Bowes Melanoma Cells Research Articles

Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression.

Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter.

A High Content Drug Screen Identifies Ursolic Acid as an Inhibitor of Amyloid β Protein Interactions with Its Receptor CD36

A pathological hallmark of Alzheimer disease (AD) is deposition of amyloid β (Aβ) in the brain. Aβ binds to microglia via a receptor complex that includes CD36 leading to production of proinflammatory cytokines and neurotoxic reactive oxygen species and subsequent neurodegeneration. Interruption of Aβ binding to CD36 is a potential therapeutic strategy for AD. To identify pharmacologic inhibitors of Aβ binding to CD36, we developed a 384-well plate assay for binding of fluorescently labeled Aβ to Chinese hamster ovary cells stably expressing human CD36 (CHO-CD36) and screened an Food and Drug Administration-approved compound library. The assay was optimized based on the cells' tolerance to dimethyl sulfoxide, Aβ concentration, time required for Aβ binding, reproducibility, and signal-to-background ratio. Using this assay, we identified four compounds as potential inhibitors of Aβ binding to CD36. These compounds were ursolic acid, ellipticine, zoxazolamine, and homomoschatoline. Of these compounds, only ursolic acid, a naturally occurring pentacyclic triterpenoid, successfully inhibited binding of Aβ to CHO-CD36 cells in a dose-dependent manner. The ursolic acid effect reached a plateau at ~20 μm, with a maximal inhibition of 64%. Ursolic acid also blocked binding of Aβ to microglial cells and subsequent ROS production. Our data indicate that cell-based high-content screening of small molecule libraries for their ability to block binding of Aβ to its receptors is a useful tool to identify novel inhibitors of receptors involved in AD pathogenesis. Our data also suggest that ursolic acid is a potential therapeutic agent for AD via its ability to block Aβ-CD36 interactions.

Camptothecin induces p53-dependent and -independent apoptogenic signaling in melanoma cells

Various DNA-targeting agents may initiate p53-dependent as well as p53-independent response and subsequent apoptosis via alternative cellular systems which include for instance p73, caspase-2 or Bcl-2 family proteins. The scope of involvement of individual molecules in this process and the mechanisms governing their potential interplay are still not entirely understood, in particular in highly aggressive cancers such as in malignant melanoma. In this work we investigated the role and involvement of both p53-dependent and -independent mechanisms in selected melanoma cell lines with differing status of p53 using a model DNA topoisomerase I inhibitor camptothecin (CPT). Here we report that CPT induced in Bowes melanoma cells apoptosis which is essentially p53 and mitochondria-dependent but with some involvement of caspase-2 and p73. Conversely, in mutant p53 melanoma cells overall levels of CPT-induced apoptosis are significantly lower, with p73 and caspase-2 signaling playing important roles. In addition, in these cells the expression of micro RNAs family 34 (miR-34) were low compared to wild-type p53 cells. The ectopic expression of wild type p53 than restored apoptotic response of cells to CPT despite the fact that the expression of miR-34 and miR-155 were not influenced. These results suggest that CPT induces multivariate cellular stress responses including activation of DNA-damage response-p53 pathway as well as p53-independent signaling and their mutual crosstalk play the decisive role in the efficient triggering of apoptosis in melanoma cells.

The Tissue-Type Plasminogen Activator Story

Milestones in the development of tissue-type plasminogen activator (t-PA) as a fibrin-specific thrombolytic agent include: purification of human t-PA from the culture fluid of the Bowes melanoma cell line, elucidation of the molecular basis of fibrin-specific plasminogen activation, first experimental animal models of thrombosis, first patient (renal allograft) treated with melanoma t-PA, pilot studies in patients with acute myocardial infarction, cloning and expression of recombinant t-PA providing sufficient amounts for large scale clinical use, and demonstration of its therapeutic benefit in large multicenter clinical trials.

Retirement of Désiré Collen.

Retirement of Désiré Collen.

