Expression of a protease-resistant insulin-like growth factor-binding protein-4 inhibits tumour growth in a murine model of breast cancer.

BACKGROUND: Insulin-like growth factor 1 (IGF1) promotes breast cancer and disease progression. Bioavailability of IGF1 is modulated by IGF-binding proteins (IGFBPs). IGFBP4 inhibits IGF1 activity but cleavage by pregnancy-associated plasma protein-A (PAPP-A) protease releases active IGF1. METHODS: Expression of IGF pathway components and PAPP-A was assessed by western blot or RT-PCR. IGFBP4 (dBP4) resistant to PAPP-A cleavage, but retaining IGF-binding capacity, was used to block IGF activity in vivo. 4T1.2 mouse mammary adenocarcinoma cells transfected with empty vector, vector expressing wild-type IGFBP4 or vector expressing dBP4 were implanted in the mammary fat pad of BALB/c mice and tumour growth was assessed. Tumour angiogenesis and endothelial cell apoptosis were assessed by immunohistochemistry. RESULTS: 4T1.2 cells expressed the IGF1R receptor and IGFBP4. PAPP-A was expressed within mammary tumours but not by 4T1.2 cells. Proliferation and vascular endothelial growth factor (VEGF) production by 4T1.2 cells was increased by IGF1(E3R) (recombinant IGF1 resistant to binding by IGFBPs) but not by wild-type IGF1. IGF1-stimulated microvascular endothelial cell proliferation was blocked by recombinant IGFBP4. 4T1.2 tumours expressing dBP4 grew significantly more slowly than controls or tumours expressing wild-type IGFBP4. Inhibition of tumour growth by dBP4 was accompanied by the increased endothelial cell apoptosis. CONCLUSION: Protease-resistant IGFBP4 blocks IGF activity, tumour growth and angiogenesis.

Robotic Mammosphere Assay for High-Throughput Screening in Triple-Negative Breast Cancer.

In order to identify novel treatment principles specifically affecting cancer stem cells in triple-negative breast cancer, we have developed a high-throughput screening method based on the mammosphere and anoikis resistance assays allowing us to screen compounds using a functional readout. The assay was validated against manual protocols and through the use of positive controls, such as the response to hypoxia and treatment with the known cancer stem cell-targeting compound salinomycin. Manual and robotic procedures were compared and produced similar results in cell handling, cell cultures, and counting techniques, with no statistically significant difference produced from either method. The variance between samples processed manually versus robotically was no greater than 0.012, while Levene's test of significance was 0.2, indicating no significant difference between mammosphere data produced manually or robotically. Through the screening of 989 FDA-approved drugs and a follow-up screen assessing the antineoplastic subgroup, we have identified three therapeutic compounds with the ability to modulate the breast cancer stem cell fraction in the triple-negative breast cancer cell line MDA-MB-231, highlighting their potential usage as stem cell-specific adjuvant treatments.

Clinical effects of a controlled trial of methylphenidate on adolescents with attention deficit disorder.

Forty-eight attention deficit disorder patients, 12 to 18 years old and without previous stimulant therapy, received a double-blind trial of methylphenidate and placebo for 3 weeks each. Stimulant treatment produced mild side effects and weight reduction. Methylphenidate significantly reduced teachers' and parents' ratings of hyperactivity, inattention, and oppositionality. In addition, patients rated themselves as clinically improved and reported elevated subjective mood during stimulant therapy. Treatment benefits were comparable for patients with and without concurrent conduct or oppositional disorder as well as those with and without past or present depressive disorders. These results support the continued effectiveness of stimulant therapy for attention deficit disorder in adolescence. However, the magnitude of clinical effectiveness reported was smaller than previously found in younger patients.

Effects of sustained-release and standard preparations of methylphenidate on attention deficit disorder.

Nineteen children with attention deficit disorder (ADD) participated in a double-blind trial consisting of four 2-week phases: sustained-release methylphenidate (MPH); standard MPH; a combination of standard and sustained-release MPH; and placebo. Pharmacological treatments were evaluated by means of parent and teacher ratings and open-ended comments, examiner ratings, and patients' performance and event-related potentials during Continuous Performance and Paired-Associate Learning Tests. Results revealed that the MPH conditions were superior to placebo and comparable to one another. Within the limited time frame of the research, the findings suggest comparable effectiveness for sustained-release and standard preparations of MPH.

Methylphenidate reduces abnormalities of stimulus classification in adolescents with attention deficit disorder.

