDCA (30 mg/kg) + WBH. Tumor growth delay, which was 1.9 days for CDDP + WBH and 2.7 days for CBDCA + WBH (P less than 0.01 compared to control), was significantly increased to 2.9 days with TNF + CDDP + WBH and 5.4 days with TNF + CBDCA + WBH (P less than 0.05). WBH, TNF, CDDP or CBDCA alone, TNF + CDDP, TNF + CBDCA, or TNF + WBH had no significant effect on tumor growth. In contrast, administration of TNF did not enhance the CDDP- or CBDCA-mediated dose limiting normal tissue toxicity. CDDP + WBH-mediated acute renal injury and CBDCA + WBH-mediated acute myelosuppression, as determined by blood urea nitrogen and peripheral blood cell counts, respectively, were not increased with the addition of TNF to either dual modality therapy. Histopathologically, addition of TNF produced no significant alterations in the kidney and the bone marrow as compared to CDDP + WBH or CBDCA + WBH. These data show that TNF enhanced the platinum + WBH-mediated antitumor effect without increasing normal tissue toxicity, suggesting that TNF may increase the therapeutic efficacy of CDDP or CBDCA combined with WBH.
Asunto(s)
Cisplatino/uso terapéutico , Fibrosarcoma/terapia , Hipertermia Inducida , Factor de Necrosis Tumoral alfa/uso terapéutico , Animales , Peso Corporal/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Cisplatino/toxicidad , Terapia Combinada , Femenino , Fibrosarcoma/patología , Hipertermia Inducida/efectos adversos , Riñón/efectos de los fármacos , Riñón/patología , Ratas , Ratas Endogámicas F344 , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
Hyperthermia can strikingly enhance tumor necrosis factor-alpha (TNF-alpha) cytotoxicity in vitro and in vivo. Other forms of TNF may have tumor therapeutic applications and their interaction with hyperthermia should also be assessed. We have compared the effect of heat on the in vitro cytotoxic response of murine L929 and EMT-6 and human T24 tumor cells to three TNF forms; recombinant human TNF-alpha, TNF-beta (lymphotoxin), and TNF-SAM2. A neutral red assay was used to measure toxicity at 18-20 h after initiating the heat treatment. TNF treatment preceded heating by 0-4 h or followed it by 2 h. Heating was done at 39 or 40.5 degrees C for 24 h, 40.5 or 42 degrees C for 1 h, or 43 degrees C for 1-1.5 h. We found that both TNF-beta and TNF-SAM2 toxicities, like that of TNF-alpha, were markedly enhanced by hyperthermia. Neither EMT-6 nor T24 cells responded consistently to any of these TNFs at heat doses up to 1 h at 43 degrees C, but an increment of only 15 min more at 43 degrees C sensitized EMT-6 cells and 1.5 h at 43 degrees C resulted in extensive EMT-6 cell killing. The T24 cells remained resistant except for variable responses at the highest TNF and heat doses. If TNF treatment was begun immediately before or 2 h after beginning to heat the EMT-6 cells, sensitization was reduced or eliminated, respectively, for all three TNF forms relative to protocols in which TNF was added 1, 2, or 4 h before heating.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Citotoxicidad Inmunológica/efectos de los fármacos , Fiebre/complicaciones , Células Tumorales Cultivadas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Estudios de Evaluación como Asunto , Humanos , RatonesRESUMEN
Thymus-derived lymphocytes undergo death after gamma-irradiation via a pathway termed apoptosis, or programmed cell death. An early step in this pathway is the production of nucleosome-sized fragments of DNA. DNA fragmentation was used as the endpoint in these investigations to examine apoptosis in lymphocytes extracted from the rat thymus and irradiated in vitro. In unirradiated thymocytes the level of DNA fragmentation rose to 15% by the first hour of culture, where it remained approximately constant until the fifth hour. In contrast, thymocytes irradiated with a dose of 2.5 Gy exhibited a large and dramatic increase in DNA fragmentation beginning 2 h postirradiation. DNA fragmentation measured 6 h after irradiation was detected after as little as 0.25 Gy and reached a maximum of 90% with 10 Gy. Metabolic control of DNA fragmentation after irradiation was evidenced by the suppression of DNA fragmentation when thymocytes were incubated with cyclohexamide or actinomycin D. When gamma-irradiated thymocytes were incubated with the Ca2+ chelator EGTA, DNA fragmentation was reduced significantly. BAPTA-AM, a highly specific intracellular Ca2+ chelator, essentially eliminated DNA fragmentation in cells irradiated with 2.5 Gy and, unlike EGTA, eliminated the background level of fragmentation in unirradiated samples. Therefore, our data are consistent with the possibility that Ca2+ serves as a second messenger to induce DNA fragmentation in irradiated thymocytes, suggesting a common pathway for cells prompted to enter apoptosis from seemingly dissimilar interval events.