Anxiolytic-like activity of the non-selective galanin receptor agonist, galnon

Galanin's influence on monoaminergic neurotransmission, together with its discrete CNS distribution in corticolimbic brain areas, points to a potential role for this neuropeptide in mediating anxiety- and depression-like responses. To evaluate this hypothesis, the non-selective galanin receptor agonist, galnon, was tested in multiple preclinical models of anxiolytic- and antidepressive-like activity. Acute administration of galnon (0.03-1mg/kg, i.p.) dose-dependently increased punished crossings in the four plate test, with magnitude similar to the effects of the endogenous ligand, galanin (0.1-1.0 microg, i.c.v.). Moreover, the effects of galnon and galanin were blocked by central administration of the non-selective galanin receptor antagonist, M35 (10 microg, i.c.v.). Interestingly, the benzodiazepine receptor antagonist, flumazenil (1mg/kg, i.p.), reversed galnon's effect in the four plate test, implicating GABAergic neurotransmission as a potential mechanism underlying this anxiolytic-like response. In the elevated zero maze, galnon (0.3-3.0mg/kg, i.p.) and galanin (0.03-0.3 microg, i.c.v.) increased the time spent in the open arms, while in the stress-induced hyperthermia model, galnon (0.3-30 mg/kg, i.p.) attenuated stress-induced changes in body temperature. Consistent with these anxiolytic-like effects, in vivo microdialysis showed that acute galnon (3mg/kg, i.p.) treatment preferentially elevated levels of GABA in the rat amygdala, a brain area linked to fear and anxiety behaviors. In contrast to the effects in anxiety models, neither galnon (1-5.6 mg/kg, i.p.) nor galanin (0.3-3.0 microg, i.c.v.) demonstrated antidepressant-like effects in the mouse tail suspension test. Galnon (1-10mg/kg, i.p.) also failed to reduce immobility time in the rat forced swim test. In vitro, galnon and galanin showed affinity for human galanin receptors expressed in Bowes melanoma cells (K(i)=5.5 microM and 0.2 nM, respectively). Galanin displayed high affinity and functional potency for membranes expressing rat GALR1 receptors (K(i)=0.85 nM; EC(50)=0.6 nM), while galnon (10 microM) failed to displace radiolabeled galanin or inhibit cAMP production in the same GALR1 cell line. Galnon (10 microM) showed affinity for NPY1, NK2, M5, and somatostatin receptors but no affinity for galanin receptors expressed in rat hippocampal membranes. Taken together, the present series of studies demonstrate novel effects of galnon in various preclinical models of anxiety and highlight the galaninergic system as a novel therapeutic target for the treatment of anxiety-related disorders. Moreover, these data indicate rodent GALR1 receptors do not mediate galnon's in vivo activity.

Structure–activity relationship study of the cell-penetrating peptide pVEC

The peptide pVEC is a recently described cell-penetrating peptide, derived from the murine vascular endothelial-cadherin protein. In order to define which part of this 18-amino acid long peptide is important for the cellular translocation, we performed a structure–activity relationship study of pVEC. Together with the l-alanine substituted peptides, the retro- pVEC, D- pVEC and the scramble pVEC are studied for comparison. The peptide analogues are labeled with carboxyfluorescein at the N-terminus for monitoring the cellular uptake into human Bowes melanoma cells with different efficacy. We show that all the Fl- pVEC analogues internalize in live Bowes melanoma cells. l-Alanine substitution of the five respective N-terminal hydrophobic amino acids significantly decreases the translocation property, while replacing of Arg 6, Arg 8 or Ser 17 by alanine enhances the uptake. The uptake of pVEC is significantly reduced by treatment with an endocytosis inhibitor wortmannin. Treatment with heparinase III, nystatin and EIPA had no effect on the peptide uptake. The data presented here show that the N-terminal hydrophobic part of pVEC is crucial for efficient cellular translocation.

