Mechanical Communication Acts as a Noise Filter.

Cells can communicate mechanically by responding to mechanical deformations generated by their neighbors. Here, we describe a new role for mechanical communication by demonstrating that mechanical coupling between cells acts as a signaling cue that reduces intrinsic noise in the interacting cells. We measure mechanical interaction between beating cardiac cells cultured on a patterned flexible substrate and find that beat-to-beat variability decays exponentially with coupling strength. To demonstrate that such noise reduction is indeed a direct consequence of mechanical coupling, we reproduce the exponential decay in an assay where a beating cell interacts mechanically with an artificial stochastic 'mechanical cell'. The mechanical cell consists of a probe that mimics the deformations generated by a stochastically beating neighboring cardiac cell. We show that noise reduction through mechanical coupling persists long after stimulation stops and identify microtubule integrity, NOX2, and CaMKII as mediators of noise reduction.

The Influence of the Timing of Cyclic Load Application on Cardiac Cell Contraction.

Cardiac cells are subjected to mechanical load during each heart-beat. Normal heart load is essential for physiological development and cardiac function. At the same time, excessive load can induce pathologies such as cardiac hypertrophy. While the forces working on the heart as an organ are well-understood, information regarding stretch response at the cellular level is limited. Since cardiac stretch-response depends on the amplitude and pattern of the applied load as well as its timing during the beating cycle, the directionality of load application and its phase relative to action potential generation must be controlled precisely. Here, we design a new experimental setup, which enables high-resolution fluorescence imaging of cultured cardiac cells under cyclic uniaxial mechanical load and electrical stimulation. Cyclic stretch was applied in different phases relative to the electrical stimulus and the effect on cardiac cell beating was monitored. The results show a clear phase-dependent response and provide insight into cardiac response to excessive loading conditions.

Arabinogalactan-folic acid-drug conjugate for targeted delivery and target-activated release of anticancer drugs to folate receptor-overexpressing cells.

Folic acid (FA) is a high affinity ligand (K(d) = 0.1-1 nM) of folate receptors (FRs) responsible for cellular uptake of folates via receptor-mediated endocytosis. FRs are frequently overexpressed in malignant epithelial cells including ovary, brain, kidney, breast, colon, and lung. FR has emerged as a target for the differential-delivery of anticancer chemotherapeutics with several FA-linked therapeutic agents currently undergoing clinical trials. Here we show that by tethering both FA and the anticancer drug methotrexate (MTX) to arabinogalactan (AG), a highly branched natural polysaccharide with unusual water solubility, a targeted biomacromolecular nanovehicle is formed, which can differentially deliver a cytotoxic cargo into FR-overexpressing cells. Moreover, by linking MTX via an endosomally cleavable peptide (GFLG), we demonstrate a target-activated release mechanism. This FA-AG-GFLG-MTX drug conjugate displayed 6.3-fold increased cytotoxic activity to FR-overexpressing cells compared to their FR-lacking counterparts. These findings establish a novel FA-tethered polymeric nanoconjugate for the targeted delivery of antitumor agents into cancer cells overexpressing FR.

A novel loss-of-function mutation in the proton-coupled folate transporter from a patient with hereditary folate malabsorption reveals that Arg 113 is crucial for function.

Hereditary folate malabsorption (HFM) patients harbor inactivating mutations including R113S in the proton-coupled folate transporter (PCFT), an intestinal folate transporter with optimal activity at acidic pH. Here we identified and characterized a novel R113C mutation residing in the highly conserved first intracellular loop of PCFT. Stable transfectants overexpressing a Myc-tagged wild-type (WT) and mutant R113C PCFT displayed similar transporter targeting to the plasma membrane. However, whereas WT PCFT transfectants showed a 22-fold increase in [(3)H]folic acid influx at pH 5.5, R113C or mock transfectants showed no increase. Moreover, WT PCFT transfectants displayed a 50% folic acid growth requirement concentration of 7 nM, whereas mock and R113C transfectants revealed 24- to 27-fold higher values. Consistently, upon fluorescein-methotrexate labeling, WT PCFT transfectants displayed a 50% methotrexate displacement concentration of 50 nM, whereas mock and R113C transfectants exhibited 12- to 14-fold higher values. Based on the crystal structure of the homologous Escherichia coli glycerol-3-phosphate transporter, we propose that the cationic R113 residue of PCFT is embedded in a hydrophobic pocket formed by several transmembrane helices that may be part of a folate translocation pore. These findings establish a novel loss of function mutation in HFM residing in an intracellular loop of PCFT crucial for folate transport.

Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators.

PPARgamma coactivator 1alpha (PGC-1alpha) is a potent stimulator of mitochondrial biogenesis and respiration. Since the mitochondrial electron transport chain is the main producer of reactive oxygen species (ROS) in most cells, we examined the effect of PGC-1alpha on the metabolism of ROS. PGC-1alpha is coinduced with several key ROS-detoxifying enzymes upon treatment of cells with an oxidative stressor; studies with RNAi or null cells indicate that PGC-1alpha is required for the induction of many ROS-detoxifying enzymes, including GPx1 and SOD2. PGC-1alpha null mice are much more sensitive to the neurodegenerative effects of MPTP and kainic acid, oxidative stressors affecting the substantia nigra and hippocampus, respectively. Increasing PGC-1alpha levels dramatically protects neural cells in culture from oxidative-stressor-mediated death. These studies reveal that PGC-1alpha is a broad and powerful regulator of ROS metabolism, providing a potential target for the therapeutic manipulation of these important endogenous toxins.

Hic-5 regulates an epithelial program mediated by PPARgamma.

PPARgamma is a dominant regulator of fat cell differentiation. However, this nuclear receptor also plays an important role in the differentiation of intestinal and other epithelial cell types. The mechanism by which PPARgamma can influence the differentiation of such diverse cell lineages is unknown. We show here that PPARgamma interacts with Hic-5, a coactivator protein expressed in gut epithelial cells. Hic-5 and PPARgamma colocalize to the villus epithelium of the small intestine, and their expression during embryonic gut development correlates with the transition from endoderm to a specialized epithelium; expression of both these factors is reduced in tumors. Forced expression of Hic-5 in colon cancer cells enhances the PPARgamma-mediated induction of several gut epithelial differentiation/maturation markers such as L-FABP, kruppel-like factor 4 (KLF4), and keratin 20. siRNA directed against Hic-5 specifically reduces PPARgamma-mediated induction of gut epithelial genes in colon cells and in an ex vivo model of embryonic gut differentiation. Finally, forced expression of Hic-5 during 3T3-L1 preadipocyte differentiation inhibits adipogenesis while inducing inappropriate expression of several mRNAs characteristic of gut epithelium in these mesenchymal cells. These results indicate that Hic5 is an important component in determining an epithelial differentiation program induced by PPARgamma.

Reduced folate carrier mutations are not the mechanism underlying methotrexate resistance in childhood acute lymphoblastic leukemia.

BACKGROUND: Although the majority of children with acute lymphoblastic leukemia (ALL) are cured with combination chemotherapy containing methotrexate (MTX), drug resistance contributes to treatment failure for a substantial fraction of patients. The primary transporter for folates and MTX is the reduced folate carrier (RFC). Impaired drug transport is a documented mechanism of MTX resistance in patients with ALL; however, to the authors' knowledge it is not known whether inactivating RFC mutations are a contributing factor. METHODS: The authors devised a genomic polymerase chain reaction-single strand conformational polymorphism assay followed by sequencing and screened the entire RFC coding region for sequence alterations in DNA from 246 leukemia specimens from patients with diverse ethnic variation, 24 at the time of recurrence and the rest at the time of diagnosis. This cohort was comprised of 203 B-precursor ALL specimens (82.5%), 32 T-lineage ALL specimens (13%), and 11 acute myeloblastic leukemia specimens (4.5%). RESULTS: Of 246 DNA samples, only 3 diagnosis B-precursor ALL specimens (1.2%) were found to harbor alterations in the RFC gene, including heterozygous single nucleotide changes resulting in D56H and D522N substitutions in the first extracellular loop and the C-terminus of this transporter, respectively. The third sample had a sequence alteration in exon 3 that could not be identified because of the lack of availability of DNA. CONCLUSIONS: Whereas inactivating RFC mutations are a frequent mechanism of MTX resistance in human leukemia cell lines and in patients with osteosarcoma, they are not common and do not appear to play any significant role in intrinsic or acquired resistance to MTX in childhood leukemia. This is the first study of RFC mutations in multiple pediatric leukemia specimens.

