STAT3 inhibition prevents lung inflammation, remodeling, and accumulation of Th2 and Th17 cells in a murine asthma model.

BACKGROUND: STAT3 drives development of Th17 cells and cytokine production by Th2 and Th17 cells, which contribute to asthma. Alternative asthma treatments are needed, especially for the Th17 phenotype. We sought to determine whether C188-9, a small-molecule STAT3 inhibitor, can block Th2 and Th17 cell expansion and cytokine production to prevent house dust mite (HDM)-induced airway inflammation and remodeling. METHODS: Three groups of C57BL/6 mice were treated intranasally (IN) and intraperitoneally (IP) daily for 3 weeks with the following: (i) vehicle 1 IN and vehicle 2 IP, (ii) HDM IN and vehicle 2 IP, or (iii) HDM IN and C188-9 IP. Sections of lung were stained with Alcian Blue/PAS and examined microscopically. Total (t) STAT3, STAT3 phosphorylated on Y705 (pSTAT3), IL-17, IL-13, IL-5, and IL-4 levels were measured in lung protein extracts and serum using Luminex beads. Frequencies of Th2-type and Th17-type lymphocytes were assessed in lungs and bronchoalveolar lavage fluid (BALF) by multiparametric flow cytometry. RESULTS: HDM inhalation markedly increased airway goblet cell numbers and thickness of the epithelium and subepithelial smooth muscle layer, which was accompanied in the whole lung by increased pSTAT3, IL-4, IL-5, IL-13, and IL-17, and % CD4+ T cells that produce IL-5, IL-13, and IL-17. HDM inhalation also increased serum IL-4 and IL-17 levels and increased BALF % CD4+ T cells that produce IL-5 and IL-13. Remarkably, treatment with C188-9 normalized each endpoint. CONCLUSION: HDM-induced airway inflammation, remodeling, and Th2/Th17-type cell accumulation involve STAT3 activation that can be prevented by C188-9 treatment.

Bacteriophages are synergistic with bacterial interference for the prevention of Pseudomonas aeruginosa biofilm formation on urinary catheters.

AIMS: We hypothesized that pretreating urinary catheters with benign Escherichia coli HU2117 plus an antipseudomonal bacteriophage (ΦE2005-A) would prevent Pseudomonas aeruginosa biofilm formation on catheters--a pivotal event in the pathogenesis of catheter-associated urinary tract infection (CAUTI). METHODS AND RESULTS: Silicone catheter segments were exposed to one of four pretreatments (sterile media; E. coli alone; phage alone; E. coli plus phage), inoculated with P. aeruginosa and then incubated up to 72 h in human urine before rinsing and sonicating to recover adherent bacteria. Pseudomonas aeruginosa adherence to catheters was almost 4 log(10) units lower when pretreated with E. coli plus phage compared to no pretreatment (P < 0.001) in 24-h experiments and more than 3 log(10) units lower in 72-h experiments (P < 0.05). Neither E. coli nor phage alone generated significant decreases. CONCLUSIONS: The combination of phages with a pre-established biofilm of E. coli HU2117 was synergistic in preventing catheter colonization by P. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a synergistic protection against colonization of urinary catheters by a common uropathogen. Escherichia coli-coated catheters are in clinical trials; adding phage may offer additional benefit.

Retinoic acid normalizes the increased gene transcription rate of TGF-alpha and EGFR in head and neck cancer cell lines.

Retinoic acid (RA) has been shown to be effective in eradicating premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck (SCCHN) in clinical trials. The basis for this effect is unclear. We have previously demonstrated that messenger RNA from tumor growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor (EGFR), is unregulated in tumors and histologically normal mucosal samples from patients with SCCHN compared with control normal mucosa from patients without cancer, implicating this ligand-receptor pair in an autocrine growth pathway early in the molecular pathogenesis of this disease. In this report, we examined the hypothesis that the action of RA on the mucosa of the upper aerodigestive tract is mediated via downregulation of steady-state TGF-alpha and/or EGFR mRNA levels. Following exposure to all-trans-RA, a series of SCCHN cell lines demonstrated a 35.4% reduction in TGF-alpha mRNA expression (P = 0.022) and 58.5% reduction in EGFR mRNA (P = 0.0027). Nuclear run-on analysis indicated that the RA-mediated reduction of TGF-alpha and EGFR steady-state mRNA levels was a result of decreased gene transcription. These results suggest that the clinical effects of RA in SCCHN patients may be due to a downmodulation of TGF-alpha and EGFR mRNA production.