In Vitro and In Vivo Pharmacological Characterization of Galanin and Galnon

The neuropeptide galanin and its receptors (GAL-R1, GAL-R2, and GAL-R3) are densely localized in areas of the brain thought to regulate stress, fear and anxiety. Several studies suggest a role for galanin in mediating anxiety-like behaviors in rodent models. Here we report that both galanin (0.1 μg, icv) and galnon (0.3, 1.0 and 3.0 mg/kg, ip) show dose-dependent anxiolytic-like activity in the mouse elevated zero maze and four-plate assays. Further, M35 (10 μg, icv), a non-selective galanin receptor antagonist, was able to block galnon’s (0.3 mg/kg, ip) effects in the four-plate model. The in vitro pharmacological basis for these behavioral effects was explored using radioligand binding and a functional calcium mobilization assay. While human galanin was able to displace 125I-galanin in GAL-R1 and human Bowes melanoma cell membranes (Ki = 0.2 nM and 0.17 nM, respectively), galnon only showed affinity for galanin receptors expressed in human Bowes melanoma cells (5.5 μM). Consistent with this finding, galnon only showed functional agonism in Bowes melanoma cells (EC50 = 109 nM). This finding suggests that galnon acts as an agonist at galanin receptors in Bowes melanoma cells but not GALR1 receptors. Taken together, these studies provide further evidence for galaninergic input in anxiety-related behaviors and suggest a functional role for galnon via GAL-R2 or GAL-R3 receptors.

Bowes melanoma cells secrete heregulin, which can promote aggregation and counteract invasion of human mammary cancer cells

Invasiveness, the ability of cancer cells to migrate beyond the normal tissue boundaries, often leads to metastasis and thereby usually turns cancer into a fatal disease. At the molecular level, the E-cadherin/catenin complex is an example of a powerful invasion suppressor in epithelial cells. Since the absence of melanocytes has been associated with disturbances in epithelial organization, we decided to investigate the influence of molecules secreted by melanocytes on the function of the E-cadherin/catenin complex. We used the Bowes melanoma cell line as a source of such molecules. The conditioned medium of Bowes melanoma stimulated aggregation of human MCF-7/6 mammary adenocarcinoma cells at short (30 min) and long (24-72 hr) notice. This effect could be inhibited by MB2, an antibody against human E-cadherin. Conditioned medium of Bowes melanoma also inhibited invasion of MCF-7/6 cells into precultured chick heart fragments. Candidate molecules such as insulin, insulin-like growth factor I, follistatin and interleukins were ruled out to be responsible for the effects, but heregulin mimicked some of the effects of the conditioned medium. Our data indicate that heregulin stimulates aggregation and inhibits invasion of MCF-7/6 cells via activation of the E-cadherin/catenin complex.

Melanoma cells secrete follistatin, an antagonist of activin-mediated growth inhibition.

Using a proteomic approach to screen for new growth factors released by melanoma cells, we identified follistatin as a major heparin-binding factor in medium conditioned by the Bowes melanoma cell line. Since follistatin is primarily studied in relation to its neutralization of activin, a member of the transforming growth factor-beta family of ligands, the expression and function of this receptor system was investigated in a panel of melanoma cell lines and melanocytes. All cell lines expressed activin receptors and showed phosphorylation of Smad signal transduction molecules upon treatment with activin. Secretion of follistatin, either native or after retroviral transduction, efficiently prevented Smad activation or activation of an activin-responsive luciferase reporter construct. In melanocytes, activin treatment led to growth inhibition and induction of apoptosis. These effects were counteracted by cotreatment with follistatin. In summary, we characterized the activin-activin receptor system in melanocytes and melanoma cell lines and found that secretion of follistatin by melanoma cells may represent an effective way to circumvent activin's negative regulatory effects.