Asunto(s)
Calcio/fisiología , Supervivencia Celular , Linfocitos T/efectos de la radiación , Animales , Cicloheximida/farmacología , ADN/metabolismo , Ácido Egtácico/farmacología , Técnicas In Vitro , Masculino , Metilprednisolona/farmacología , Ratas , Ratas EndogámicasRESUMEN
We have reported on the effect of heat in C127 cells having various basal levels of the Ca(2+)-binding proteins calmodulin (CaM) or parvalbumin [Evans, Simonette, Rasmussen, Means, and Tomasovic, J. Cell. Physiol. 142, 615-627 (1990)]. These studies suggested that induction of the synthesis of 26-kDa heat-shock protein (hsp-26) depended on increased intracellular free Ca2+ [Ca2+]i and that induction was abrogated by increased Ca(2+)-binding capacity. To evaluate further the role of [Ca2+]i in mediating the response to hyperthermia and the potential for Ca(2+)-buffering to affect these processes, we loaded C127 parental cells with the Ca2+ chelators BAPTA or quin-2 (5 microM for 60 min) and then immediately heated the cells (30 min at 43 degrees C) and labeled them (3 h at 37 degrees C) with [3H]leucine. Measurements of [Ca2+]i with quin-2 and fura-2 showed that an increase in [Ca2+]i occurred with this heat dose, but that the quin-2 buffered that increase. Two-dimensional gels showed that cells loaded with BAPTA and quin-2 had a reduced rate of synthesis of the most basic (nonphosphorylated) hsp-26a isoform. The apparent synthesis of the more acidic isoforms (hsp-26b, hsp-26c) was less affected, but labeling studies with 32P showed this reflected continued accumulation of these phosphorylated isoforms, especially the most highly phosphorylated hsp-26c. Although it reduced hsp-26a synthesis, the temporary buffering of [Ca2+]i did not alter the subsequent expression of heat killing or the extent of thermotolerance significantly, possibly because phosphorylated hsp-26 was still generated. These data support the hypothesis that perturbations of [Ca2+]i directly modulate induction of hsp-26a synthesis.
Asunto(s)
Calcio/fisiología , Proteínas de Choque Térmico/biosíntesis , Aclimatación/efectos de los fármacos , Aclimatación/fisiología , Aminoquinolinas , Animales , Tampones (Química) , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Quelantes , Dimetilsulfóxido/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Fura-2 , CalorRESUMEN
Murine bacillus Calmette-Guerin activated macrophages release several monokines when triggered by the bacterial endotoxin lipopolysaccharide (LPS); this has recently been reported to be strongly influenced by the sequence of hyperthermic and LPS treatments. In the work reported here, it was found that LPS treatment markedly modulated the rate of synthesis of proteins in the heat stress protein (HSP) 70 family in these macrophages. The rate of synthesis of the HSP 70 family was slightly reduced if the cells were incubated with LPS 4 h prior to heating at 43 degrees C for 1 h, but was greatly reduced as the triggering time approached the initiation of heating and was nearly completely abrogated if the LPS triggering immediately preceded or followed heating. Near-normal rates of HSP 70 synthesis occurred if the triggering was delayed until 1-2 h after the heating ended. The LPS-triggered release of tumour necrosis factor (TNF) was also reduced as the time of LPS addition approached the heating time, but this depressed release preceded the effects on HSP 70 synthesis and did not recover for up to 3 h after heating. The effects of LPS on HSP 70 synthesis also occurred in a murine monocytic cell line, PU5-1.8, which releases TNF in response to LPS, and in a murine fibroblast cell line, NIH/3T3. This indicates that these effects are not restricted to cells of monocyte or macrophage lineage. The nature of the transcriptional or translational mechanisms controlling these responses is unknown, but these data may contribute to the understanding of (1) the regulation of the HSP 70 family and (2) TNF processing in stressed cells.
