Evidence that membrane transduction of oligoarginine does not require vesicle formation

Experimental Cell Research 307 (2005) 164 – 173 www.elsevier.com/locate/yexcr

Evidence that membrane transduction of oligoarginine does not require vesicle formation Jennica L. Zaro, Wei-Chiang ShenT University of Southern California, School of Pharmacy, Department of Pharmaceutical Sciences, 1985 Zonal Avenue, PSC 404B, Los Angeles, CA 90033-1039, USA Received 28 September 2004, revised version received 17 February 2005 Available online 29 March 2005

Abstract The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 168C as compared to the 378C control, and was only partially inhibited at 48C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITCdextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine. D 2005 Elsevier Inc. All rights reserved. Keywords: Membrane transduction; Endocytosis; Oligoarginine; Membrane transduction peptides; Cell-penetrating peptides; Quantitative measurement

Introduction Recently, peptides characterized as membrane transduction peptides (MTPs), or cell-penetrating peptides (CPPs), are of interest due to their ability to translocate

Abbreviations: a-MEM, a-minimum essential medium; AEBSF, 4-(2aminoethyl)-benzenesulfonyl fluoride hydrochloride; CHO, Chinese hamster ovary; CPP, cell-penetrating peptide; E-64, (2S,3S)-3-(N-{(S)-1-[N-(4guanidinobutyl)carbamoyl]3-methylbutyl}carbamoyl)-oxirane-2-carboxylic acid; EDTA, ethylene-di-amine-tetra-acetic acid; E-64, trans-(epoxy-succinyl)-l-leucylamino-(4-guanidino)butane; FITC, fluorescein 5V-isothiocyanate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HIV-1, human immunodeficiency virus type 1; MTP, membrane transduction peptide; NH4Cl, ammonium chloride; PBS, phosphate-buffered saline; PMSF, phenyl-methylsulfonyl fluoride; RFU, relative fluorescent units. T Corresponding author. Fax: +1 323 442 1390. E-mail address: [email protected] (W.-C. Shen). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.02.024

across cellular membranes directly into the cytoplasm of cells in a novel mechanism termed membrane transduction. This membrane transduction process is of tremendous importance in the delivery of macromolecular drugs, including peptides, oligonucleotides, and genes, with cytoplasmic and nuclear targets [1–3]. Molecules capable of membrane transduction are short sequences of amino acids, usually from 9–30 amino acids, containing an abundance of arginine and lysine residues (recently reviewed in [4]). However, whether these MTPs are able to gain access to the cytoplasmic compartment directly via the plasma membrane, or across intracellular vesicles following internalization via endocytosis is still a subject of controversy [5,6]. Early studies of MTPs such as HIV-1 Tat-(47–57) [7], Antennapedia (43–58) [8], and small oligoarginine peptides [9,10], across cell membranes showed that the mechanism was temperature, energy, and

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cell-type independent. These findings suggested an exclusion of endocytosis as a mechanism of membrane transduction. On the other hand, recent studies have implicated the involvement of several different types of endocytosis, including macropinocytosis, in the internalization of MTPs with varying results [5,11–15]. Most recently, it has been proposed that the guanidinium headgroups of argininecontaining peptides form bidentate hydrogen bonds on the cell surface, resulting in the migration across the lipid bilayer into the cytosol [16]. However, this hypothesis does not preclude endosomes as the location that the transmembrane transport occurs. The discrepancy regarding the mechanism of membrane transduction is mostly due to problems with the experimental methods being used to measure the peptide internalization. The high plasma membrane surface binding and artifactual re-distribution following fixation have made many of the conclusions from previous studies questionable [17–19]. Moreover, the in vitro methods previously used may not quantitatively measure the membrane transduction efficiency of these MTPs since it is likely that membrane transduction occurs concurrently with endocytosis [16,20]. Many studies on the MTPs were based on the measurement of cytoplasmic activity of cargo macromolecules in the cell. However, the biological activity of cargo macromolecules may not accurately measure the membrane transduction efficiency, because the attachment of large macromolecules with varying chemical properties alters the internalization properties [21–24], possibly changing transduction and endocytosis to different extents. Consequently, there is no established quantitative correlation between the expression of biological activity of the cargo macromolecules and the transduction efficiency of the MTPs. In order to obtain unambiguous results to determine the involvement of these two transport processes, we have recently developed a quantitative method to measure the membrane transduction efficiency, separately from endocytosis. The technique separates the vesicular versus cytoplasmic compartments, which allows for the calculation of the membrane transduction and the characterization of the structural requirements for several MTPs. Using this method, we have previously shown that while oligoarginine is primarily transduced into CHO cells, oligolysine is internalized predominantly by endocytosis [20]. This report describes the differential effect of temperature, pH, and several other inhibitors on internalization via endocytosis or membrane transduction of oligoarginine and oligolysine, and the subsequent nuclear localization of these oligopeptides.