A suppressor lymphokine produced by human T leukemia cell lines. Partial characterization and spectrum of activity against normal and malignant hemopoietic cells.

Human T leukemia cell lines spontaneously release into their medium a suppressor lymphokine, T leukemia-derived suppressor lymphokine (TLSL), able to inhibit proliferation, DNA synthesis, and colony formation in a variety of malignant hemopoietic cell lines, as well as in normal myelomonocytic progenitor cells from bone marrow and peripheral blood. Titration curves indicated that the inhibitory activity in the crude supernatant preparations ranged from 10(-3)-10(-9): the supernatants from CCRF/CEM, HUT-78, and MOLT-4 cell lines were the most active, those from HPB-ALL, JM, and CCRF/HSB2 displayed an intermediate activity, and the Jurkat supernatant was the least active. Target cell lines of B cell origin (Burkitt lymphomas) were more sensitive than granulocytic, monocytic, erythroid, and T cell lines. Partial purification by ammonium sulfate precipitation and column chromatography demonstrated that TLSL is a protein with an Mr of 88,000, as determined by gel filtration. A high Mr form (greater than 300,000) was produced in serum-free medium by one of the most active producer cell lines (CCRF/CEM), and appeared to be an aggregate of the 88,000 Mr form. Neither the partially purified fractions obtained nor the crude supernatant preparations displayed antiviral activity or contained interleukin 2. Unlike lymphotoxin and tumor necrosis factor, TLSL is cytostatic: maximal inhibition of proliferation was observed 4-5 d after addition of crude supernatant to the target cells, and was not accompanied by a significant loss in cell viability. The antiproliferative capacity of TLSL was manifested both in suspension and methylcellulose cultures. Treated target cells accumulated either in the G1 or in the S phase of the cell cycle. The effect of TLSL on the target cells is irreversible: even brief (1 h) incubation of sensitive cells with TLSL resulted in inhibition of proliferation measured 5 d later. Although TLSL is produced by leukemic T cell lines, this lymphokine inhibits proliferation of normal peripheral blood T cells in response to mitogens or alloantigens: T lymphocyte activation was inhibited by all of the T cell supernatants tested. In contrast, when T cell lines were used as targets, no inhibition of proliferation was detected with two exceptions: the low producer Jurkat cell line was sensitive to all the T cell-derived supernatants, and the intermediate producer CCRF/HSB2 cell line was sensitive only to the three most active supernatants, CCRF/CEM, MOLT-4, and HUT-78. The possible significance of TLSL and its relationship with other suppressor lymphokines previously described in other systems is discussed.

Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock.

Resuscitation from hemorrhagic shock induces profound changes in the physiologic processes of many tissues and activates inflammatory cascades that include the activation of stress transcriptional factors and upregulation of cytokine synthesis. This process is accompanied by acute organ damage (e.g., lungs and liver). We have previously demonstrated that the inducible nitric oxide synthase (iNOS) is expressed during hemorrhagic shock. We postulated that nitric oxide production from iNOS would participate in proinflammatory signaling. Using the iNOS inhibitor N6-(iminoethyl)-L-lysine or iNOS knockout mice we found that the activation of the transcriptional factors nuclear factor kappaB and signal transducer and activator of transcription 3 and increases in IL-6 and G-CSF messenger RNA levels in the lungs and livers measured 4 h after resuscitation from hemorrhagic shock were iNOS dependent. Furthermore, iNOS inhibition resulted in a marked reduction of lung and liver injury produced by hemorrhagic shock. Thus, induced nitric oxide is essential for the upregulation of the inflammatory response in resuscitated hemorrhagic shock and participates in end organ damage under these conditions.

Decreased intranuclear mobility of acute myeloid leukemia 1-containing fusion proteins is accompanied by reduced mobility and compartmentalization of core binding factor beta.