Membrane Type-1 Matrix Metalloproteinase Promotes Human Melanoma Invasion and Growth

Membrane type-I metalloproteinase (MT1-MMP) is a transmembrane metalloproteinase that is critical for tumor cell invasion. MT1-MMP can degrade extracellular matrix (ECM) proteins directly and/or indirectly by activating soluble MMPs such as pro-MMP-2. Although MT1-MMP is upregulated in malignant melanoma, the biological consequences of elevated MT1-MMP expression for tumor progression are not entirely understood. In the current study, we have utilized the Bowes melanoma line for evaluating MT1-MMP in invasion and growth. Our studies extend the earlier observations to demonstrate that MT1-MMP expression in Bowes melanoma cells promotes selective invasion into matrigel but not matrices consisting of type-I collagen. Furthermore, MT1-MMP expressing melanoma cells exhibit increased migration in response to laminin 1 but not to type-I or type-IV collagen. MT1-MMP expression results in enhanced 3 dimensional growth in agarose gels and in long-term cultures within matrigel. The hydroxymate inhibitor BB94 inhibits MT1-MMP enhanced invasion and growth in 3 dimensional culture systems, but had no effect on increased motility. We demonstrated that MT1-MMP expression significantly facilitated tumorigenicity and growth by intradermal injection. The results suggest a more general role for elevated MT1-MMP in promoting both the selective invasion and increased growth of malignant melanoma in vivo.

A Role for Nicotinic Acetylcholine Receptors in Alzheimer's Disease

Neurofibrillary tangles, which contain a phosphorylated form of the microtubule-binding protein tau, and amyloid plaques, which contain the β-amyloid peptide Aβ 1-42 , are common in the brains of Alzheimer's disease patients. Wang et al. showed that cells that express α 7 nicotinic acetylcholine receptors (α 7 nAChRs) exhibit a dose-dependent Aβ 1-42 stimulation of tau phosphorylation at all three residues commonly phosphorylated in tau found in neurofibrillary tangles. Specifically, cortical and hippocampal synaptosomes and human neuroblastoma SK-N-MC cells responded to Aβ 1-42 , whereas cells that did not express α 7 nAChRs, human Bowes melanoma cells and rat striatum synaptosomes, did not exhibit increased tau phosphorylation. The stimulation of tau phosphorylation was only observed in response to the Aβ 1-42 fragment and not other fragments of Aβ. Furthermore, tau phosphorylation in response to Aβ 1-42 was inhibited by pharmacological treatments that selectively blocked α 7 nAChRs or inhibition of α 7 nAChR expression by oligonucleotide antisense treatment. Interestingly, Aβ 1-42 exhibits inhibitory activity toward α 7 nAChR with respect to calcium mobilization and acetylcholine release; however, Wang et al. reported that Aβ 1-42 stimulated extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase 1 (JNK-1). Furthermore, stimulation of these kinases was inhibited by antisense oligonucleotide treatment to decrease α 7 nAChR. JNK-1 and ERK1/2 were able to phosphorylate tau in vitro. Thus, Aβ 1-42 may contribute to tau phosphorylation through an α 7 nAChR pathway, further supporting a role for α 7 nAChRs in Alzheimer's disease progression. H.-Y. Wang, W. Li, N. J. Benedetti, D. H. S. Lee, α 7 Nicotinic acetylcholine receptors mediate β-amyloid peptide-induced tau protein phosphorylation. J. Biol. Chem. 278 , 31547-31553 (2003). [Abstract] [Full Text]