Asunto(s)
Proteínas de Choque Térmico/biosíntesis , Lipopolisacáridos/toxicidad , Animales , Células Cultivadas , Calor , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The interaction of calmodulin (CaM) with heat-shock and other binding proteins was studied in rat adenocarcinoma cells. Cells were equilibrium-labeled for 48 h prior to heating for 1 h at 43 degrees C, or pulse-labeled for 2 h at 37 degrees C after heating, to monitor the effect of heat on the affinity of CaM-binding proteins synthesized under these conditions. A CaM antagonist shown to sensitize to heat killing, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], was used in competition assays to help monitor any changes in affinity. We found that heating tended to reduce the CaM-binding of proteins synthesized before heating relative to their 37 degrees C controls and proteins synthesized after heating tended to have increased binding relative to their respective controls. Members of the heat-shock protein (hsp) 90-, 70-, and 26-kDa families were among the proteins that bound to CaM and were eluted by W-7. The peak elution fractions for the hsp's and other cellular proteins varied, but hsp-70 eluted in the early fractions. The hsp-70 family was also found to be among a number of W-7-binding proteins. We conclude that the assumption that CaM antagonists potentiate killing of heated cells solely by competing nonspecifically for CaM-binding protein sites on CaM does not explain the process completely. These antagonists could also act by competing for CaM-binding sites with specific proteins whose interaction with CaM is important for survival following heating, or by directly binding to other proteins whose function is important for survival and inhibiting their activity. We do not have sufficient data to discern the predominant mechanism among these possibilities, but we believe all are likely to occur in heated cells and speculate that inhibition of the functions of the hsp-70 family is important in several of these antagonist actions.
Asunto(s)
Proteínas de Unión a Calmodulina/aislamiento & purificación , Proteínas de Choque Térmico/aislamiento & purificación , Calor , Células Tumorales Cultivadas , Adenocarcinoma/patología , Animales , Calmodulina/antagonistas & inhibidores , Supervivencia Celular/efectos de los fármacos , Cromatografía de Afinidad , Técnicas In Vitro , Neoplasias Mamarias Experimentales/patología , Ratas , Sulfonamidas/farmacologíaRESUMEN
Using a bovine papilloma virus-based vector, mouse mammary adenocarcinoma cells have been transformed to express elevated amounts of functional calmodulin (CaM) (Rasmussen and Means, 1987) and another Ca2(+)-binding protein, parvalbumin (PV) (Rasmussen and Means, 1989) that is not normally synthesized in these cells. Parental cells (C127) and cells transformed by the vector alone (BPV-1), the vector containing a CaM gene (CM-1), or the vector containing parvalbumin (PV-1) were used to study the effect of increased synthesis of Ca2(+)-binding proteins on heat-stress protein (HSP) synthesis and cell survival following heating at 43 degrees C. The induction, stability, and repression of the synthesis of most HSPs after 43 degrees C heating was not significantly affected by increased amounts of Ca2(+)-binding proteins, but the rate of synthesis of all three isoforms of the 26-kDa HSP (HSP26) was greatly reduced. C127 cells, which have about one half as much CaM as do BPV-1 cells, synthesized the most HSP26. CM-1 cells, which have more than fourfold higher levels of CaM than do BPV-1 cells, had a rate of synthesis of HSP26 approaching that of unheated cells. BPV-1 cells, with a two-fold increase in CaM, were intermediate in HSP26 synthesis. This effect on HSP26 synthesis may be largely related to the Ca2(+)-binding capacity of CaM rather than to a specific CaM-regulated function, since PV-1 cells also showed reduced rates of HSP26 synthesis. Survival experiments showed that reduced HSP26 synthesis in cells with increased amounts of Ca2(+)-binding proteins did not significantly alter intrinsic resistance to continuous 43 degrees C heating. Thermotolerance was not reduced and appeared to develop more rapidly in CM-1 and PV-1 cells. These results suggest that (1) the signal for HSP26 synthesis can be largely abrogated by elevated Ca2+ binding protein levels, and (2) if these HSPs are involved in thermotolerance development, that function may be associated with intracellular Ca2+ homeostasis.