Forty-eight adolescents with attention deficit disorder (ADD) received placebo and methylphenidate (M = 35.21 mg/day) for 3 consecutive weeks each. ADD patients who received placebo in the first phase of treatment were compared with unmedicated normal adolescents. ADD and normal adolescents did not differ in slope of reaction time as a function of memory load in a Sternberg (1969) memory task. These results may be interpreted as reflecting normal rates of memory search in ADD. However, in comparison with normal subjects, ADD subjects made disproportionately more errors to targets and lacked faster latencies of the P3b component of event-related potentials for targets than nontargets. These findings suggest abnormalities in stimulus classification. Methylphenidate did not affect ADD patients' rates of memory search, but it did reduce misclassifications of targets at high memory loads. The drug also evoked the normal pattern of slower P3b latencies for nontargets by shortening latencies for targets. Thus the stimulant reduced ADD adolescents' abnormalities in stimulus classification.

Clinical and cognitive effects of methylphenidate on children with attention deficit disorder as a function of aggression/oppositionality and age.

Children diagnosed with attention deficit disorder (ADD; n = 44), ADD plus aggression/oppositionality (ADD/O; n = 34), and as not meeting ADD criteria (NC; n = 29) received methylphenidate and placebo for 21 consecutive days each. Parents and teachers rated all groups improved under medication, but teachers reported less improvement for NC than for ADD/O children. Methylphenidate and chronological age had generally similar effects in a Sternberg task: greater accuracy and speed (especially for nontargets at low memory loads), larger P3b waves of event-related potentials, more pronounced slowing of P3b latency by memory load, and a greater trend of earlier peaks for targets than for nontargets. Both methylphenidate and maturation promoted more efficient strategies involving differentiated evaluation of targets and nontargets. These results were comparable among ADD groups.

A comparison of intra- and interpersonal interlimb coordination: coordination breakdowns and coupling strength.

Intra- and interpersonal interlimb coordination of pendulums swung from the wrist was investigated. For both kinds of coordination, the steady state and breakdown of bimanual rhythmic coordination as indexed by the time series of the relative phase angle phi were studied under the manipulation of coordination mode, frequency of oscillation, and the difference in the eigenfrequencies (preferred tempos) of the individual oscillating limbs. The properties observed for both intra- and interpersonal coordination were those predicted by a dynamical model of rhythmic coordination that considers the coordinated limbs coupled to be nonlinear oscillators. Using a regression method, the coupling strengths of the coupled system were recovered. As predicted by the dynamical model, the strength of the dynamic was generally greater for the in-phase than the anti-phase mode and decreased with increasing frequency. Further, the strength of the interpersonal interlimb coupling was weaker than that of intrapersonal interlimb coupling.

Methylphenidate speeds evaluation processes of attention deficit disorder adolescents during a continuous performance test.

Forty-six Attention Deficit Disorder (ADD) adolescents took a Continuous Performance Test (CPT) under placebo and methylphenidate (35.33 mg/day). The task required pressing one button for targets (p = .133), and another button for nontargets. Subjects displayed a strong bias to make the more frequent negative response before completely evaluating stimuli. Consistent with this assumption, subjects responded faster (by an average of 87 ms) to nontargets than to targets. Methylphenidate increased accuracy and speeded reaction times (RTs) to targets. The drug also increased the amplitude of the P3b component of the event-related potential for nontargets and shortened the latency of P3b for both targets and nontargets. These results suggest increased capacity allocation to and faster evaluation of task stimuli. Finally, the stimulant lengthened relative motor processing time (RT-P3b latency) for nontargets, a finding implying that response processing was accomplished with the benefit of earlier completion of evaluation processes for these stimuli.

Methylphenidate slows reactions of children with attention deficit disorder during and after an error.

A Sternberg memory search task was administered under placebo and methylphenidate to 42 children with cross-situational attention deficit disorder (ADD), 31 children with cross-situational ADD plus oppositional features, and 25 patients with marginal ADD. Overall, stimulant medication enhanced accuracy and speed. In addition, patients reacted faster on correct responses not preceded by an error than on errors (especially false alarms) or on correct responses following an error. The slowness during error reactions may reflect decreased confidence or confusion during stimulus classification. This uncertainty may also lead subjects to respond with greater caution, hence more slowly, on correct responses following errors. Notably, methylphenidate increased the slowing of reactions on error trials as well as on correct reactions following an error. Stimulant medication may augment subjects' persistence when they are uncertain or confused, thereby heightening caution and promoting accuracy on succeeding trials. Consistent with previous reports of the generality of enhancement of performance by stimulant medication, the impact of methylphenidate was comparable for the three subtypes of ADD studied.