Materials and methods Peptide synthesis and labeling YG(R)9, and YG(K)9 were synthesized by Genemed (South San Francisco, CA). The tyrosine moiety was labeled

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with Na-125I (ICN, Irvine, CA) using the Chloramine-T method [25]. The labeled oligopeptides were purified by size exclusion chromatography using Sephadex G-15 (Sigma, St. Louis, MO) gel matrix. Cell culture Chinese hamster ovary (CHO) cells (ATCC, Manassas, VA) were grown in a-minimum essential medium (a-MEM) (GIBCO-BRL, Carlsbad, CA) containing 10% fetal bovine serum (GIBCO-BRL). The cells were replenished with fresh medium the day before confluence at which time the localization assays were performed. Cytoplasmic localization assays The measurement of cytoplasmic localization of MTPs was performed with a previously reported procedure [20]. Briefly, confluent CHO monolayers grown in T75 flasks (Corning, Acton, MA) were incubated in serum-free medium containing 5 Ag/mL of 125I-labeled oligopeptide, 0.1 mg/mL FITC-Dextran (70 kDa) (FD) (Sigma), and protease inhibitor cocktail (PI) (Sigma) containing 4 AM AEBSF, 2 AM EDTA, 0.3 AM bestatin, 30 nM E-64, 2 AM leupeptin, and 0.6 nM aprotinin. After treatment for 1 h at either 4, 16, or 378C, the monolayers were washed three times with cold PBS, detached by treatment with trypsinEDTA at 378C for 5 min, and the isolated cell pellets washed with 0.5 mg/mL heparin-PBS followed by PBS. The cell pellets were then homogenized in buffer (HB) containing 0.25 M sucrose, 2 mM EDTA, and 10 mM HEPES, pH 7.4 using the Balch cell press (H & Y Enterprises, Redwood City, CA) [26]. The cell homogenate was centrifuged at 600  g at 48C for 10 min, and the post-nuclear supernatant was fractionated using Sephacryl S-500 (Amersham, Piscataway, NJ) size exclusion chromatography (1  13 cm column dimensions) with HB as the eluting buffer. 1-mL fractions were collected and assayed for 125I-oligopeptides using a Gamma counter (Packard, Downers Grove, IL), for FD using fluorescence spectroscopy (Hitachi, Tokyo, Japan) (Ex 494 nm Em 519 nm), and for protein content using the Pierce protein assay kit. The amount of oligopeptide internalized by endocytosis versus transduction was calculated using the equations previously described [20]. For the endocytosis inhibitor assays, the localization assay method was performed after pre-treating CHO cell monolayers at 378C with serum-free medium containing either 0.3 M sucrose for 15 min, or 50 mM NH4Cl, 1.5 AM filipin, or 100 AM amiloride for 30 min, followed by a 1-h coincubation with 125I-oligopeptide and FD and the respective inhibitors. The internalization of 125I-transferrin (Sigma) (3 Ag/mL) and BODIPY-lactosylceramide (Molecular Probes) (2 AM) was measured following the above pre-treatments with sucrose and filipin as positive controls for clathrin- and caveolar-mediated endocytosis, respec-