Disruption of transport activity in a D93H mutant thiamine transporter 1, from a Rogers Syndrome family.

Rogers syndrome is an autosomal recessive disorder resulting in megaloblastic anemia, diabetes mellitus, and sensorineural deafness. The gene associated with this disease encodes for thiamine transporter 1 (THTR1), a member of the SLC19 solute carrier family including THTR2 and the reduced folate carrier (RFC). Using transient transfections into NIH3T3 cells of a D93H mutant THTR1derived from a Rogers syndrome family, we determined the expression, post-translational modification, plasma membrane targeting and thiamine transport activity. We also explored the impact on methotrexate (MTX) transport activity of a homologous missense D88H mutation in the human RFC, a close homologue of THTR1. Western blot analysis revealed that the D93H mutant THTR1 was normally expressed and underwent a complete N-glycosylation. However, while this mutant THTR1 was targeted to the plasma membrane, it was completely devoid of thiamine transport activity. Consistently, introduction into MTX transport null cells of a homologous D88H mutation in the hRFC did not result in restoration of MTX transport activity, thereby suggesting that D88 is an essential residue for MTX transport activity. These results suggest that the D93H mutation does not interfere with transporter expression, glycosylation and plasma membrane targeting. However, the substitution of this negatively charged amino acid (Asp93) by a positively charged residue (His) in an extremely conserved region (the border of transmembrane domain 2/intracellular loop 2) in the SLC19 family, presumably inflicts deleterious structural alterations that abolish thiamine binding and/or translocation. Hence, this functional characterization of the D93H mutation provides a molecular basis for Rogers syndrome.

APC-dependent suppression of colon carcinogenesis by PPARgamma.

Activation of PPARgamma by synthetic ligands, such as thiazolidinediones, stimulates adipogenesis and improves insulin sensitivity. Although thiazolidinediones represent a major therapy for type 2 diabetes, conflicting studies showing that these agents can increase or decrease colonic tumors in mice have raised concerns about the role of PPARgamma in colon cancer. To analyze critically the role of this receptor, we have used mice heterozygous for Ppargamma with both chemical and genetic models of this malignancy. Heterozygous loss of PPARgamma causes an increase in beta-catenin levels and a greater incidence of colon cancer when animals are treated with azoxymethane. However, mice with preexisting damage to Apc, a regulator of beta-catenin, develop tumors in a manner insensitive to the status of PPARgamma. These data show that PPARgamma can suppress beta-catenin levels and colon carcinogenesis but only before damage to the APC/beta-catenin pathway. This finding suggests a potentially important use for PPARgamma ligands as chemopreventative agents in colon cancer.

Genetic analysis of adipogenesis through peroxisome proliferator-activated receptor gamma isoforms.

Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor that is a key regulator of adipogenesis and is present in two isoforms generated by alternative splicing, PPARgamma1 and PPARgamma2. Studies of the ability of each isoform to stimulate fat differentiation have yielded ambiguous results, in part because PPARgamma stimulates its own expression. We have thus undertaken a formal genetic analysis using PPARgamma-null fibroblast cell lines to assess the specific role of each individual isoform in adipogenesis. We show here that both PPARgamma1 and PPARgamma2 have the intrinsic ability to stimulate robust adipogenesis. Adipose cells stimulated by either PPARgamma1 or PPARgamma2 express a similar gene profile and show similar responses to insulin. However, in response to low ligand concentrations, PPARgamma2 shows a quantitatively greater ability to induce adipogenesis. Analyses involving coactivator binding and transcriptional assays indicate that PPARgamma2 has an enhanced ability to bind components of the DRIP/TRAP complex, coactivators required for fat differentiation.

Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis.

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.