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Proteínas de Unión al Calcio/metabolismo , Proteínas de Choque Térmico/biosíntesis , Animales , Calmodulina/fisiología , Línea Celular , Supervivencia Celular , Electroforesis en Gel Bidimensional , Calor , Ratones , Peso Molecular , Parvalbúminas/fisiología , TransfecciónRESUMEN
Hyperthermia in the febrile (less than or equal to 41 degrees C) or tumor therapeutic (greater than or equal to 42 degrees C) ranges is known to alter tumor-host interactions: there are reports of either inhibitory or enhancing effects on tumor metastasis and various host defense mechanisms. Historically, this has been an area of conflicting and often anecdotal reports, and there are still significant gaps in our knowledge of the effects of temperature on tumor-host interactions. However, we believe that the tools are now available to further our understanding of the complex relationships between febrile episodes or therapeutically applied heat and various tumor-host cytotoxic mechanisms, and that potentially important and exploitable relationships can be defined. In this review we give an overview of the current status of this field and the factors that have shaped it. We also describe our recent experimental work with macrophages and their monokines, primarily tumor necrosis factor (TNF), which we feel offers new scientific and clinical opportunities for future studies.
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Hipertermia Inducida , Macrófagos/citología , Neoplasias Experimentales/patología , Neoplasias/patología , Animales , Comunicación Celular , Fiebre/inmunología , Humanos , Macrófagos/inmunología , Ratones , Neoplasias/inmunología , Neoplasias Experimentales/inmunologíaRESUMEN
Hyperthermia modulated the cytotoxic activities of murine macrophages cocultured with EMT-6 tumor cells, altered the production of the monokines RIF (respiratory inhibition factor) and FeRF (iron-releasing factor) by these effector cells, and perturbed the activities of these monokines against EMT-6 cells. Cytotoxic activities of heated murine macrophages activated by Bacillus Calmette-Guérin were inhibited by heat doses of 40.5 degrees C for greater than or equal to 1 h if heating preceded triggering by the endotoxin lipopolysaccharide; however, cytotoxic activities were better retained if triggering preceded heating by 2 h. Treatment-sequence dependencies were also found in the secretion of some monokines that participate in these macrophage cytotoxic effects. Secretion of both RIF and FeRF by macrophages was slightly augmented or at least better retained if triggering of macrophages occurred several hours before heating for 1 h at 40.5-43 degrees C or for 24 h at 39-40.5 degrees C. If heating was nearly simultaneous with triggering or preceded it by several hours, there was a dose-dependent decrease in monokine secretion. The sensitivity of tumor cell targets to these monokines was also modified by the treatment sequence. When EMT-6 cells were treated with RIF- or FeRF-containing macrophage conditioned supernatants 1 h before heating at 40.5-43 degrees C, the RIF-treated cells became sensitized and the FeRF-treated cells retained better cytotoxic response than if cells had been treated 1 h after heating. Chronic heating for 24 h at 39-40.5 degrees C showed less dependence of tumor cell response on treatment sequence. Similar observations have recently been made in our laboratory for the interaction of tumor necrosis factor cytotoxic pathways with hyperthermia (Klostergaard et al., J. Biol. Response Modif., in press, 1989; Tomasovic et al., Int. J. Hyperthermia, in press, 1989). Those results and the present observations with the monokines RIF and FeRF support our hypothesis that the cytotoxic actions of macrophages and the cytotoxicity of endogenously added monokines can be augmented by appropriately constructed sequences combining hyperthermia with either macrophage priming/triggering or monokine treatment of tumor cells.