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tively, as described [27,28]. For measurement of total FD internalization, CHO monolayers grown in 6-well culture plates (Corning, Acton, MA) were incubated in serum-free medium containing 5 Ag/mL oligopeptide and 0.5 mg/mL FITC-Dextran (70 kDa) for 1 h at 378C. The cell pellets were washed with PBS, isolated following treatment with trypsin-EDTA, washed with PBS, and dissolved in 0.4% Triton X-100. The amount of FD internalized was measured by fluorescence spectroscopy (Ex 494 nm Em 519 nm), and the total cell protein content determined by the Bradford assay method. Enzyme activity assays The fractions obtained from the cytoplasmic localization assay were further analyzed for the markers of plasma membrane (alkaline phosphatase) and lysosomes (h-hexosaminidase) [29]. Alkaline phosphatase enzyme activity was determined using U-nitrophenyl phosphate Liquid Substrate System (Sigma). 100 AL of each fraction sample was incubated with 200 AL substrate solution for 1 h at 378C. The absorbance at 405 nm was measured. h-hexosaminidase activity was determined using 4-methyl umbelliferyl Nacetyl-h-d-glucosaminide (Sigma). 30 AL of each fraction was diluted with 50 AL distilled water and incubated with 50 AL substrate solution containing 7.5 mM 4-methyl umbelliferyl N-acetyl-h-d-glucosaminide in basic buffer solution (133 mM Na citrate, 133 mM NaCl, pH 4.3) for 30 min at room temperature in the dark, and subsequently assayed by fluorescence spectroscopy (Ex 360 Em 465). Nuclear isolation The confluent CHO cell monolayers were treated with 5 Ag/mL of 125I-oligopeptide at 15-min, 1-h, or 2-h time points. The nuclei were isolated following a slightly modified method by Beck et al. [30]. Briefly, the labeled cells were washed three times with cold PBS, and detached by treatment with trypsin-EDTA for 5 min at 378C. After two additional washes with cold PBS, the cells were resuspended in 300 AL TKM buffer (50 mM Tris–HCl, pH 7.5, 25 mM KCl, 5 nM MgCl2) containing 1 mM phenyl-methylsulfonylchloride (PMSF) and PI (TKM/ PMSF/PI) and allowed to swell on ice for 15 min. 10% (w/v) sodium deoxycholate in 1% Triton X-100 was added to a final concentration of 0.4% to the cells while vortexing for 15 s, and the cells were incubated for an additional 10 min. An equal volume of 500 mM sucrose in TKM/PMSF/ PI was added drop wise and the nuclei were pelleted by centrifugation for 10 min at 640  g. The nuclei were resuspended in 250 mM sucrose in TKM/PMSF/PI followed by the addition of sodium deoxycholate in Triton X-100 to a final concentration of 0.4% while vortexing for 30 s, and pelleted by centrifugation. This wash procedure was repeated twice, followed by two washes with 250 mM sucrose in TKM buffer containing PI. The nuclear pellet was

assayed for radioactivity using a Gamma Counter, and the amount of nuclear protein was determined using the Pierce protein assay kit. Fluorescence spectroscopy assays CHO cell monolayers were grown overnight in 6-well culture plates (Corning, Acton, MA) to 50% confluence, after which time the monolayers were incubated in serumfree medium for 20 min followed by incubation in serumfree medium containing 1 mg/mL FITC-dextran (70 kDa) for 15 min at either 168C or 378C in the presence and absence of 25 Ag/mL YG(R)9. The cell monolayers were washed with cold PBS, fixed in 3.7% formaldehyde and quenched in 50 mM NH4Cl. The microslides were mounted in Prolong Antifade solution (Molecular Probes, Eugene, OR), stored at 48C overnight, and imaged using a fluorescence microscope at 570 nm.

Results Characterization of cytoplasmic localization measurement To validate the cytoplasmic localization assay as described in our previous report [20], the fractions obtained from the size-exclusion chromatograph were further characterized. CHO cell monolayers grown to confluence were treated with serum-free medium containing 5 Ag/mL of 125I-YG(R)9, 0.1 mg/mL FITC-Dextran (FD), and protease inhibitor cocktail for 1 h at 378C. The cell monolayers were detached with trypsin-treatment, washed with PBS followed by heparincontaining buffer, and then homogenized using a Balch cell press. A Dounce homogenizer can be used to substitute Balch cell press; however, the recovery rate of the post-nuclear supernatant (PNS) appeared to be low and inconsistent. The PNS was then isolated by centrifugation, and fractionated on a Sephacryl S-500 column (Fig. 1A). The resultant elution profile shows the oligoarginine and FD localized in the vesicular (Peak 1) and cytoplasmic compartments (Peak 2, fraction No. 8 for FD and No. 11 for 125I-YG(R)9). To verify that Peak 1 was not due to non-specific binding to membrane components after the cell homogenization, the elution profile was compared to that of PNS spiked with either 125Ioligoarginine or FD. As shown in Fig. 1B, neither oligoarginine nor FD can be detected at the void volume of the column (Fig. 1A, Peak 1) when spiked in the PNS, and both elute in fractions overlapping with their respective Peak 2 in Fig. 1A. Another concern for this analytical method is the possibility of not completely removing the surface-bound oligopeptide following the extensive washes with heparincontaining buffer. Therefore, following homogenization of cell monolayers, the isolated PNS and post-nuclear pellet (PNP) were further analyzed for the presence of plasma membrane marker, alkaline phosphatase, and lysosomal enzyme marker, h-hexosaminidase. As expected, the major-