Self-administered intelligibility practice for esophageal speakers.

Eight esophageal speakers used multiple-choice intelligibility training materials in a self-administered practice regimen. "Before" and "after" test tapes were made and scored for intelligibility. A naive group and the speakers themselves served as listeners. Both sets of scores showed proportionally similar improvements in intelligibility.

Computer-assisted modeling of subtilisin enantioselectivity in organic solvents.

In order to rationalize our discovery of a marked dependence of subtilisin's enantioselectivity on the organic solvent used as the reaction medium, we employed the X-ray crystal structure of the enzyme and the means of interactive computer modeling to construct the structures of the reactive enzyme-substrate complexes. For subtilisin-catalyzed trans-esterifications between vinyl butyrate and S and R enantiomers of chiral secondary alcohols XCH(OH)Y, the computer simulation data clearly explain a higher reactivity of the former enantiomer on the basis of severe steric hindrances experienced by the latter enantiomer in the active site of subtilisin. The models of binding derived by computer modeling also successfully predicted changes in subtilisin enantioselectivity as a function of the sizes of the X and Y substituents in the nucleophile and upon addition of certain inhibitors.

X-ray crystal structure of cross-linked subtilisin Carlsberg in water vs. acetonitrile.

The crystal structure of subtilisin Carlsberg lightly cross-linked with glutaraldehyde was solved in aqueous solution by X-ray crystallography at 2.3 A resolution. It was found to be virtually identical to the recently determined (Fitzpatrick, P.A., Steinmetz, A.C.U., Ringe, D.A. & Klibanov, A.M. (1993) Proc. Natl. Acad. Sci. USA 90, 8653) structure of the cross-linked enzyme in anhydrous acetonitrile. The latter structure was found to be significantly more rigid than in water, as reflected by their average B factors. The numbers of subtilisin-bound water molecules in the two structures are similar (114 and 99 in water and in acetonitrile, respectively), but the locations of some half of these bound waters are distinct.

Enzyme crystal structure in a neat organic solvent.

The crystal structure of the serine protease subtilisin Carlsberg in anhydrous acetonitrile was determined at 2.3 A resolution. It was found to be essentially identical to the three-dimensional structure of the enzyme in water; the differences observed were smaller than those between two independently determined structures in aqueous solution. The hydrogen bond system of the catalytic triad is intact in acetonitrile. The majority (99 of 119) of enzyme-bound, structural water molecules have such a great affinity to subtilisin that they are not displaced even in anhydrous acetonitrile. Of the 12 enzyme-bound acetonitrile molecules, 4 displace water molecules and 8 bind where no water had been observed before. One-third of all subtilisin-bound acetonitrile molecules reside in the active center, occupying the same region (P1, P2, and P3 binding sites) as the specific protein inhibitor eglin c.

Functional implications of the modeled structure of maspin.

The tumor suppressor maspin (mammary-specific serpin) is an unstable serpin that does not undergo the stressed to relaxed transition typical of proteinase inhibitory serpins and, consequently, is not likely to function as a serine proteinase inhibitor. This suggests that the positioning and configuration of the reactive site loop (RSL) of maspin are likely to resemble those of ovalbumin, the best studied non-inhibitory serpin. Accordingly, the tertiary structure of maspin has been modeled on the crystal structure of native ovalbumin. Biochemical data and the modeled theoretical structure of maspin reveal the absence of disulfide bonds in the molecule and the presence of an unstable RSL that adopts a distorted helical structure. We confirm that the RSL is extremely sensitive to limited proteolysis and suggest that this may provide a structural basis for the proteolytic inactivation of maspin, a process that is likely to modulate the activity of maspin in biological systems.

Receptor-mediated phagocytosis of rat macrophages is regulated differentially for opsonized particles and non-opsonized particles containing beta-glucan.

