Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

Background  

Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA.     

Development of a regenerable cell culture system that senses and releases dead cells

We developed a rapidly regenerable cell culture system in which the cell culture substrate detects cell death and selectively releases the dead cells. This culture material was achieved by combining a detector that responds to the signal from the dead cells and an actuator to release the dead cells. Benzo-18-crown-6-acrylamide (BCAm) with a pendant crown ether receptor was used as the sensor to recognize cellular signals and N-isopropylacrylamide (NIPAM) was used as the actuator. This copolymer of NIPAM and BCAm can respond to potassium ions and change its nature from hydrophobic to hydrophilic at the culture temperature of 37 degrees C. Living cells concentrate potassium ion internally; when cells die, potassium ions are released. The polymer surface recognizes the potassium ions released from the dead cells, the NIPAM hydrates, and the dead cells are selectively detached. This in vitro culture system is a novel one in which artificial culture materials work cooperatively with cellular metabolism by responding to this signal from the cells, thereby realizing in vitro tissue regeneration partly mimicking the mechanisms of in vivo homeostasis.     

Epigallocatechin-3-gallate inhibits paracrine and autocrine hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the cell-based screening assay, (-)epigallocatechin-3-gallate (EGCG) inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering and uPA activation (IC50=15.8 microgram/ml). Further analysis revealed that EGCG at low doses specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met but not epidermal growth factor (EGF)-induced phosphorylation of EGF receptor (EGFR). On the other hand, high-dose EGCG decreased both Met and EGFR proteins. We also found that EGCG did not act on the intracellular portion of Met receptor tyrosine kinase, i.e., it inhibited InlB-dependent activation of Met but not NGF-induced activation of Trk-Met hybrid receptor. This inhibition decreased HGF-induced migration and invasion by parental or HGF/SF-transfected B16F10 melanoma cells in vitro in either a paracrine or autocrine manner. Furthermore, EGCG inhibited the invasion/metastasis of HGF/SF-transfected B16F10 melanoma cells in mice. Our data suggest the possible use of EGCG in human cancers associated with dysregulated paracrine or autocrine HGF/SF-Met signaling.     

Presence of autocrine hepatocyte growth factor-Met signaling and its role in proliferation and migration of SNU-484 gastric cancer cell line

Autocrine stimulation via coexpression of hepatocyte growth factor (HGF) and its receptor (Met) has been reported in many human sarcomas, but few in carcinomas. In this report, we found that one gastric cancer cell line, SNU-484, among 11 gastric cell lines tested has an autocrine HGF- Met stimulation. RT-PCR, ELISA and scattering assay using MDCK cells revealed that SNU-484 cells secreted a significant amount of active HGF (about 1.25 +/- 0.41 ng/24 h/10(6) cells) into conditioned medium. Resultantly, Met in this cell line was constitutively phosphorylated. Neutralizing antibodies against HGF reduced the tyrosine phosphorylation of Met, resulting in the inhibition of cell proliferation and migration (P <0.005). To the best of our knowledge, this is the first report on autocrine HGF-Met signaling in a gastric cancer cell line. Our observations with SNU-484 cells suggest that HGF is involved in the development and/or progression of some gastric carcinoma through an autocrine mechanism.     

B cell activation factor (BAFF) is a novel adipokine that links obesity and inflammation

B cell activation factor (BAFF) is a novel member of the TNF ligand superfamily, mainly produced by myeloid cells. BAFF has been shown to participate in B-cell survival and B- and T-cell maturation. BAFF expression in adipocytes has been recently demonstrated. In the current study, we verified that BAFF expression is increased during adipocyte differentiation. BAFF expression was augmented by TNF-α treatment and was decreased by rosiglitazone treatment. BAFF secretion in lean and in ob/ob mice sera were compared and smaller amount of BAFF was secreted in ob/ob mice. mRNA and protein expression were different between epididymal and visceral adipose tissue. BAFF expression was also increased in ob/ob mouse adipose tissue. We sought to identify known BAFF receptors (BAFF-R, BCMA, and TACI) in adipocytes, and determined that all three were present and upregulated during adipocyte differentiation. However, the expression of TACI was distinct from that of BAFF-R and BCMA under TNF-α and BAFF ligand treatment. BAFF-R and BCMA expression levels were upregulated under pro-inflammatory conditions, but TACI was reduced. Conversely, BAFF-R and BCMA expression levels were downregulated by rosiglitazone treatment, but TACI was increased. Taken together, our results suggest that BAFF may be a new adipokine, representing a link between obesity and inflammation.     

Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells

Resveratrol has been reported to possess cancer preventive properties. In this study, we analyzed anti-tumor activity of a newly synthesized resveratrol analog, cis-3,4'',5-trimethoxy-3''-hydroxystilbene (hereafter called 11b) towards breast and pancreatic cancer cell lines. 11b treatments reduced the proliferation of human pancreatic and breast cancer cells, arrested cells in the G2/M phase, and increased the percentage of cells in the subG1/G0 fraction. The 11b treatments also increased the total levels of mitotic checkpoint proteins such as BubR1, Aurora B, Cyclin B, and phosphorylated histone H3. Mechanistically, 11b blocks microtubule polymerization in vitro and it disturbed microtubule networks in both pancreatic and breast cancer cell lines. Computational modeling of the 11b-tubulin interaction indicates that the dimethoxyphenyl group of 11b can bind to the colchicine binding site of tubulin. Our studies show that the 11b treatment effects occur at lower concentrations than similar effects associated with resveratrol treatments and that microtubules may be the primary target for the observed effects of 11b. These studies suggest that 11b should be further examined as a potentially potent clinical chemotherapeutic agent for treating pancreatic and breast cancer patients.     

Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display

Functional antibody fragments may be displayed on the surface of filamentous bacteriophage by introducing variable region genes into the viral genome at a gene encoding a viral coat protein. “Phage display” enables the isolation of antibody genes from large libraries according to the binding specificities they encode. We have constructed a new phage-display vector encoding a polyhistidine tag that has been used for rapid purification of soluble antibody fragments. An antibody library derived from immunized mice was cloned into this vector. This library was panned against the transition state analog RT3, and a high proportion of binders isolated after two rounds of panning. PCR analysis revealed that there were 24 different pattern groups. Sequencing of 15 clones within the major pattern group revealed 10 related clones with a range of point mutations. Thus, phage display can provide a large diverse repertoire of candidate catalytic antibodies based on TSA selection and screening.     

p53 overexpression represses androgen-mediated induction of NKX3.1 in a prostate cancer cell line

Prostate cancer is a disease involving complicated multiple-gene alterations. Both NKX3.1 and p53 are related to prostate cancer and play crucial roles in prostate cancer progression. However, little is known about the relationships and interactions between p53 and NKX3.1 in prostate cancer. We found that NKX3.1 expression is down-regulated by over-expression of wild type (wt) p53 in prostate cancer LNCaP cells. NKX3.1 is down-regulated at both the mRNA and protein levels by p53 over- expression due to either transient transfection of exogenous p53 or induction of endogenous p53. p53 over-expression represses androgen-induced transactivation of NKX3.1 by inhibiting the promoter of the androgen acceptor (AR) gene and by blocking AR-DNA binding activity. In addition, transfection with the p21 expression vector (pPSA-p21) showed that p21 does not reduce NKX3.1 expression, indicating that NKX3.1 expression is not the result of nonspecific effects of cell growth arrest. Our results provide biochemical and cellular biologic evidence that NKX3.1 is down-regulated by p53 over-expression in prostate cancer cells.     

Sulfonated molecules that bind a partially structured species of beta2-microglobulin also influence refolding and fibrillogenesis

Human beta2-microglobulin (beta2-m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of beta2-m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.     

Simulation of proliferation of neural stem cells on a surface with emphasis on spatial constraints on cell division

We present Monte Carlo lattice simulations of proliferation of cells on a surface in the situation when the cell-cell adhesion is relatively strong and the cells may form islands and/or flattened hemispheres. The model parameters were chosen to mimic proliferation of adult rat neural stem cells (or, more specifically, adult hippocampal progenitor cells) deposited on polyornithine and laminin coated polystyrene. The results obtained show that the spatial constraints on cell division may result in slowdown of the exponential growth. Depending on the rules used for cell division, this effect may be either nearly negligible or appreciable. In the latter case, the scale of the deviations from the exponential growth is comparable with that observed in our experiments. In the simulations, the slowdown of the growth starts however somewhat earlier and occurs in a less abrupt manner. This seems to indicate that the spatial constraints on division of cells are not the main factor behind the experimentally observed termination of the growth.     