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Fig. 1. Cytoplasmic localization assay. CHO cell monolayers grown to confluence were treated with serum-free medium containing 5 Ag/mL of 125I-YG(R)9 (closed circles), 0.1 mg/mL FD (open circles), and protease inhibitor cocktail for 1 h at 378C. The cell monolayers were detached with trypsin-treatment, washed with PBS followed by heparin-containing buffer, and then homogenized using a Balch cell press. The post-nuclear supernatant (PNS) was isolated by centrifugation, and fractionated on an S-500 column (A). The isolated PNS of CHO cell monolayers was spiked with either 125I-oligoarginine or FITC dextran (FD) and eluted on an S-500 column (B). The isolated PNS of CHO cell monolayers was eluted on an S-500 column and the fractions obtained were assayed for activity of alkaline phosphatase using U-nitrophenyl phosphase substrate (C) or for h-hexosaminidase activity using 4-methyl umbelliferyl N-acetyl-h-dglucosaminide (D).

ity of the plasma membrane material is found in the PNP fraction following centrifugation (76%) in comparison to the PNS (24%). The PNS was then run on the Sephacryl S-500 column. As seen in Fig. 1C, alkaline phosphatase activity was found in the void volume and in the fractionation range. The later peak elutes slightly before that of free 125Ioligopeptide. The small amount eluting in the void volume (11% of total activity) does not significantly contribute to the measurement of vesicular-bound 125I-oligoarginine. Additionally, the fractions were also assayed for the lysosomal enzyme, h-hexosaminidase, to verify that Peak 1 was due to intact vesicular components. As seen in Fig. 1D, the majority of the h-hexosaminidase activity was found in the void volume of the column. There is a slight amount of h-hexosaminidase detected in the fractionation range of the column due to the rupturing of the lysosomes during the homogenization process.

temperature, the internalization of FITC-dextran at 168C was much less than internalization at 378C, as measured by fluorescence microscopy (Fig. 4). Membrane transduction of oligoarginine at 48C was inhibited by approximately 40% as compared to the 378C control (Fig. 2).

Effect of temperature on membrane transduction

Fig. 2. Effect of temperature on transduction. CHO cultured cell monolayers were incubated with serum-free medium containing FITCDextran, 5 Ag/mL 125I-YG(R)9, and protease-inhibitor cocktail at the indicated temperatures for 1 h. The cell monolayers were detached by treatment with trypsin, and the cell pellets were washed with heparincontaining PBS, followed by PBS, and homogenized using a Balch Cell press. The PNS was isolated by centrifugation and separated on an S-500 gel filtration column. The resultant fractions were assayed for radioactivity using a Gamma counter, and for fluorescence using a Fluorescence Spectrophotometer. The amount internalized by endocytosis (closed bars), or transduction (open bars) was calculated as described in the Materials and methods section.

To determine the effects of reduced temperature on internalization of oligoarginine, cells were treated with 5 Ag/ mL 125I-YG(R)9 for 1 h at 4, 16, and 378C (Fig. 2). The amount of oligoarginine internalized via endocytosis was inhibited by about 50% at both 4 and 168C. The internalization via membrane transduction, however, was not changed at 168C incubation as compared to the 378C control. To verify the inhibition of endocytosis at this

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Investigation of endosomolytic activity of oligoarginine The possible endosomolytic property of oligoarginine was investigated by the comparison of the elution profiles of FITC-Dextran (FD) in the presence and absence of oligoarginine. The amount of FD retained in the endosomes (RE) was calculated as described in the Materials and methods section and compared after incubation in the presence and absence of 25 Ag/mL YG(R)9. The results presented in Table 1 demonstrate that the amount of FD located in the cytoplasmic fraction is not higher in the presence of oligoarginine, indicating that oligoarginine is not released into the cytoplasm due to endosomolytic properties. Inhibition of endosomal acidification by ammonium chloride treatment The effect of the inhibition of the endosomal acidification on oligoarginine and oligolysine internalization was determined following treatment with 50 mM NH4Cl. The results presented in Fig. 3 demonstrate that transduction of YG(R)9 and YG(K)9 is not inhibited following treatment, while endocytosis was inhibited by 27% and 62%, respectively. In fact, transduction of both oligoarginine and oligolysine was increased following treatment with the lysosomotropic amine (16 and 50%, respectively). Inhibition of endocytic internalization To investigate the involvement of several types of endocytosis on the internalization of oligoarginine, the transduction of 5 Ag/mL YG(R)9 was measured following treatment with inhibitors of endocytosis including incubation in hypertonic medium, an inhibitor of clathrinmediated endocytosis, or filipin, an inhibitor of caveolarmediated endocytosis. As a positive control, the inhibition of clathrin- and caveolar-mediated endocytosis was measured using 125I-transferrin and BODIPY-lactosylceramide, respectively. As seen in Table 2, internalization of oligoarginine via transduction is not significantly affected by any of these treatments, while the respective positive controls are inhibited. These results further demonstrate that transduction does not involve vesicular-formation processes. Table 1 Endosomolytic activity of oligoarginine Sample

% REa

Control +25 Ag/mL YG(R)9

62 F 1 54 F 2

a

CHO cells were incubated in serum-free medium containing 1 mg/mL FITC-Dextran in the absence and presence of 25 Ag/mL YG(R)9 for 1 h, 378C. The amount of FITC-Dextran released from the endosomes (% RE) was calculated as described. Data are presented as average F standard deviation with n = 3.

