Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors.

In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53-HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.

Mechanism of inhibition of ribonucleotide reductase with motexafin gadolinium (MGd).

Motexafin gadolinium (MGd) is an expanded porphyrin anticancer agent which selectively targets tumor cells and works as a radiation enhancer, with promising results in clinical trials. Its mechanism of action is oxidation of intracellular reducing molecules and acting as a direct inhibitor of mammalian ribonucleotide reductase (RNR). This paper focuses on the mechanism of inhibition of RNR by MGd. Our experimental data present at least two pathways for inhibition of RNR; one precluding subunits oligomerization and the other direct inhibition of the large catalytic subunit of the enzyme. Co-localization of MGd and RNR in the cytoplasm particularly in the S-phase may account for its inhibitory properties. These data can elucidate an important effect of MGd on the cancer cells with overproduction of RNR and its efficacy as an anticancer agent and not only as a general radiosensitizer.

Tumor suppressor Fhit protein interacts with protoporphyrin IX in vitro and enhances the response of HeLa cells to photodynamic therapy.

Fhit, the product of tumor suppressor fragile histidine triad (FHIT) gene, exhibits antitumor activity of still largely unknown cellular background. However, it is believed that Fhit-Ap(3)A or Fhit-AMP complex might act as a second class messenger in cellular signal transduction pathway involved in cell proliferation and apoptosis. We demonstrate here for the first time that the photosensitizer, protoporphyrin IX (which is a natural precursor of heme) binds to Fhit protein and its mutants in the active site in vitro. Furthermore, PpIX inhibits the enzymatic activity of Fhit. Simultaneously, PpIX shows lower binding capacity to mutant Fhit-H96N of highly reduced hydrolase activity. In cell-based assay PpIX induced HeLa cell death in Fhit and Fhit-H96N-dependent manner which was measured by means of MTT assay. Moreover, HeLa cells stably expressing Fhit or mutant Fhit-H96N were more susceptible to protoporphyrin IX-mediated photodynamic therapy (2J/cm(2)) than parental cells.

Membrane perturbation effects of peptides derived from the N-termini of unprocessed prion proteins.

Peptides derived from the unprocessed N-termini of mouse and bovine prion proteins (mPrPp and bPrPp, respectively), comprising hydrophobic signal sequences followed by charged domains (KKRPKP), function as cell-penetrating peptides (CPPs) with live cells, concomitantly causing toxicity. Using steady-state fluorescence techniques, including calcein leakage and polarization of a membrane probe (diphenylhexatriene, DPH), as well as circular dichroism, we studied the membrane interactions of the peptides with large unilamellar phospholipid vesicles (LUVs), generally with a 30% negative surface charged density, comparing the effects with those of the CPP penetratin (pAntp) and the pore-forming peptide melittin. The prion peptides caused significant calcein leakage from LUVs concomitant with increased membrane ordering. Fluorescence correlation spectroscopy (FCS) studies of either rhodamine-entrapping (REVs) or rhodamine-labeled (RLVs) vesicles, showed that addition of the prion peptides resulted in significant release of rhodamine from the REVs without affecting the overall integrity of the RLVs. The membrane leakage effects due to the peptides had the following order of potency: melittin>mPrPp>bPrPp>pAntp. The membrane perturbation effects of the N-terminal prion peptides suggest that they form transient pores (similar to melittin) causing toxicity in parallel with their cellular trafficking.

Ligand-receptor interactions in live cells by fluorescence correlation spectroscopy.

