The glucose-regulated protein (GRP) system in mammalian cells is induced by glucose deprivation, anoxia, the calcium ionophore A23187, and 2-deoxyglucose. In Chinese hamster ovary cells the major 97, and 170 kDa.Removal of each of these four GRP-inducing stresses leads to the coordinate repression of GRPs and induction of the major heat shock proteins at 70 and 89 kDa. The application of each of these four GRP-inducing conditions leads to a significnt induction of resistance to the drug doxorubicin. Removal of each GRPinducing condition results in the rapid disappearance of this resistance in a manner that correlates with the repression of the GRPs. 2). In addition to glucose deprivation, this system can be induced by 2-deoxyglucose (3), the calcium ionophore A23187 (4), chronic anoxia (5), and viral infection (6). The GRP system represents a subset of a group of stress proteins that also includes the major heat shock proteins. In the case of the heat shock proteins, it has been shown that the application of a heat shock proteininducing stress results in concomitant expression of a heatresistant state referred to as thermotolerance (7-11). GRPs, however, are not associated with thermotolerance (12).Independent of these studies, it is also recognized that two conditions that induce GRPs, chronic anoxia and 2-deoxyglucose, lead to resistance to the drug doxorubicin (13-16). Based on these studies we consider here the hypothesis that GRP induction confers resistance to this chemotherapeutic agent in analogy to studies relating heat shock proteins and heat resistance. To test this hypothesis, we first used glucose deprivation, the most common method of GRP induction. We report that this treatment as well as A23187 induce resistance to doxorubicin. Finally, with all four inducers, there is a good temporal correlation between the application of the GRP-inducing stress and the induction of cellular resistance to doxorubicin and between the removal of the GRP-inducing stress and repression of cellular resistance to doxorubicin.The development of drug resistance is a major limiting factor in determining the success of cancer chemotherapy, and the investigation of mechanisms of resistance has attracted considerable attention (17-19). Since hypoxia and nutrient deprivation can occur during tumor development, this information suggests an alternative approach to the study of the mechanism(s) by which resistance to this chemotherapeutic agent may occur and connects this protective phenomenon with conditions associated with the expression of a major stress protein system. METHODSChinese hamster ovary (CHO) cells initially obtained from Los Alamos National Laboratory were maintained as monolayer cultures at 370C in Ham's F-10 medium (GIBCO) supplemented with 15% (vol/vol) newborn calf serum. The RIF-1 cell line was cultured in the a modification of minimal essential medium with 10% (vol/vol) fetal calf serum as described (20,21). In situ studies were performed by collecting cultured cells and then intradermally inocula...

The acquisition of radioresistance by esophageal squamous carcinoma (ESC) cells during radiotherapy may lead to cancer recurrence and poor survival. Previous studies have demonstrated that ionizing radiation (IR) induces epithelial–mesenchymal transition (EMT) of ESC cells accompanied by increased migration, invasion, and radioresistance. However, the underlying molecular mechanisms of IR-induced EMT and radioresistance are not well established, hampering the development of potential solutions. To address this issue, we investigated the role of the IL-6/STAT3/TWIST signaling pathway in IR-induced EMT. We found not only the pathway was activated during IR-induced EMT but also STAT3 inhibition or Twist depletion reversed the EMT process and attenuated radioresistance. These results improve our understanding of the underlying mechanisms involved in IR-induced EMT and suggest potential interventions to prevent EMT-induced acquisition of radioresistance.

A rabbit antiserum has been prepared using as antigen the 110,000-dalton mammalian heat-shock protein. This protein was purified for injection by two-dimensional PAGE of heat-shocked Chinese hamster ovary cells. Characterization by immunoautoradiography and immunoprecipitation reveals that the antiserum is specific for the 110,000-dalton protein.Both techniques also reveal that the protein against which the antiserum is directed is induced by heat shock. Indirect immunofluorescence shows that the antigen is primarily localized at or near the nucleolus in cultured cells and numerous murine tissues. Treatment of cultured cells with deoxyribonuclease destroys the organization of staining within the nucleus while ribonuclease appears to completely release the antigen from the nucleus. A binding of the antiserum to cytoplasmic structures is also observed by immunofluorescence. This association with nucleoli may have implications in the regulatory aspects of the heat-shock response.A hyperthermic challenge to a cell can result in a reorganization at translational and transcriptional levels (7, 23) and at the same time protect the cell from additional applications of the same stress (8), probably through the intervention of heatshock proteins (14,15,17,18,27,28). Despite the implications of this, it is important to recognize that the function of heat-shock proteins remains a mystery. However, reports indicate that at least some heat-shock proteins my localize in the nucleus (2,3,13,16,31,32), suggesting a direct role in the regulatory changes associated with this response.To learn more regarding the function of heat-shock proteins and their role in regulation and protection, we investigated mammalian heat-shock proteins using an immunologic approach. We report here on the characterization of an antiserum against the major l l0,000-dalton mammalian heatshock protein (HSP 110). ~ It is demonstrated by indirect immunofluorescence that this protein is localized at or near the nucleolus and is released from the nucleus by treatment with ribonuclease. This suggests that aspects of nucleolar function may be important in the protective and regulatory properties of the heat-shock response. mented with 15% heat-inactivated newborn calf serum. In heat-shock experiments: flasks were immersed horizontally into a constant temperature water bath (Haake FK-2) for the indicated times at 45"C + 0.1°C. 10TY2 mouse embryo fibroblasts were originally obtained from Dr. John Bertram of this Institute. These cells are grown in Eagles basal medium with Earle's salts (Gibco Laboratories) supplemented with 10% heat-inactivated fetal calf serum. Frozen sections were obtained from tissues of BALB/c CR mice supplied by the West Seneca Laboratories of this Institute. MATERIALS AND METHODS Cells Preparation of Antigen and Immunoautoradiography:Heat-shock proteins were identified by autoradiography of two-dimensional O'Farrel gels, as previously modified and described (10,19), by applying a [35S]methionine pulse during the peak induction peri...