Fig. 3. Effect of ammonium chloride on transduction and endocytosis. CHO cultured cell monolayers were incubated with serum-free medium containing FITC-Dextran, 5 Ag/mL 125I-YG(R)9 (closed bars) or 125I-YG(K)9 (open bars) at 378C for 1 h in the absence (Control) or presence of 50 mM NH4Cl. The amount internalized by endocytosis (A), or transduction (B) was determined using the subcellular fractionation method as described in the Materials and methods section.

Involvement of macropinocytosis on oligoarginine transduction Several studies were done to investigate the involvement of macropinocytosis on oligoarginine transduction. The total amount of FD recovered from the size-exclusion chromatography data in the presence and absence of oligoarginine was compared to determine the effect of the MTP on the stimulation of fluid-phase endocytosis in CHO cells. Treatment with YG(R)9 resulted in the total internalization of 1.379 F 0.284 RFU/mg cell protein of FD, which was not statistically significant from the control, 1.305 F 0.052 RFU/mg cell protein (Table 3). This result was further verified by incubation of CHO Table 2 Effect of endocytosis inhibitors on oligoarginine transduction Treatment

YG(R)9 transductiona, % control

Tf endocytosisa, % control

LacCer endocytosisa, % control

Control Hypertonic medium Filipin

100 F 15 99 F 19 90 F 12

100 F 4 53 F 9 105 F 11

100 F 8 86 F 3 61 F 3

a

CHO cultured cell monolayers were pre-incubated with serum-free medium containing 0.3 M sucrose, or 1.5 AM filipin, followed by incubation with serum-free medium containing the respective inhibitors and either 0.1 mg/mL FITC-Dextran, 5 Ag/mL 125I-YG(R)9 and proteaseinhibitor cocktail; 3 Ag/mL 125I-transferrin (Tf); or 2 AM BODIPYlactosylceramide (LacCer). The amount transduced to the cytoplasm or endocytosed was determined as described in the Materials and methods section. Data are presented as average F standard deviation with n = 4.

J.L. Zaro, W.-C. Shen / Experimental Cell Research 307 (2005) 164–173 Table 3 Involvement of macropinocytosis on oligoarginine internalization in CHO cells Treatment

Oligoarginine transduceda, % control

FD internalizationa, % control

YG(R)9 + FITC-dextran (control) YG(R)9 + FITC-dextran + amiloride FITC-dextran

100 F 15

100 F 18

85 F 7

121 F 43

NA

105 F 5

a

CHO cells were incubated in serum-free medium containing either FITCDextran (FD) and YG(R)9 (Control); FD, YG(R)9, and amiloride following pre-treatment with amiloride; or FD alone. The amount oligoarginine transduced and the total amount of FD internalization was determined as described in the Materials and methods section. Data are presented as average F standard deviation with n = 4.

cells grown on glass coverslips with FD in the presence and absence of YG(R)9 at 16 and 378C, followed by analysis using fluorescence microscopy. As seen in Fig. 4, the intracellular fluorescence of FD in the presence and absence of oligoarginine was not changed qualitatively at either temperature. Furthermore, large FD-containing vesicles, or macropinosomes, could not be detected in this cell line. These results indicate that treatment with oligoarginine does not stimulate fluid-phase endocytosis in CHO cells. Additionally, the effect of the specific macropinocytosis inhibitor, amiloride, on oligoarginine transduction was determined. As seen in Table 3, treatment

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with amiloride did not inhibit oligoarginine transduction to a great affect. Furthermore, the inhibition did not have a significant effect on the internalization of FD, again indicating that macropinocytosis does not occur in this cell line. Investigation of nuclear transport of oligoarginine and the relation to endocytosis vs. transduction The kinetics of internalization of 125I-YG(R)9 via transduction was measured in CHO cells using the subcellular fractionation method as described. As seen in Fig. 5A, transduction is a rapid process, occurring within 10–15 min of incubation. The amount of oligoarginine transduced reaches a constant level around 60 min, after which time the transduction of oligoarginine begins to decrease. The kinetics of 125I-YG(R)9 transported into the nucleus was also measured at 15-, 60-, and 120-min time points and compared to the transduction data. The results indicate the nuclear transport begins to dramatically increase after the 60-min time point. This increase in nuclear localization overlaps with the decrease in cytoplasmic localization with the labeled oligoarginine. The effect of ammonium chloride on nuclear transport of 125 I-YG(R)9 was also investigated. CHO cells were treated with 50 mM NH4Cl followed by incubation with 5 Ag/mL 125 I-YG(R)9. The nuclei were isolated as described in the Materials and methods section, and the total amount of 125I-