Acute myeloid leukemia 1 (AML1) gene on chromosome 21 is involved in several chromosomal translocations, including t(8;21) and t(16;21), that produce chimeric fusion proteins AML1-eight twenty-one (ETO) and AML-myeloid transforming gene chromosome 16 (MTG16), which contribute to leukemogenesis. The molecular basis for the leukemogenic effects of these fusion proteins is incompletely understood. Using gel-shift assay, we showed that AML1-ETO and AML1-MTG16 bound to a series of AML1 consensus DNA-binding sites with different affinities. Using fluorescence recovery after photobleaching (FRAP), we demonstrated that a fusion of AML1 with ETO or MTG16 exhibits reduced intranuclear mobility compared with wild-type AML1 or either fusion partner. The dimerization domain (nervy homology region 2) of ETO is responsible for the reduced mobility of AML1-ETO. Dual FRAP studies revealed that CBFbeta colocalized with AML1-ETO within the nucleus, resulting in reduced mobility of CBFbeta. Therefore, AML1 fusion proteins may interfere with normal AML1 function due to aberrant nuclear dynamics, which leads to spatial and temporal sequestration of CBFbeta and perhaps other coregulators critical for myeloid differentiation.

Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor- mediated cell growth In vitro.

Stimulation of epidermal growth factor receptor (EGFR) by ligand(s) leads to activation of signaling molecules including Stat1 and Stat3, two members of the signal transducers and activators of transcription (STAT) protein family. Activation of Stat1 and Stat3 was constitutive in transformed squamous epithelial cells, which produce elevated levels of TGF-alpha, and was enhanced by the addition of exogenous TGF-alpha. Targeting of Stat3 using antisense oligonucleotides directed against the translation initiation site, resulted in significant growth inhibition. In addition, cells stably transfected with dominant negative mutant Stat3 constructs failed to proliferate in vitro. In contrast, targeting of Stat1 using either antisense or dominant-negative strategies had no effect on cell growth. Thus, TGF-alpha/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat3 but not Stat1.

Levels of TGF-alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival.

BACKGROUND: The most accurate predictor of disease recurrence in patients treated for head and neck squamous cell carcinoma is, at present, the extent of regional lymph node metastasis. Since elevated levels of epidermal growth factor receptor (EGFR) and of its ligand, transforming growth factor-alpha (TGF-alpha), have been detected in primary tumors of patients with head and neck squamous cell carcinoma, we determined whether tumor levels of these proteins were of prognostic importance. METHODS: Monoclonal antibodies specific for EGFR and TGF-alpha were used for immunohistochemical detection of each protein in tissue sections of primary tumors from 91 patients who were treated by surgical resection. Levels of immunoreactive EGFR and TGF-alpha were quantified by use of a computerized image analysis system and were normalized to appropriate standards. The logrank test and proportional hazards regression analysis were used to calculate the probability that EGFR and TGF-alpha levels were associated with disease-free survival (i.e., no recurrence of cancer) and cause-specific survival (i.e., patients do not die of their disease). All P values were two-sided. RESULTS: When tumor levels of EGFR or TGF-alpha were analyzed as continuous variables, disease-free survival and cause-specific survival were reduced among patients with higher levels of EGFR (both P = .0001) or TGF-alpha (both P = .0001). In a multivariate analysis, tumor site, tumor level of EGFR, and tumor level of TGF-alpha were statistically significant predictors of disease-free survival; in a similar analysis, regional lymph node stage and tumor levels of EGFR and of TGF-alpha were significant predictors of cause-specific survival. CONCLUSION: Quantitation of EGFR and TGF-alpha protein levels in primary head and neck squamous cell carcinomas may be useful in identifying subgroups of patients at high risk of tumor recurrence and in guiding therapy.

Inhibition of human squamous cell carcinoma growth in vivo by epidermal growth factor receptor antisense RNA transcribed from the U6 promoter.