Aims:  To determine the prevalence of Pseudomonas aeruginosa among dogs with suspected soft tissue infections and to characterize these isolates. Methods and Results:  Swabs were taken from infected soft tissues of 402 dogs. Pseudomonas aeruginosa strains were confirmed phenotypically and tested for susceptibility to 11 antimicrobial agents and genotyped by SpeI pulsed‐field gel electrophoresis (PFGE). The genetic basis of fluoroquinolone (FQ) resistance and the presence of integrons were also characterized. A total of 27 (6·7%) dogs tested positive for Ps. aeruginosa. Fourteen different SpeI patterns were observed in 25 typeable strains. Among the β‐lactams, three isolates presented resistance to ticarcillin and carbenicillin, while only one isolate exhibited resistance to ceftazidime. Among the aminoglycosides (AGs), three strains showed resistance to amikacin, and four strains exhibited resistance to gentamicin and tobramycin. Four strains with mutations that led to the substitution of Thr at position 83 with Ile in GyrA and the exchange of Ser at position 87 with Leu in ParC displayed resistance to all tested FQs. These strains also carried class 1 integrons and showed resistance to between 6 and 10 antimicrobials. These integrons included four different gene cassettes (aacA4‐aadA1, blaOXA‐31‐aadA2, aadA1‐arr‐3‐catB3 and cmlA5‐cmlA‐aadA1). Conclusions:  A small proportion of infected dogs treated in two animal hospitals in Beijing, China carried Ps. aeruginosa isolates. Low levels of resistance to anti‐pseudomonal agents were observed in these strains. Significance and Impact of the Study:  This study is the first report on the antimicrobial resistance profiles of Ps. aeruginosa isolated from infected canine origin in China. Additionally, this is the first report of the oxacillin resistance gene blaOXA‐31 in a canine Ps. aeruginosa isolate.

Chronic anoxia, glucose starvation, low pH, and numerous other conditions induce the glucose-regulated system of stress proteins (GRPs), whose principal members are observed at 78, 94, and 170 kDa. These stresses may be expected to occur during growth in untreated tumors. To examine the possibility that GRPs are correspondingly induced, we have examined the protein profiles of small (< 0.1 g), intermediate (0.2-0.8 g), and large (> 1.8 g) radiation-induced fibrosarcoma (RIF) tumors grown on C3H mice. One and two-dimensional gel electrophoresis indicate that the principal GRPs at 78 and 94 are coordinately and substantially increased in large tumor masses, relative to the small, and may be partially increased in the intermediate tumors. Necrotic material removed from large tumors exhibited an identical pattern of GRP induction with no visible indication of protein degradation and also contained a significant fraction of viable cells. Western blot analysis using rabbit antisera raised against the 78 and 170 kDa GRPs also demonstrated the enhanced accumulation of these proteins in the large tumors. The antibody against the 170 kDa GRP was also capable of detecting the induction of this stress protein in large tumors by indirect immunofluorescence analysis. Northern blot studies using a probe for the GRP 78 gene also showed an increase in GRP 78 message in large tumors as well as in RIF cells exposed to anoxic stress in vitro. Two-dimensional gel electrophoresis indicated that the major heat shock proteins at 70 and 90 kDa were not increased in the larger tumors, and the amount of the 90 kDa species was reduced. Finally, the quantity of vimentin and its degradation products is significantly diminished in large tumors and in anoxic cells. This study demonstrates that RIF tumor cells undergo a glucose regulated stress response in situ during tumor growth.