Receptor binding studies most often require the use of radioactively labeled ligands. In certain cases, the numbers of receptors are few per cell and no specific binding is detected because of a high background. Specific interactions between certain ligands (e.g. peptides, hormones, natural products) and their receptors are, therefore, often overlooked by the conventional binding technique. Fluorescence correlation spectroscopy (FCS) allows detection of the interaction of ligands with receptors in their native environment in live cells in a tiny confocal volume element (0.2 fl) at single-molecule detection sensitivity. This technique permits the identification of receptors which were not possible before to detect by isotope labeling. The beauty of the FCS technique is that there is no need for separating an unbound ligand from a bound one to calculate the receptor bound and free ligand fractions. This review will show FCS as a sensitive and a rapid technique to study ligand-receptor interaction in live cells and will demonstrate that the FCS-analysis of ligand-receptor interactions in live cells fulfils all the criteria of a ligand binding to its receptor i.e. it is able to provide information on the affinity and specificity of a ligand, binding constant, association and dissociation rate constants as well as the number and mobility of receptors carrying a fluorescently labeled ligand. This review is of pharmaceutical significance since it will provide insights on how FCS can be used as a rapid technique for studying ligand-receptor interactions in cell cultures, which is one step forward towards a high throughput drug screening in cell cultures.

Ligand-macromolecule interactions in live cells by fluorescence correlation spectroscopy.

The receptor concept is the primary theoretical basis for modern pharmacology. Drugs, hormones, neurotransmitters, toxin, and other biologically active substances are referred to as ligands. Ligands exert their actions by way of interaction with receptors/macromolecules. The resulting receptor/macromolecule-ligand complexes produce alterations in physiological processes. Receptor/macromolecule-binding studies most often require the use of radioactively labeled ligands. When the numbers of receptors/macromolecules are few per cell, it is impossible to detect the specific binding because of a high background. Specific interactions between certain ligands and their receptors/macromolecules are, therefore, often overlooked by the conventional binding technique. Fluorescence correlation spectroscopy (FCS) allows detection a ligand-macromolecule interaction in live cells in a tiny confocal volume element (0.2 femtoliter (fL)) at single-molecule detection sensitivity. FCS permits the identification of macromolecules that were not possible to detect before by isotope labeling. The beauty of the FCS technique is that there is no need for separating an unbound ligand from a bound one to calculate the macromolecule bound and free ligand fractions. This study will demonstrate FCS as a sensitive and a rapid technique to study ligand-macromolecule interaction in live cells using fluorescently labeled ligands (Fl-L). This study is of pharmaceutical significance since FCS assay of ligand-macromolecule interactions in live cells is one step forward toward a high throughput drug screening in cell cultures.

Binding of the Alzheimer amyloid beta-peptide to neuronal cell membranes by fluorescence correlation spectroscopy.

The deposition of the Alzheimer amyloid beta-peptide (Abeta) fibrils in brain is a key step in Alzheimer's disease. The aggregated Abeta is found to be toxic to neurons since cells die when the aggregated Abeta is added to the cell culture medium. However, target of action of Abeta to cells is unknown. We have applied the fluorescence correlation spectroscopy (FCS) technique to study the existence of a receptor or target molecule for the Alzheimer amyloid beta-peptide (Abeta) in cultured human cerebral cortical neurons. FCS measurement of the fluorophore rhodamine-labeled Abeta (Rh-Abeta) shows diffusion times: 0.1 ms, 1.1 ms and 5.9 ms. Thus, 0.1 ms corresponds to the unbound Rh-Abeta, and 1.1 ms and 5.9 ms correspond to slowly diffusing complexes of Rh-Abeta bound to a kind of receptor or target molecule for Abeta. Addition of excess non-labeled Abeta is accompanied by a competitive displacement, showing that the Abeta binding is specific. Full saturation of the Abeta binding is obtained at nanomolar concentrations, indicating that the Abeta binding is of high affinity. The notion that using FCS we have found a kind of receptor or target molecule for Abeta makes an important point that Abeta kills cells possibly by affecting cell membranes via a receptor or target molecule. This study is of highly significance since it suggests that Abeta possibly affects neuronal cell membranes of Alzheimer patients via a receptor or target molecule.

Protoporphyrin IX interacts with wild-type p53 protein in vitro and induces cell death of human colon cancer cells in a p53-dependent and -independent manner.