A Role for Nicotinic Acetylcholine Receptors in Alzheimer's Disease

Neurofibrillary tangles, which contain a phosphorylated form of the microtubule-binding protein tau, and amyloid plaques, which contain the β-amyloid peptide Aβ1-42, are common in the brains of Alzheimer's disease patients. Wang et al. showed that cells that express α7 nicotinic acetylcholine receptors (α7nAChRs) exhibit a dose-dependent Aβ1-42 stimulation of tau phosphorylation at all three residues commonly phosphorylated in tau found in neurofibrillary tangles. Specifically, cortical and hippocampal synaptosomes and human neuroblastoma SK-N-MC cells responded to Aβ1-42, whereas cells that did not express α7nAChRs, human Bowes melanoma cells and rat striatum synaptosomes, did not exhibit increased tau phosphorylation. The stimulation of tau phosphorylation was only observed in response to the Aβ1-42 fragment and not other fragments of Aβ. Furthermore, tau phosphorylation in response to Aβ1-42 was inhibited by pharmacological treatments that selectively blocked α7nAChRs or inhibition of α7nAChR expression by oligonucleotide antisense treatment. Interestingly, Aβ1-42 exhibits inhibitory activity toward α7nAChR with respect to calcium mobilization and acetylcholine release; however, Wang et al. reported that Aβ1-42 stimulated extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase 1 (JNK-1). Furthermore, stimulation of these kinases was inhibited by antisense oligonucleotide treatment to decrease α7nAChR. JNK-1 and ERK1/2 were able to phosphorylate tau in vitro. Thus, Aβ1-42 may contribute to tau phosphorylation through an α7nAChR pathway, further supporting a role for α7nAChRs in Alzheimer's disease progression. H.-Y. Wang, W. Li, N. J. Benedetti, D. H. S. Lee, α7 Nicotinic acetylcholine receptors mediate β-amyloid peptide-induced tau protein phosphorylation. J. Biol. Chem. 278, 31547-31553 (2003).[Abstract][Full Text]

Latent Transforming Growth Factor-β-binding Protein 2 Is an Adhesion Protein for Melanoma Cells

Of the four latent transforming growth factor (TGF)-beta-binding proteins (LTBPs), LTBP-2 is different in the respect that it does not bind small latent forms of TGF-beta. LTBP-2 is therefore likely to have other roles in the extracellular matrix. LTBP-2 contains an RGD putative integrin recognition site, suggesting a role in cell adhesion. We carried out a study on cell attachment to LTBP-2. Purified recombinant LTBP-2 was used as substratum in cell adhesion and migration studies. We found that, unlike most adherent cell lines, all of the melanoma cell lines tested adhered to LTBP-2 very efficiently and in a concentration-dependent manner. Bowes melanoma cells bound most efficiently to LTBP-2 and were used for further characterization. Cell adhesion assays with recombinant LTBP-2 fragments indicated that the adhesive site is located in an N-terminal region of LTBP-2. The attachment of melanoma cells to LTBP-2 was prevented with monoclonal antibody against beta1 integrin in a concentration-dependent manner, suggesting an important role for beta1 integrin in the process. Antibodies against integrin subunits alpha3 and alpha6 decreased melanoma cell adhesion as well. The beta1 and alpha3 integrins were localized on the cell surface, especially in lamellipodia, as observed by immunofluorescence. In addition to integrin antagonists, heparin also markedly decreased melanoma cell adhesion. LTBP-2 also supported Bowes cell migration in modified Boyden chamber assays in a manner similar to the migration on fibronectin. Current data indicate that LTBP-2 can play a role in melanoma cell adhesion.

Dermal nevus cells from congenital nevi cannot penetrate the dermis in skin reconstructs.

Congenital nevi are composed of pigment cells bearing common features with melanocytes but showing altered differentiation which leads to nesting and dermal involvement. Using a dead de-epidermized dermis seeded with a combination of keratinocytes and various sources of pigment cells (normal melanocytes, dermal nevus cells from congenital nevi, Bowes melanoma cells), we have studied the formation of nests and the dermal migration of pigment cells together with their secretion profiles of matrix metalloproteinases (MMP). Dermal fibroblasts were also used as control cells in epidermal reconstructs. Besides their morphologic features, the absence of pigment donation to keratinocytes was the major characteristic of dermal nevus cells. A positive correlation was established between the increasing percentage of seeded nevus cells and the patchy pigmentation of reconstructs, as well as the clustering of cells in junctional nests. However, the presence of nevus cells in the dermis of reconstructs was never detected, whereas melanoma cells and dermal fibroblasts could invade the dermis during the time span of the experiments. MMP9 was never expressed in congenital dermal nevus cells but pro-MMP2 was constitutively expressed by all strains of congenital nevus cells and dermal fibroblasts. Melanocytes produced comparable amounts of both pro-MMP2 and pro-MMP9, and Bowes melanoma cells secreted a marginal level of pro-MMP2. In view of their three-dimensional behaviour and secretion of MMPs, we propose that dermal congenital nevus cells correspond to an intermediate status of differentiation between normal melanocytes and melanoma cells. Activation of MMPs by a cofactor or the activation of another signalling pathway seems necessary to induce the dermal passage of nevus cells.

