mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer.

The mammalian target of rapamycin (mTOR) is a central regulator of G1 cell cycle protein synthesis that precedes commitment to normal cellular replication. We have studied the effect of cell cycle inhibitor-779 (CCI-779), a rapamycin ester that inhibits mTOR function, on the proliferation of a panel of breast cancer cell lines. Six of eight lines studied were sensitive (IC(50)< or = 50 nM) and two lines were resistant (IC(50)>1.0 microM) to CCI-779. Sensitive lines were estrogen dependent (MCF-7, BT-474, T-47D), or lacked expression of the tumor suppressor PTEN (MDA-MB-468, BT-549), and/or overexpressed the Her-2/neu oncogene (SKBR-3, BT-474). Resistant lines (MDA-MB-435, MDA-MB-231) shared none of these properties. CCI-779 (50 nM) inhibited mTOR function in both a sensitive and a resistant line. In nu/nu mouse xenografts, CCI-779 inhibited growth of MDA-MB-468 (sensitive) but not MDA-MB-435 resistant tumors. Treatment of sensitive lines with CCI-779 resulted in a decrease in D-type cyclin and c-myc levels and an increase in p27(kip-1) levels. There was good correlation between activation of the Akt pathway and sensitivity to CCI-779. Amplification of mTOR-regulated p70S6 kinase, which is downstream of Akt, may also have conferred CCI-779 sensitivity to MCF-7 cells. Taken together, the data suggest that mTOR may be a good target for breast cancer therapy, especially in tumors with Akt activation resulting from either growth factor dependency or loss of PTEN function.

Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR.

Recent evidence places the FRAP/mTOR kinase downstream of the phosphatidyl inositol 3-kinase/Akt-signaling pathway, which is up-regulated in multiple cancers because of loss of the PTEN tumor suppressor gene. We performed biological and biochemical studies to determine whether PTEN-deficient cancer cells are sensitive to pharmacologic inhibition of FRAP/mTOR by using the rapamycin derivative CCI-779. In vitro and in vivo studies of isogenic PTEN(+/+) and PTEN(-/-) mouse cells as well as human cancer cells with defined PTEN status showed that the growth of PTEN null cells was blocked preferentially by pharmacologic FRAP/mTOR inhibition. Enhanced tumor growth caused by constitutive activation of Akt in PTEN(+/+) cells also was reversed by CCI-779 treatment, indicating that FRAP/mTOR functions downstream of Akt in tumorigenesis. Loss of PTEN correlated with increased S6 kinase activity and phosphorylation of ribosomal S6 protein, providing evidence for activation of the FRAP/mTOR pathway in these cells. Differential sensitivity to CCI-779 was not explained by differences in biochemical blockade of the FRAP/mTOR pathway, because S6 phosphorylation was inhibited in sensitive and resistant cell lines. These results provide rationale for testing FRAP/mTOR inhibitors in PTEN null human cancers.

Internalization of Staphylococcus aureus by endothelial cells induces cytokine gene expression.

The ability of the vascular endothelium to elaborate cytokines in response to gram-positive sepsis has received limited attention. This study examined cytokine expression by human umbilical vein endothelial cells (EC) following infection with a gram-positive bacterial pathogen, Staphylococcus aureus. S. aureus infection of EC resulted in the production of interleukin-6 (IL-6) and IL-1 beta. For IL-6, message was detected at 3 h after infection, protein was present at 24 h, and both message and protein persisted for 72 h. IL-1 beta message was detected at 12 h, IL-1 beta protein was detected at 24 h, and both persisted for 72 h. Message for colony-stimulating factor 1 remained unaltered. UV-killed S. aureus also elicited IL-1 beta and IL-6 message and protein expression at 24 and 48 h. Twenty-one clinical isolates of S. aureus were tested, and all induced IL-6 release by 48 h. However, the laboratory strain 8325-4 did not induce cytokine expression at any time point and was internalized by EC 1,000-fold less than other strains were. Internalization of latex beads by EC did not induce IL-6 gene expression. Furthermore, cytochalasin D treatment of the EC prevented IL-1 and IL-6 induction by S. aureus but not by tumor necrosis factor alpha or lipopolysaccharide. These results indicate that S. aureus is a potent inducer of IL-1 and IL-6 in EC and that internalization of S. aureus by EC is necessary for their cytokine expression. Thus, our data suggest that the vascular endothelium may play an important role in the pathogenesis of septicemia caused by gram-positive organisms.

Biochemical and immunological properties of two forms of pertactin, the 69,000-molecular-weight outer membrane protein of Bordetella pertussis.