BACKGROUND: Squamous cell carcinomas of the head and neck (SCCHN), unlike normal mucosal squamous epithelial cells, overexpress epidermal growth factor receptor (EGFR) messenger RNA and protein. EGFR protein is required to sustain the proliferation of SCCHN cells in vitro. To determine whether EGFR expression contributes to tumor growth, we investigated the effect of suppressing EGFR expression in tumor xenografts through in situ expression of antisense oligonucleotides. METHODS: Intratumoral cationic liposome-mediated gene transfer was used to deliver plasmids capable of expressing sense or antisense EGFR sequences into human head and neck tumors, which were grown as subcutaneous xenografts in nude mice. The oligonucleotides were expressed under the control of the U6 RNA promoter. RESULTS: Direct inoculation of the EGFR antisense (but not the corresponding sense) plasmid construct into established SCCHN xenografts resulted in inhibition of tumor growth, suppression of EGFR protein expression, and an increased rate of apoptosis (programmed cell death). Sustained antitumor effects were observed for up to 2 weeks after the treatments were discontinued. CONCLUSION: These results suggest that interference with EGFR expression, using an antisense-based gene therapy approach, may be an effective means of treating EGFR-overexpressing tumors, including SCCHN.

Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer.

The squamous mucosa of patients who develop head and neck cancer is "condemned" or predisposed to disregulated growth as reflected by the high incidence of synchronous and metachronous primary tumors. We hypothesized that transformed and nontransformed mucosa from head and neck cancer patients would produce increased levels of transforming growth factor alpha (TGF-alpha) and its cell surface receptor, the epidermal growth factor receptor (EGFR), thereby contributing to this predisposition. Using molecular biological techniques, we examined the incidence and mechanism of TGF-alpha and EGFR overproduction in tumors and histologically normal mucosa excised from patients with squamous cell carcinoma of the head and neck (SCCHN) to test this hypothetical mechanism of field cancerization. Northern blot hybridization was used to evaluate the frequency of increased TGF-alpha and EGFR mRNA production in tissue excised from 24 patients with SCCHN and 10 cell lines compared with 7 control patients without cancer or a history of alcohol and tobacco use. Southern blot hybridization was used to examine for gene amplification. In patients with SCCHN, TGF-alpha mRNA was elevated by a mean of 5-fold in 95% of histologically "normal" mucosa samples (P = 0.001) and by a mean of 5-fold in 87.5% of tumors (P = 0.0001) while EGFR mRNA was elevated by a mean of 29-fold in 91% of histologically normal mucosa specimens (P = 0.0005) and by a mean of 69-fold in 92% of tumors (P = 0.0005), compared with mRNA levels in control normal mucosa. In 10 SCCHN cell lines, TGF-alpha mRNA was increased by a mean of 16-fold and EGFR mRNA levels were increased by a mean of 77-fold. Increased production of TGF-alpha and EGFR mRNA in the histologically normal mucosa of patients at risk for a primary or secondary head and neck cancer may serve both as a marker for malignant transformation and as a target for preventive therapies.

Inhibition of growth of mouse leukemic cell lines in vitro and in vivo by a monoclonal antibody that recognizes an interleukin 3 receptor-associated protein.

Myeloid cell lines that have achieved leukemic transformation may also have acquired the ability to produce hematopoietic growth factors. In certain instances, neutralizing antibodies directed against the growth factor have inhibited growth, supporting an autocrine mechanism in their transformation. The effect of anti-receptor antibodies on their growth and transformed phenotype has not been reported. We have developed a monoclonal antibody, 4G8, directed against a protein that is associated with the IL-3 receptor complex; 4G8 inhibits IL-3 binding and signal transduction in nonleukemic IL-3-dependent cell lines. In this study, we examined the effect of 4G8 on the growth in vitro and in vivo of leukemic cell lines, including WEHI-3B, which spontaneously produces IL-3, and NFS-60, an IL-3-dependent cell line. Our results demonstrate that the 4G8 antigen can be detected in both WEHI-3B and NFS-60 cells by flow cytometry and Western blotting; 4G8 inhibits the autonomous growth of WEHI-3B and the IL-3-dependent growth of both WEHI-3B and NFS-60. In addition, s.c. administration of 4G8 prolonged the survival of syngeneic mice given s.c. injections of WEHI-3B. These results support the conclusion that an autocrine mechanism involving IL-3 and its receptor plays a critical role in the growth and transformed phenotype of WEHI-3B and raises the possibility that anti-IL-3 receptor antibodies may be useful in the treatment of some leukemias.

A mix of S and ΔS variants of STAT3 enable survival of activated B-cell-like diffuse large B-cell lymphoma cells in culture.

Activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) is characterized by increased expression and activator of signal transducer and activator of transcription 3 (STAT3). ABC DLBCL cells require STAT3 for growth in culture. In ABC DLBCL cells, eosinophils and perhaps all cells, four variant STAT3 mRNAs (Sα, ΔSα, Sß and ΔSß) are present as a result of two alternative splicing events, one that results in the inclusion of a 55-residue C-terminal transactivation domain (α) or a truncated C-terminal domain with 7 unique residues (ß) and a second that includes (S) or excludes (ΔS) the codon for Ser-701 in the linker between the SH2 and C-terminal domains. A substantial literature indicates that both α and ß variants are required for optimal STAT3 function, but nothing is known about functions of ΔS variants. We used a knockdown/re-expression strategy to explore whether survival of ABC DLBCL cells requires that the four variants be in an appropriate ratio. No single variant rescued survival as well as STAT3Sα-C, Sα with activating mutations (A661C and N663C) in the SH2 domain. Better rescue was achieved when all four variants were re-expressed or Sα and ΔSα or Sß and ΔSß were re-expressed in pairs. Rescue correlated with expression of STAT3-sensitive genes NFKBIA and NFKBIZ. We consider a variety of explanations why a mix of S and ΔS variants of STAT3 should enable survival of ABC DLBCL cells.

Inhibition of epidermal growth factor receptor gene expression and function decreases proliferation of head and neck squamous carcinoma but not normal mucosal epithelial cells.

Previous reports have shown that fresh tissues and cell lines from patients with squamous cell carcinoma of the head and neck (SCCHN) overexpress transforming growth factor alpha (TGF-alpha) and its receptor, the epidermal growth factor receptor (EGFR) at both the mRNA and protein levels. Protein localization studies confirm that TGF-alpha and EGFR are produced by the same epithelial cells in tissues from head and neck cancer patients further supporting an autocrine growth pathway. Using three strategies, we examined the hypothesis that downmodulation of EGFR would reduce the proliferation of SCCHN cells. We targeted EGFR mRNA using antisense oligonucleotides and the mature EGFR protein at two sites, the ligand-binding domain and the kinase domain, and determined the effects of this targeting on SCCHN proliferation. Treatment of several SCCHN cell lines with a pair of antisense oligodeoxynucleotides directed against the translation start site and first intron-exon splice junction of the human EGFR gene resulted in decreased EGFR protein production and inhibited growth by 86% compared to a 13% reduction in cells treated with sense oligonucleotides (P=0.03). Growth inhibition was specific for carcinoma cells since the same EGFR antisense oligonucleotides had no effect on the proliferation of normal mucosa cells harvested from non-cancer patients. Two monoclonal antibodies which block ligand binding to EGFR (MAbs 425 and 528) inhibited the growth of several SCCHN cell lines by up to 97% which suggests that EGFR is participating in an autocrine pathway in SCCHN that is, at least in part, external. An EGFR-specific tyrosine kinase inhibitor (PD 153035) was found to inhibit EGFR phosphorylation in SCCHN cell lines and to reduce growth by 68% although it had no effect on the growth rate of normal mucosal epithelial cells. These experiments indicate that EGFR gene expression and function is critical for SCCHN cell growth but not for growth of normal mucosa cells and therefore may serve as a tumor-specific target for preventive and therapeutic strategies in head and neck cancer.

Increased expression of the differentiation-defective granulocyte colony-stimulating factor receptor mRNA isoform in acute myelogenous leukemia.

Granulocyte colony-stimulating factor (G-CSF) critically affects all stages of granulopoiesis by activating a signaling cascade initiated by dimerization of its receptor (G-CSFR). Five human G-CSFR isoforms have been identified (classes I-V). A quantitative polymerase chain reaction (Q-PCR) technique was used to examine the expression of these five isoforms in normal and leukemic myeloid cells. We demonstrated that neutrophils expressed predominantly the class I isoform and low levels of class IV isoform (IV/I = 0.037 +/- 0.005). No expression of the class II, class III, or class V isoform was detected. In contrast, all AML cell lines and acute myelogenous leukemia (AML) patient samples expressed increased relative amounts of the class IV isoform (IV/I = 0.047-0.350). When compared to normal immature myeloid cells, as represented by the CD34+ fraction of adult bone marrow (ABM) cells, three of eight AML cell lines and three of six AML patient samples expressed significantly increased levels of the class IV isoform relative to class I. This suggests that the increase in the relative expression of the class IV isoform seen in a considerable portion of AML cell samples is related to their leukemic phenotype. Given the inability of the class IV G-CSFR to drive myeloid maturation, the relative increase in class IV expression in AML cells may contribute to their aberrant response to G-CSF.