VE-Cadherin-Derived Cell-Penetrating Peptide, pVEC, with Carrier Functions

Cell-penetrating peptides, CPPs, have been shown to translocate into living cells by a receptor-independent mechanism and to carry macromolecules over the plasma membrane. This article reports studies of the internalization of pVEC, an 18-amino acid-long peptide derived from the murine sequence of the cell adhesion molecule vascular endothelial cadherin, amino acids 615–632. Fluorophore-labeled pVEC entered four different cell lines tested: human aortic endothelial cells, brain capillary endothelial cells, Bowes melanoma cells, and murine brain endothelial cells. In order to evaluate the translocation efficiency of pVEC, we performed a side-by-side comparison with penetratin, a well-characterized CPP. The cellular uptake of pVEC was highest for murine brain endothelial cells. All cell lines tested contained equal or slightly higher concentrations of pVEC than penetratin. pVEC mainly accumulated in nuclear structures but was also found throughout the cells. Furthermore, pVEC functioned as a transporter of both a hexameric peptide nucleic acid molecule of 1.7 kDa and a 67-kDa protein, streptavidin–FITC, and cellular uptake of fluorophore-labeled pVEC took place at 4°C, suggesting a nonendocytotic mechanism of translocation. In conclusion, our results indicate that pVEC is efficiently and rapidly taken up into cells and functions as a potent carrier peptide.

Biphasic response to human galanin of extracellular acidification in human Bowes melanoma cells

The metabolic response of galanin GAL1 receptor subtype, endogenously expressed in human Bowes melanoma (HBM) cells, was investigated. Cytosensor microphysiometry was used to determine the extracellular acidification rate. A biphasic response, consisting of a rapid increase in the extracellular acidification rate followed by a decrease below the basal level, was observed after perfusion with human galanin. The magnitude and the rate of onset of both phases were dependent on the galanin concentration. The increase in the extracellular acidification rate (maximum of 25% of basal level; −log(EC 50)=7.23±0.14) was transient, whereas the following decrease (maximum of 40% of basal level; −log(EC 50)=7.77±0.23) was sustained. The EC 50 values for the increase and decrease were in a similar range. After consecutive galanin administration, the magnitude of the response was the same as for the unexposed cells, indicating the absence of galanin receptor desensitization or internalization in HBM cells. Responses were blocked by pretreatment with pertussis toxin and phorbol-12-myristate-13-acetate (PMA), indicating a G-protein/protein kinase C signalling pathway. Our microphysiometry results show a biphasic response of the extracellular acidification rate mediated by the galanin receptor expressed in HBM cells which has not been described previously for any other endogenously expressed neuropeptide receptor.

Deletion analogues of transportan

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

Translocation properties of novel cell penetrating transportan and penetratin analogues.

Novel analogues of the cell-penetrating peptides penetratin and transportan were synthesized. The distribution of the biotin-labeled peptides in Bowes melanoma cell line has been investigated by indirect fluorescence with fluorescein-streptavidin detection. The time course of uptake of (125)I-labeled transportan analogues has been characterized in the same cell line. Molecular modeling was used to analyze the penetration and the orientation of molecules in a simulated biological membrane. The results, both from molecular modeling and fluorescence studies, imply that penetratin and transportan do not enter the cells by related mechanisms and that they do not belong to the same family of translocating peptides.

Deletion analogues of transportan

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.