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Calor , Activación de Macrófagos , Macrófagos/inmunología , Monocinas/metabolismo , Adenocarcinoma , Animales , Línea Celular , Supervivencia Celular , Células Cultivadas , Cromo/metabolismo , Femenino , Hierro/metabolismo , Cinética , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Masculino , Neoplasias Mamarias Experimentales , Ratones , Ratones Endogámicos , Monocinas/inmunología , Mycobacterium bovis/inmunologíaRESUMEN
Hyperthermia in the therapeutic (greater than or equal to 42-43 degrees C) and febrile (less than or equal to 39-40.5 degrees C) ranges modulated the cytotoxic activities of macrophages cocultured with tumour cells and of the monokine tumour necrosis factor (TNF) against tumour cells. These modulatory interactions had clear treatment-sequence dependencies, and some sequences markedly augmented cytotoxic activities. Heated murine bacillus Calmette-Guerin-activated macrophages retained their cytotoxic activities better in coculture with unheated tumour cells if triggering with the endotoxin lipopolysaccharide preceded 1 h of heating at 42 or 43 degrees C than they did if heating to the same extent was concomitant with, or preceded, triggering. Retention of cytotoxicity in coculture during 24 h of 39 or 40.5 degrees C heating was less dependent on pre-heating triggering. The triggering/heating sequence also had modulatory effects on the secretion by heated macrophages of TNF which is involved in cytotoxic manifestations in coculture. Production of TNF by macrophages heated for 1 h at 40.5-43 degrees C or 24 h at 39 or 40.5 degrees C was augmented 1.5- to 6-fold (depending on the heat dose) when triggering preceded heating, whereas sequences in which heating was concomitant with triggering or preceded triggering were detrimental to TNF secretion. Profound treatment-sequence dependencies were also seen when timing of the addition of recombinant human TNF was varied in relation to the heat treatment of tumour cells. Sensitization of TNF-responsive L-929 and TNF-resistant EMT-6 tumour cells occurred if monokine addition preceded heating, whereas the reverse treatment-sequence reduced or eliminated sensitization. Both tumour cell types were also sensitized to TNF if monokine treatment preceded 24 h heating at 40.5 degrees C. These results support the hypothesis that appropriately constructed sequences for either macrophage priming/triggering or monokine treatment of tumour cells, combined with hyperthermia, could augment the cytotoxic actions of macrophages and the cytotoxicity of endogenously added monokines.
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Calor/uso terapéutico , Macrófagos/metabolismo , Células Tumorales Cultivadas/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Células Cultivadas , Citotoxicidad Inmunológica , Activación de Macrófagos , Macrófagos/inmunología , Ratones , Mycobacterium bovis/inmunología , Factores de Tiempo , Células Tumorales Cultivadas/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The role of calmodulin (CaM) in hyperthermic cell killing, and the development of thermotolerance in rat 13762NF mammary adenocarcinoma cells, was investigated by using the CaM antagonists W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and W-13 [N-(4-aminobutyl)-2-chloro-naphthalenesulphonamide] and their less active analogues W-5 [N-(6-aminohexyl)-1-naphthalenesulphonamide] and W-12 [N-(4-aminobutyl)-2-naphthalenesulphonamide]. The CaM antagonists W-7 and W-13 potentiated 43 degrees C cell killing (and the less active analogues did not) at a concentration compatible with CaM inhibition, thus hyperthermic perturbation of CaM-regulated processes may contribute to cellular lethality. The potentiation of hyperthermic killing by antagonists appeared to be temperature-dependent, sensitizing much more effectively to 43 degrees C than to 42 degrees C killing. The effect may be related to differing primary mechanisms of hyperthermic killing activated at the two temperatures, or simply to differences in incorporation or localization of the antagonists. The presence of the CaM antagonists throughout fractionated 42 degrees C or 43 degrees C heating, or during continuous 42 degrees C heating, did not significantly inhibit or potentiate the triggering and development of thermotolerance or alter the rates of heat stress protein (HSP) synthesis. Studies using CaM-agarose isolation of CaM-binding proteins indicated that binding of some HSP to CaM-agarose occurred and was Ca2+-dependent. The specificity and physiological relevance of these HSP binding to CaM was not clear, since their affinity was not high in these cells. Presumably W-7 would perturb any physiologically relevant CaM-protein interactions in cells but W-7 concentrations that reduced HSP and other protein binding to CaM-agarose columns by 50 per cent or more, had no effect on thermotolerance development in cells. These observations, combined with the studies that showed little effect of CaM antagonists on HSP synthesis at concentrations which potentiated cell killing, suggested that events leading to triggering or developing thermotolerance were not strongly dependent on any putative HSP binding to CaM. These studies also suggest some targets of hyperthermic cell killing at 43 degrees C are different from those that lead to the triggering and development of thermotolerance.