Fig. 4. Internalization of FITC-Dextran at 16 and 378C and the effect of YG(R)9. CHO cultured cell monolayers grown on glass coverslips to 50% confluence were pre-incubated with serum-free medium at 168C (A, C) or 378C (B, D) for 20 min, followed by incubation in medium containing 1 mg/mL FITC-dextran for 15 min in the absence (A, B) and presence of 25 Ag/mL YG(R)9 (C, D). The cells were rinsed with PBS, fixed in 3.7% formaldehyde and mounted in Prolong Antifade solution. The cells were imaged by fluorescence microscopy at 570 nm. Scale bar, 20 Am.

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Fig. 5. Kinetics of internalization via transduction and nuclear transport (A) and effect of ammonium chloride (B). CHO cultured cell monolayers were treated for the indicated time points with serum-free medium containing 5 Ag/mL 125I-YG(R)9, protease-inhibitor cocktail, and 0.1 mg/mL FITCDextran (for transduction measurement assay) for the indicated time points. The ng internalized by transduction (solid circles) was measured using the subcellular fractionation method, and the ng located in the nucleus (open circles) was determined following nuclei isolation from the cell monolayers harvested following treatment with trypsin as described in the Materials and methods section (A). To determine the effect of ammonium chloride on nuclear transport, CHO cultured cell monolayers were treated with serumfree medium containing 5 Ag/mL 125I-YG(R)9 for 1 h at 378C (Control), and for 1 h at 378C in the presence of 50 mM NH4Cl. The nuclei were isolated from the harvested cell monolayers following treatment with trypsin, and the amount of oligopeptide/nuclear protein extract was determined as described in the Materials and methods section (B).

YG(R)9 was compared to that of the non-treated cells. The results presented in Fig. 5B show that NH4Cl does not have an effect on nuclear transport of 125I-YG(R)9, indicating that the nuclear transport is not mediated via endocytosis.

Discussion The internalization of oligoarginine was investigated using a recently developed quantitative method to measure the membrane transduction efficiency, exclusively from endocytosis. One concern with this method is the interference of MTP bound to plasma membrane components or vesicles, which is not extensively removed following the wash with heparin-containing buffer and PBS. The location of the plasma membrane and vesicular components on the size-exclusion chromatograph was determined using the plasma membrane marker, alkaline phosphatase, and lysosomal enzyme marker, h-hexosaminidase. The majority of

the plasma membrane marker was found in the post-nuclear pellet (74%) following centrifugation of homogenized cell pellet. The remaining alkaline phosphatase activity found in the PNS elutes in the void volume of the column, and overlaps with the lysosomal enzyme marker (Figs. 1C and D). This minimal activity (8% of total alkaline phosphatase activity) could possibly be a non-specific activity of other phosphatases found in the lysososome, but does not interfere with the measurement of free 125I-oligoarginine. Fig. 1D shows that the majority of the lysosome fraction elutes in the void volume of the column. A small amount of the lysosomal enzyme can be detected in the later fraction, corresponding to the molecular weight of the enzyme (60 kDa), due to the rupture of the lysosomes during the homogenization. The minimal activity of free alkaline phosphatase as shown in Fig. 1C indicates that the large amount of 125I-YG(R)9 at Peak 2 in Fig. 1A was not a result of the release of surface-bound peptides from membrane due to membrane dissociation during the homogenization process. Additionally, 125I-YG(R)9 and FD when spiked in the PNS following homogenization elute in one peak, which overlaps the later peaks, Peak 2, obtained from the subcellular fractionation assay (Figs. 1A and B). This result indicates that both oligoarginine and dextran do not bind to any vesicle/membrane components in PNS; therefore, Peaks 1 and 2 in Fig. 1A represent truly the vesicle-associated and cytosolic 125I-YG(R)9 and FD in the intact cells. The subcellular fractionation method was used to further distinguish internalization of oligoarginine via endocytosis and membrane transduction. Endocytosis is known to be a highly temperature-sensitive process. Temperature-dependence assays are generally carried out at 48C to achieve the maximum inhibition of internalization through endocytosis, which also significantly alters the membrane fluidity. Temperatures below 208C have been shown to inhibit intracellular vesicular fusion events and transport of endocytosed material to the lysosomes [31]. The effect of temperature on membrane transduction, however, is not clearly defined. Cellular uptake assays of 125I-YG(R)9 were compared for 4, 16, and 378C incubation temperatures (Fig. 2). The amount of oligoarginine internalized via endocytosis was inhibited similarly at both 4 and 168C incubation. On the other hand, membrane transduction at 168C was not statistically significantly different from the control, while incubation at 48C inhibited transduction by approximately 40% of control. The lack of inhibition at 168C indicates that transduction does not depend on vesicular fusion events, and is not likely to be due to an endocytic process. Since the transduction mechanism is thought to occur through the direct interaction of the MTPs with the plasma membrane, it is conceivable that such an inhibition of membrane fluidity at 48C can also inhibit transduction at this temperature. Since the transduction was not inhibited at 168C, an alternative route of cytoplasmic entry for the MTPs is via endosomolysis through the early endosomes. The possibility that the MTPs are entering the cytoplasm via endosomal