Experiments were conducted to test the hypothesis that opsonic and non-opsonic phagocytic capacities are differentially regulated by resting and wound-derived macrophages. Furthermore, the phagocytosis of non-opsonized zymosan and beta-glucan particles was quantified to determine whether cells differentially regulate non-opsonic lectinophagocytosis in accordance with the carbohydrate composition of the ligand. In that regard, wound macrophages exhibited profound differential regulation in lectinophagocytosis with a seven-fold increase in phagocytosis of beta-glucan particles following overnight culture but with a relatively modest increase in internalization of mannan-containing zymosan. Cultured peritoneal macrophages increased uptake of both particles similarly. Upon activation with interferon-gamma/lipopolysaccharide (IFN-gamma/LPS), wound macrophages selectively suppressed beta-glucan ingestion, while phagocytosis of zymosan particles was unaffected. Lectinophagocytosis was decreased in activated peritoneal macrophages regardless of particle composition and was due in part to a nitric oxide-dependent mechanism which was without a role in regulation of wound macrophage lectinophagocytosis. Overnight culture of wound macrophages suppressed their capacity for opsonic-dependent phagocytosis independently of activation, whereas suppression of phagocytosis by peritoneal macrophages was activation-dependent. Regulation of all three phagocytic pathways was achieved distinctly by peritoneal and wound-derived macrophages, with changes found in the percentage of resident peritoneal macrophages capable of phagocytosis, whereas the phagocytic capacity of wound macrophages was primarily affected by the number of particles ingested by individual cells. Taken together, these findings demonstrate that the differential regulation of phagocytic pathways encompasses the nature of the phagocytic particle, the site from which macrophages are obtained, their response to activating agents and the mechanism through which the cell population alters its phagocytic potential.

SARPs: a family of secreted apoptosis-related proteins.

Quiescent mouse embryonic C3H/10T1/2 cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T1/2 cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T1/2 cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated sarp1 (secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T1/2 cells. Hybridization studies with sarp1 revealed two additional family members. Cloning and sequencing of sarp2 and sarp3 revealed 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with sarp2 displayed increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of beta-catenin, suggesting that SARPs interfere with the Wnt-frizzled proteins signaling pathway.

The tumor suppressor maspin does not undergo the stressed to relaxed transition or inhibit trypsin-like serine proteases. Evidence that maspin is not a protease inhibitory serpin.

The role of tumor suppressor proteins in the development of malignancy has made the understanding of their molecular mechanisms of action of great importance. Maspin is a tumor suppressor produced by a number of cell types of epithelial origin. Exogenous recombinant maspin has been shown to block the growth, motility, and invasiveness of breast tumor cell lines in vitro and in vivo. Although belonging to the the serine proteinase inhibitor (serpin) superfamily of proteins, the molecular mechanism of maspin is currently unknown. Here we show that the reactive site loop of maspin exists in an exposed conformation that does not require activation by cofactors. The reactive site loop of maspin, however, does not act as an inhibitor of proteinases such as chymotrypsin, elastase, plasmin, thrombin, and trypsin but rather as a substrate. Maspin is also unable to inhibit tissue and urokinase type plasminogen activators. Stability studies show that maspin cannot undergo the stressed-relaxed transition typical of proteinase-inhibitory serpins, and the protein is capable of spontaneous polymerization induced by changes in pH. It is likely, therefore, that maspin is structurally more closely related to ovalbumin and angiotensinogen, and its tumor suppressor activity is independent of a latent or intrinsic trypsin-like serine proteinase-inhibitory activity.

Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression.

Interferon (IFN)-gamma increases the sensitivity of tumor cell lines, many of which are p53 mutants, to tumor necrosis factor-alpha-mediated and anti-Fas antibody-mediated cell death. To better understand the mechanism of IFN-gamma action in modulating the cell death response independently of p53 function, we analyzed the death of the human colon adenocarcinoma cell line, HT-29, following treatment with IFN-gamma and various cytotoxic agents. Here we show that IFN-gamma modulates cell death by sensitizing the cells to killing by numerous pro-apoptotic stimuli but not pro-necrotic stimuli. Furthermore, we show that select genes from several important apoptosis-related gene families are induced by IFN-gamma, including the apoptosis-signaling receptors CD95 (Fas/APO-1) and TNFR 1 and interleukin-1beta-converting enzyme (Ice) family members Ice, CPP32 (Yama, apopain), ICErel-II (TX, Ich-2), Mch-3 (ICE-LAP3, CMH-1), Mch-4, and Mch-5 (MACH, FLICE). Of the bcl-2 family members, IFN-gamma directly induced bak but notably not bax, which is activated by p53. The IFN-responsive transcriptional activator interferon regulatory factor-1 was also strongly induced and translocated into the nucleus following IFN-gamma treatment. We propose that IFN-gamma modulates a p53-independent apoptotic pathway by both directly and indirectly inducing select apoptosis-related genes.