Two apparent isoforms of the virulence-associated 69,000-molecular-weight protein pertactin were purified from Bordetella pertussis. Mass spectrometry showed a difference of 2,060 Da, which may result from differential C-terminal cleavage of a larger precursor. Both forms were protective in a mouse model, eliciting bactericidal antibodies and reducing respiratory tract colonization.

Lower antibody response to tetanus toxoid associated with higher auto-anti-idiotypic antibody in old compared with young humans.

The production of anti-tetanus toxoid (TT) antibody and anti-anti-TT (auto-anti-Id) antibody has been measured in three young and three old persons. Both antibodies were measured using ELISA. We found that: (i) the anti-TT response of old subjects was lower than that of young subjects, (ii) serum auto-anti-Id antibody concentration was higher in old compared with young humans before boosting with TT, and (iii) anti-TT antibodies from different humans shared Id cross-reactivity when tested with a rabbit anti-Id antibody. Thus, the serum anti-TT antibody response to boosting was inversely correlated with the serum level of auto-anti-Id. This conclusion is consistent with the view that the higher level of auto-anti-antibody in older subjects contributes to their impaired antibody response to TT.

Testicular circulatory isolation: a phase I study.

Antineoplastic agents may damage germinal epithelium. Testicular circulatory isolation (TCI) is a regional drug exclusion technique designed to minimize this. We evaluated the technical and anaesthetic aspects of TCI in 10 patients who underwent bilateral orchiectomy immediately thereafter. A modified aortic clamp was placed trans-scrotally across the left testicular pedicle without pre-medication or anaesthesia and left in place for 1 h, occluding testicular blood flow. Minimal pain and anxiety were reported during the procedure. There were no complications related to TCI in any patient. This study supports the institution of trials of TCI in young men about to receive fertility-threatening chemotherapy.

Pluronic F 127 liquid sensitizes mice to low doses of Escherichia coli lipopolysaccharide.

BACKGROUND AND METHODS: In murine models of endotoxemia, large amounts of lipopolysaccharide have to be administered to induce mortality. If mice are pretreated with D-galactosamine, the amount of lipopolysaccharide required to induce mortality is significantly lowered. Pluronic F 127 liquid is a relatively non-toxic copolymer that exhibits reverse gelation properties. Thus, it is a liquid at cold temperature and a gel at body temperature. The present studies were performed to ascertain whether the reverse gelation properties of Pluronic F 127 liquid could be used in devising a model of septic shock where a sustained delivery of lipopolysaccharide occurred. In evaluating this model, dose-response studies were conducted with lipopolysaccharide when a) it was administered intraperitoneally in saline or in Pluronic F 127 liquid, and b) it was administered intravenously to mice that had been pretreated with saline or Pluronic F 127 liquid. Mortality was followed for up to 72 hrs. RESULTS: Various doses of Escherichia coli lipopolysaccharide dissolved in saline or in Pluronic F 127 liquid were administered intraperitoneally to mice. The lethal dose of lipopolysaccharide required to kill 50% of the mice (LD50) administered in Pluronic F 127 liquid was approximately ten- to 15-fold less than the values obtained for lipopolysaccharide administered in saline. This decrease in the LD50 of lipopolysaccharide was also observed if the mice were treated intraperitoneally with Pluronic F 127 liquid and challenged 6 hrs later with iv lipopolysaccharide. The concentrations of tumor necrosis factor and interleukin-6 in the plasma were significantly higher when a low dose of lipopolysaccharide was administered to mice that had been pretreated with Pluronic F 127 liquid. While there was no effect on the liver enzymes, Pluronic F 127 liquid caused an increase in the plasma triglycerides. CONCLUSIONS: The data reported in this paper indicate that the LD50 of lipopolysaccharide is significantly decreased if it is administered in Pluronic F 127 liquid or administered to mice that have been pretreated with the Pluronic F 127 liquid. Thus, Pluronic F 127 liquid appears to sensitize mice to low levels of lipopolysaccharide. Unlike the D-galactosamine model, lipopolysaccharide can be administered as late as 6 hrs after treatment with Pluronic F 127 liquid. While the mechanisms by which Pluronic F 127 liquid sensitizes mice is not known, plasma triglycerides were increased in mice treated with this agent, suggesting that tissues responsible for the synthesis and/or degradation of triglycerides play a role in this sensitization process.

RSD score: criteria for the diagnosis of reflex sympathetic dystrophy and causalgia.