Asynchronous modulation of transforming growth factor alpha and epidermal growth factor receptor protein expression in progression of premalignant lesions to head and neck squamous cell carcinoma.

The development of head and neck squamous cell carcinoma occurs as a result of the accumulation of genotypic and phenotypic alterations in the upper aerodigestive tract mucosa. Up-regulation of epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor alpha (TGF-alpha), have been identified previously as early events in head and neck carcinogenesis. To determine the timing of increased TGF-alpha and EGFR protein expression in the development of head and neck cancer, we examined progressive mucosal dysplasias from three distinct and complimentary patient groups: (a) samples from patients with lesions demonstrating different degrees of dysplasia (n = 22) compared with mucosa samples from gender and age-matched controls (n = 8); (b) patients with lesions demonstrating different degrees of dysplasia at a single time point (n = 3); and (c) patients who progressed over several years to invasive cancer at the site of dysplasia (n = 7). Immunohistochemical analysis with monoclonal antibodies specific for TGF-alpha and EGFR were used to detect protein expression in all specimens. Protein levels were further quantitated using a computerized image analysis system. In all three groups, we found that TGF-alpha protein levels were elevated in mild dysplasia compared with control normal mucosa and were not further modulated with increasing degrees of dysplasia. In contrast, EGFR levels were relatively low in mild dysplasia and increased with higher degrees of dysplasia. These findings indicate that up-regulation of TGF-alpha and EGFR are distinct events both chronologically and, possibly, mechanistically in the pathogenesis of head and neck squamous cell carcinoma.

Bcl-2 but not Bax expression is associated with apoptosis in normal and transformed squamous epithelium.

Aberrant regulation of apoptosis may contribute to tumorigenesis. Relative levels of apoptosis regulatory proteins, such as Bcl-2 and Bax as well as interactions of these proteins with other gene products, may contribute to the rate of apoptosis in neoplasia. We examined Bcl-2 expression in 104 squamous cell carcinomas of the head and neck, as well as histologically normal mucosa several centimeters away from the tumor, and in control normal mucosa from patients without cancer. Immunohistochemistry and immunoblotting demonstrated Bcl-2 expression in 30% (31 of 104) of squamous cell carcinoma, with an increase in Bcl-2 protein levels compared with control normal mucosa from noncancer patients. Bcl-2-positive tumors demonstrated a 5-fold decrease in the number of apoptotic cells compared with Bcl-2-negative tumors. Bcl-2 protein expression was associated with poorly differentiated tumor grade but was not correlated with Bax expression or patient survival. These findings demonstrate that Bcl-2 contributes to apoptosis in normal and transformed squamous epithelium.

Granulocyte colony-stimulating factor activates a 72-kDa isoform of STAT3 in human neutrophils.

Granulocyte colony-stimulating factor (G-CSF) signaling involves activation of STATs, proteins that serve the dual function of signal transduction and activation of transcription. We previously demonstrated that G-CSF activated a distinct Stat3-like protein in immature and mature normal myeloid cells, StatG. StatG in normal immature human myeloid cells, i.e. adult CD34+ bone marrow cells, was composed of Stat3beta. This investigation was undertaken to determine the composition of StatG in mature normal human myeloid cells, i.e. polymorphonuclear neutrophilic granulocytes (PMN). These studies revealed that the major protein in extracts of PMN activated by G-CSF to bind the high-affinity serum-inducible element (hSIE) is a 72-kDa protein that cross-reacts with Stat3 monoclonal antibody, which we have designated Stat3gamma. Stat3gamma is derived from Stat3alpha by limited proteolysis and lacks the carboxyl-terminal portion of Stat3alpha. Because this region of Stat3alpha is involved in transcriptional activation, our findings suggest the possibility that Stat3gamma may be transcriptionally inactive and may compete with Stat3alpha for Stat3 binding sites in these terminally differentiated myeloid cells.