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rupture was investigated by the comparison of the elution profiles of the fluid-phase endocytosis marker, FD. The elution profile of the FD contains two peaks, the first being the amount of FD localized in the intracellular vesicles, and the second peak being the amount of FD that leaked into the soluble fraction due to vesicular rupture by shear forces during the homogenization process. The elution profile of FD in cell preparations containing endosomolytic agents should show a higher amount of FD localized in the cytoplasmic fraction. Therefore, the elution profiles of FD in the presence and absence of oligoarginine were compared. As shown in Table 1, the amount of FD retained in the vesicles while co-incubated with 25 Ag/mL YG(R)9 is actually slightly higher than that without the MTP (54 vs. 62%, respectively). This increase in the FD retention could either be due to the stabilization of the vesicles, or the increase of the intensity of FD in the presence of oligoarginine. In either case, the amount used in this experiment was in 5-fold excess of that used in the other experiments, so the results obtained from other experiments would not likely be affected to the same extent. The lack of increase in the amount of FD released to the cytoplasm in the presence of oligoarginine indicates that the MTP is not endosomolytic and does not reach this cytoplasm via this process. Many ligands internalized via fluid-phase and receptormediated endocytosis are subjected to the acidification of the endosomal compartment [32–34]. Lysosomotropic amines such as ammonium chloride (NH4Cl) accumulate in the acidic subcellular compartments and produce an elevation in organelle pH for lysosomes, endosomes, and trans-Golgi apparatus [35]. Such an inhibition has been shown to prevent the intracellular processing and vesicular trafficking of this pH-dependent pathway. To investigate the effect of the pH on the internalization of oligoarginine and oligolysine, cells were treated with 50 mM ammonium chloride followed by incubation with 5 Ag/mL of either 125IYG(R)9 or 125I-YG(K)9. Oligolysine was used in the experiment since it has been previously shown to be internalized primarily by endocytosis [20]. The results show that while endocytosis of both oligoarginine and oligolysine is significantly inhibited, transduction is actually increased. The percent increase of transduction of oligolysine is greater than that of oligoarginine (50 vs. 16%, respectively). This increase in transduction could be due to several possibilities, such as the increased stability and half-life of the MTP due to the inhibition of endosomal acidification. However, the results indicate that transduction is not subjected to pH processing events similar to that of endocytosis. To further investigate the involvement of vesicular formation in membrane transduction, inhibitors of different types of endocytosis were used. Several different types of endocytic pathways have been implicated in the involvement of MTP internalization, including clathrin-mediated, caveolar-mediated, and macropinocytosis [5,12–15]. However, since the quantitative methods used for these inves-