No standardized criteria for the diagnosis of reflex sympathetic dystrophy (RSD) are in common use. An RSD score scoring system of diagnostic criteria is proposed. Diagnosis of reflex sympathetic dystrophy by RSD score correlated well with clinical diagnosis in a group of 25 patients who had treatment for chronic upper extremity pain. Standardization of diagnostic criteria with the RSD score should enhance comparison of the outcomes of studies concerning the treatment of RSD.

Interscalene blocks for chronic upper extremity pain.

A study of 25 patients was carried out to determine the efficacy of interscalene block (ISB) for the treatment of chronic upper extremity pain. An RSD score was used to categorize these patients. Seventeen of the 25 patients had less pain after ISB, and 14 also had increased range of motion of the affected limb. Patients with reflex sympathetic dystrophy (RSD)/causalgia, as well as other chronic pain conditions, improved. ISB was compared with stellate ganglion block (SGB) in patients undergoing both treatments. ISB seemed to be at least as effective as SGB for treatment of RSD/causalgia and may have some advantages over SGB. The role of somatic and sympathetic blockade is discussed.

A comparison of two epidural alpha 2-agonists, guanfacine and clonidine, in regard to duration of antinociception, and ventilatory and hemodynamic effects in goats.

Epidural clonidine produces analgesia in humans with acute and chronic pain. Its use is limited because of short-lasting analgesia, hemodynamic depression, sedation, and respiratory depression. Intrathecal guanfacine has a longer duration of action than intrathecal clonidine. The present study compares these two drugs administered epidurally. Pulmonary artery, carotid artery, and epidural catheters were inserted into five goats. Each animal received guanfacine 5 mg/10 mL, clonidine 750 micrograms/10 mL, or a 10-mL saline control solution on separate occasions. Antinociception (tested via a point pressure stimulation device), arterial blood pressure, heart rate, cardiac output, pulmonary capillary wedge pressure, and arterial and mixed venous blood gases were measured every 30 min for 8 h. Guanfacine produced a longer duration of antinociception (guanfacine = 8 h vs clonidine = 5.5 h). Increases in PaCO2 were more pronounced in the clonidine group. There were no marked hemodynamic differences between the two drugs. Pretreatment with epidural idazoxan, an alpha 2-antagonist, blocked the antinociceptive effects of guanfacine. Because of a longer duration of action and less respiratory depression, epidural guanfacine may be superior for postoperative analgesia and chronic pain syndromes.

GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus.

The human gene GLVR1 has been shown to render mouse cells sensitive to infection by gibbon ape leukemia virus. This indication that the GLVR1 protein acts as a virus receptor does not reveal the protein's normal physiological role. We now report that GLVR1 is homologous to pho-4+, a phosphate permease of Neurospora crassa, at a level sufficiently high to predict that GLVR1 is also a transport protein, although the substrate transported remains unknown. To characterize the gene further, we have cloned cDNA for the mouse homolog of the gene, Glvr-1. The sequence of the murine protein differs from that of the human protein in 10% of residues, and it may be presumed that some of these differences are responsible for the inability of gibbon ape leukemia virus to infect mouse fibroblasts. Glvr-1 RNA is most abundant in mouse brain and thymus, although it is present in all tissues examined. The pattern of RNA expression found in mouse tissues was also found in rat tissues, in which the RNA was expressed at high levels in all compartments of the brain except the caudate nucleus and was expressed most abundantly early in embryogenesis. Thus, high-level expression of Glvr-1 appears to be restricted to specific tissues and may have developmental consequences.

Antiinflammatory and antiarthritic properties of a substituted quinoline carboxylic acid: CL 306,293.

CL 306,293, a substituted quinoline carboxylic acid at a daily oral dose between 1.5 and 3.0 mg/kg suppressed the inflammation and joint destruction (radiological criteria) associated with both developing and established adjuvant arthritis. When a weekly oral dosing regimen was used, joint destruction was attenuated when this agent was administered at a dose of 50 to 200 mg/kg. Inflammation associated with a delayed type hypersensitivity reaction in dogs was suppressed at a daily dose of 0.25 mg/kg or a weekly dose of 1 mg/kg. At efficacious doses, CL 306,293 had no effects on cyclooxygenase or lipoxygenase activities nor did it have an effect on carrageenin induced paw edema. In acute tests, the compound was not ulcerogenic. The above observations indicate that the antiinflammatory effects of CL 306,293 are distinct from those observed with nonsteroidal antiinflammatory agents. Mechanistic studies conducted and to be published indicate that CL 306,293 down regulates T cell function and this mechanism may account, at least in part, for the antiinflammatory and antiarthritic properties observed in animal models of inflammation and joint destruction.