Photodynamic therapy (PDT) of cancer is an alternative treatment for tumors resistant to chemo- and radiotherapy. It induces cancer cell death mainly through generation of reactive oxygen species by a laser light-activated photosensitizer. It has been suggested that the p53 tumor suppressor protein sensitizes some human cancer cells to PDT. However, there is still no direct evidence for this. We have demonstrated here for the first time that the photosensitizer protoporphyrin IX (PpIX) binds to p53 and disrupts the interaction between p53 tumor suppressor protein and its negative regulator HDM2 in vitro and in cells. Moreover, HCT116 colon cancer cells exhibited a p53-dependent sensitivity to PpIX in a dose-dependent manner, as was demonstrated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and fluorescence-activated cell sorter (FACS) analysis of cell cycle profiles. We have also observed induction of p53 target pro-apoptotic genes, e.g. puma (p53-up-regulated modulator of apoptosis), and bak in PpIX-treated cells. In addition, p53-independent growth suppression by PpIX was detected in p53-negative cells. PDT treatment (2 J/cm2) of HCT116 cells induced p53-dependent activation of pro-apoptotic gene expression followed by growth suppression and induction of apoptosis.

Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers.

Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine prion protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (DeltapH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of DeltapH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of DeltapH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: DeltapH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission.

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.

Denaturation of dsDNA by p53: fluorescence correlation spectroscopy study.

p53 activates transcription through interaction with specific DNA sequences in gene promoters. It also regulates DNA replication, recombination, and repair apparently through interactions with DNA intermediates of these reactions. Biochemical activities relevant for these functions of p53 include binding to the ends and internal segments of single-stranded DNA molecules, catalysis of DNA renaturation, and strand exchange. We report a novel activity of p53, its ability to denature double-stranded DNA molecules aggregated by basic peptides. Stable complexes of coiled single-stranded DNA molecules with basic peptides are formed in this reaction. Thus, complementary to the ability to catalyze DNA renaturation, p53 denatures double-stranded DNA when the latter reaction is thermodynamically favorable. This p53 activity, along with its ability to interact physically with DNA helicases, may be relevant for resolving double-stranded DNA intermediates and inhibition of DNA recombination, which is critical for guarding of the genome.

Visualization of a functionally enhanced GFP-tagged galanin R2 receptor in PC12 cells: constitutive and ligand-induced internalization.

Trafficking of the galanin R2 receptor (GALR2) fused with enhanced GFP (EGFP) was studied by using confocal fluorescence microscopy. The fusion protein was predominantly localized on the plasma membrane with some intracellular fluorescent structures (vesicles), mainly in the perinuclear region. Incubation with galanin resulted in a concentration-dependent increase in intracellular Ca2+ concentration levels, suggesting that the GALR2-EGFP conjugate is functional. After blocking endocytosis with methyl-beta-cyclodextrin GALR2-EGFP expression was increased on the surface and decreased in the cytoplasm. Blocking endocytic recycling with monensin caused an increase of intracellular GALR2-EGFP accumulation and a decrease of fluorescence on the plasma membrane. GALR2-EGFP on the plasma membrane was internalized within 5-10 min after treatment with galanin or AR-M1896, a selective GALR2 agonist, with a dramatic reduction in plasma membrane localization and appearance in intracellular vesicles. Neither M35 nor M40, two galanin analogues with putative antagonistic action, prevented GALR2 agonist-induced internalization of GALR2-EGFP, suggesting that they are not antagonists at this receptor under the present circumstances. Galanin stimulation at low temperature caused GALR2-EGFP aggregation and clustering on the surface but no translocation to cytoplasm. After coincubation with galanin the GALR2-EGFP was colocalized with internalized Texas red-transferrin, a marker of the clathrin endocytic pathway. Hyperosmotic sucrose inhibited internalization of GALR2-EGFP. Taken together these findings indicate that GALR2 undergoes constitutive endocytosis and recycling and that both ligand-independent and ligand-dependent internalization use the clathrin-dependent endocytic recycling pathway.