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tigations measured total internalization of MTP via transduction and endocytosis, the differential effect on transduction alone has not been demonstrated. Therefore, the transduction of oligoarginine was measured following treatment with hypertonic medium, filipin, and amiloride. Treatment with hypertonic medium inhibits clathrin-mediated endocytosis, via several postulated mechanisms such as prevention of surface aggregation of receptors, or prevention of vesicular movement [36]. The sterol binding agent filipin, which sequesters cholesterol from the plasma membrane and prevents formation of caveolae, inhibits caveolar-mediated endocytosis [37]. As seen in Table 2, neither of these inhibitors showed a significant inhibition of transduction of oligoarginine. Taken together with the other results presented here, transduction of oligoarginine does not depend on vesicle formation events, and is likely to occur directly via the plasma membrane. The possibility of stimulation of macropinocytosis by oligoarginine [12] was also investigated. It has been shown that stimulators of macropinocytosis are known to increase the internalization of fluid-phase markers by 5- to 10-fold [38]. Therefore, the affect of oligoarginine on the total internalization of FD was evaluated to determine the potential stimulation of these processes. Table 3 shows that the total internalization of FD was not statistically significantly different in the presence and absence of oligoarginine. This result was further verified qualitatively by fluorescence microscopy. In addition to the lack of increase in FD internalization in the presence of oligoarginine, there are no large FD filled vesicles that could be detected (Fig. 4), which are indicative of macropinosome formation [39]. The lack of stimulation of FD internalization by oligoarginine is an indication that the MTP does not stimulate these processes in CHO cells. In addition to the increase of fluidphase markers, internalization via macropinocytosis can also be investigated by the response to drugs such as amiloride, an inhibitor of Na+/H+ exchange, that act on the intracellular pH [40]. As presented in Table 3, the presence of amiloride does not greatly inhibit oligoarginine transduction. Additionally, this inhibitor does not inhibit FD internalization, further indicating that FD internalization does not depend on membrane-ruffling events, which are required for macropinocytosis [39]. The results obtained for the involvement of macropinocytosis on internalization of oligoarginine in CHO cells may be different in other cell types. Macropinocytosis is known to vary greatly among cell lines. Different cells ruffle to different extents, the intracellular fate and regulation of macropinosomes vary among cell type and the effect of inhibitors differ in constitutive versus induced processes [39,41–43]. However, although oligoarginine may be endocytosed in different extents in various cell lines via this pathway, the data presented here indicate that membrane transduction is not likely to occur via this process in CHO cells. It is also of interest whether the capability of many arginine-containing oligopeptides for nuclear transport and

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localization [7,9,10,44,45] is mediated via endocytosis or membrane transduction. The kinetics of transduction and nuclear transport presented in Fig. 5 show that the increase in nuclear transport coincides with the decrease in cytoplasmic localization. It is of interest to notice the lag in the nuclear transport following transduction, since transduction occurs rapidly, within 10–15 min, while nuclear transport is much later, after 60 min. There are several possible reasons for this observation. The mechanism of membrane transduction may involve binding to some cell-surface component, such as heparan sulfate proteoglycans [14,46]. The subsequent nuclear transport of the MTP may require dissociation from this component. Another possibility for the delay in nuclear transport is the other intracellular processing pathways of MTP likely following cytoplasmic localization. As seen in Fig. 5, the increase of nuclear localization can only be considered a part of the decrease of cytoplasmic localization of MTP, suggesting other processing pathways may also be involved following membrane transduction. These other pathways, such as degradation, or reverse transduction, may need to be saturated before nuclear transport occurs. Alternatively, it is also possible that oligoarginine is not an efficient NLS, and therefore the nuclear transport is slow. To further determine the role of endocytosis and membrane transduction in nuclear transport of these oligopeptides, selective inhibition of endocytosis was tested. Since temperature is known to affect nuclear transport [47], inhibition of endocytosis with ammonium chloride rather than low temperature was investigated. The results show that nuclear transport of oligoarginine is not affected by this treatment. The lack of dependence on the acidification of the endosomes, which inhibited the endocytosis of oligoarginine (Fig. 3), is an indication that nuclear transport is mediated via transduction, and not endocytosis. The application of membrane transduction peptides in cytosolic and nuclear drug delivery is significant. Although various intracellular pathways in endocytosis have been extensively investigated, very little information is available for the possible fates following membrane transduction. The results presented here show that transduction and endocytosis are indeed two distinct processes of cellular entry, and the subsequent nuclear transport of oligoarginine occurs following membrane transduction, and not endocytosis. It has been shown previously that the ratio of internalization of the oligopeptide under investigation via transduction versus endocytosis differs with the size of the MTP and cationic residue composition [20]. In addition, it has been implicated that the ratio of transduction versus endocytosis may also be altered by the size and hydrophobicity of the cargo attached [21–24]. For example, it is likely that fluorescently labeled peptides may be internalized differently than the 125I-labeled peptides. This may be an additional reason why the results presented here are in disagreement with other studies which have measured the internalization of fluorescently labeled oligopeptides [5,12,13]. However, using the 125I-label is

more suitable to truly study the internalization properties of oligoarginine since it is likely to have much less of an effect on the internalization than larger, hydrophobic molecules. Therefore, although the results presented here indicate that endocytosis is not a prerequisite for membrane transduction of oligoarginine, crossing of the endosomal membrane may occur with other macromolecules. Since these MTPs are entering the cell via both processes concurrently, a quantitative measurement of membrane transduction without the interference from endocytosis is essential for elucidating the underlying mechanisms of this novel transport process.

Acknowledgments J.L. Zaro is a recipient of the American Foundation for Pharmaceutical Education pre-doctoral fellowship and the Charles and Charlotte Krown Fellowship.

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