Designed, functionalized helix-loop-helix motifs that bind human carbonic anhydrase II: a new class of synthetic receptor molecules

Polypeptides designed to fold into helix-loop-helix motifs and to dimerize to form four-helix bundles were functionalized by the introduction of a sulfonamide derivative known to bind human carbonic anhydrase II (HCAII) and one or both of the dansyl- and methoxycoumarin fluorescent probes. The 42-residue sequence DC that carries all three substituents in solvent-exposed positions was found to bind HCAII with a dissociation constant of 5 nM in aqueous solution at pH 7. At 2 muM concentration, DC was mainly dimeric in aqueous solution but bound HCAII as a monomer. Upon addition of a large excess of a helix-loop-helix motif without a high-affinity ligand, KE2-Q, a ternary complex was formed between HCAII, DC, and KE2-Q. Hydrophobic interactions between DC and HCAII and coordination of the sulfonamide group to the zinc ion of HCAII contributed cooperatively to binding in a demonstration of the usefulness of folded polypeptide-small organic molecule chimera as novel protein receptors. The DC homodimer was found to be a very sensitive biosensor component due to intermolecular quenching of its fluorescence that was inhibited upon binding to HCAII.     

Purification to single isoforms of a secreted epidermal growth factor receptor in a multicompartment electrolyzer with isoelectric membranes.

A purified, size-homogeneous (100 kDa), desialylated form of a truncated, soluble form of the epidermal growth factor receptor secreted by A431 human tumor cells has been found, by isoelectric focusing in immobilized pH gradients, to consist of two major isoforms (with pIs of 6.96 and 6.71), one intermediate form (pI 6.45) and a number (> 10) of minor components. The two major components have been purified to charge homogeneity by isoelectric focusing in a multicompartment electrolyzer with buffering isoelectric membranes having the following pI values: 5.90, 6.63, 6.76, 6.92, 7.05 and 7.35. Such single pI species are presently used for attempts at crystal growing.     

Soluble mediators from mesenchymal stem cells suppress T cell proliferation by inducing IL-10

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naïve or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naïve and pre-activated T cells in which IL-10 and IDO play important roles.     

Identification of compounds that bind mitochondrial F1F0 ATPase by screening a triazine library for correction of albinism

A triazine-based combinatorial library of small molecules was screened in albino murine melanocytes to identify compounds that induce pigmentation. Six compounds (of 1536 screened) produced at least 3-fold increases in pigmentation. Immunohistochemical studies demonstrated that the compounds conferred correct routing of the mistrafficked enzyme tyrosinase, which is critical to normal melanogenesis. Affinity matrices of the immobilized compounds allowed the cellular target to be identified as the mitochondrial F1F0-ATP synthase. Oligomycin and aurovertin B, small molecules known to inhibit the mitochondrial ATP synthase, were shown to compete with the triazine-based compounds for their cellular target in albino melanocytes and confer similar effects on pigmentation and tyrosinase rerouting. This is the first demonstration of the mitochondrial ATP synthase as a potential therapeutic target for restoring pigmentation in albino melanocytes.     

LYRM1, a gene that promotes proliferation and inhibits apoptosis during heart development

Congenital heart disease (CHD) is the most common type of birth defect, but its underlying molecular mechanisms remain unidentified. Previous studies determined that Homo sapiens LYR motif containing 1 (LYRM1) is a novel nucleoprotein expressed at the highest level in adipose tissue and in high levels in heart tissue. The LYRM1 gene may play an important role in the development of the human heart. This study was designed to identify the biological characteristics of the LYRM1 gene in heart development. On the basis of expression-specific differentiation markers identified with quantitative real-time RT-PCR and the morphology of LYRM1-overexpressing cells during differentiation, ectopic expression was not found to significantly affect differentiation of P19 cells into cardiomyocytes. MTT assays and cell cycle analysis showed that LYRM1 dramatically increases the proliferation of P19 cells. Furthermore, data from annexin V-FITC binding and caspase-3 activity revealed that LYRM1 can inhibit the apoptosis of P19 cells. Our data suggest that LYRM1 might have the potential to modulate cell growth, apoptosis, and heart development.