Modulation of human G-CSF receptor mRNA and protein in normal and leukemic myeloid cells by G-CSF and retinoic acid.

Granulocyte colony-stimulating factor (G-CSF) is produced by several cell types throughout the body and has a variety of effects on neutrophils and their precursors, which are mediated by binding to its receptor. It is not yet known what physiologic factors modulate G-CSF receptor mRNA expression in these cells. We studied the effect of G-CSF on freshly isolated neutrophils and bone marrow cells from normal human subjects and on myeloid leukemic cell lines. We found that G-CSF receptor mRNA levels were maintained by G-CSF in neutrophils but not in bone marrow cells. Of the leukemic cell lines tested, K562 and BV173, both of which contain the bcr-abl translocation, neither expressed G-CSF receptor mRNA. Whereas G-CSF did not affect mRNA levels for its receptor in myeloid leukemic cell lines, exposure of the acute promyelocytic cell line, NB4, to all-trans retinoic acid induced a striking increase in G-CSF receptor mRNA expression and resulted in increased G-CSF receptor surface expression. The effect of retinoic acid on G-CSF receptor mRNA on NB4 cells occurred early, before morphologic evidence of differentiation, and required protein synthesis. All-trans retinoic acid also upregulated G-CSF receptor mRNA in the myeloid leukemia cell line HL-60. Thus, maintenance of G-CSF receptor on neutrophils by G-CSF may extend the duration of ligand responsiveness. Furthermore, the ability of retinoic acid to up-regulate G-CSF receptor may account for the synergistic effect of G-CSF and retinoic acid in differentiation induction of acute promyelocytic leukemia.

G-CSF instillation into rat lungs mediates neutrophil recruitment, pulmonary edema, and hypoxia.

Activated neutrophils (PMN) have been implicated in the pathogenesis of adult respiratory distress syndrome (ARDS). Granulocyte colony-stimulating factor (G-CSF) is essential for PMN production and activation of PMN functions. We have recently shown that levels of G-CSF mRNA in a rat model of hemorrhagic shock correlated with severity of shock, PMN infiltration, pulmonary edema, and hypoxia. To determine whether increased tissue levels of G-CSF contribute to PMN recruitment and PMN-mediated injury, we instilled G-CSF into the lungs by intratracheal injection. Animals treated with G-CSF became hypoxic, hypocapnic, and alkalotic and demonstrated increased BAL fluid cellularity compared with control animals. The wet-to-dry ratio increased significantly after G-CSF instillation and peaked at 12 h. Histological examination of the lungs from G-CSF-treated rats revealed marked edema and increased PMN within the interstitium and alveoli. These results indicate that the presence of G-CSF alone in the lung can lead to recruitment of PMN, lung injury, and impaired pulmonary function, suggesting that local production of G-CSF may contribute to the development of lung damage and possibly ARDS in the setting of resuscitated hemorrhagic shock.

Drug-repositioning screening identified piperlongumine as a direct STAT3 inhibitor with potent activity against breast cancer.

Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been used to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescence microscopy search strategy to identify compounds in a drug-repositioning library (Prestwick library) that block ligand-induced nuclear translocation of Stat3 and identified piperlongumine (PL), a natural product isolated from the fruit of the pepper Piper longum. PL inhibited Stat3 nuclear translocation, inhibited ligand-induced and constitutive Stat3 phosphorylation, and modulated expression of multiple Stat3-regulated genes. Surface plasmon resonance assay revealed that PL directly inhibited binding of Stat3 to its phosphotyrosyl peptide ligand. Phosphoprotein antibody array analysis revealed that PL does not modulate kinases known to activate Stat3 such as Janus kinases, Src kinase family members or receptor tyrosine kinases. PL inhibited anchorage-independent and anchorage-dependent growth of multiple breast cancer cell lines having increased pStat3 or total Stat3, and induced apoptosis. PL also inhibited mammosphere formation by tumor cells from patient-derived xenografts. PL's antitumorigenic function was causally linked to its Stat3-inhibitory effect. PL was non-toxic in mice up to a dose of 30 mg/kg/day for 14 days and caused regression of breast cancer cell line xenografts in nude mice. Thus, PL represents a promising new agent for rapid entry into the clinic for use in treating breast cancer, as well as other cancers in which Stat3 has a role.