HeLa Cell Nuclei Research Articles

Amphiphilic polycarbonate conjugates of doxorubicin with pH-sensitive hydrazone linker for controlled release

Novel amphiphilic polycarbonates-graft-doxorubicin (mPEG-b-P(ATMC-co-DTC)-g-DOX) were successfully designed and synthesized for pH-triggered intercellular drug release in cancer cells. The amphiphilic block copolymer, mPEG-b-P(ATMC-co-DTC), was synthesized in bulk using immobilized porcine pancreas lipase (IPPL) as the catalyst. After allyl epoxidation of ATMC units, DOX was covalently conjugated to the hydrophobic polycarbonates block through a hydrazone linkage. The resulting mPEG-b-P(ATMC-co-DTC)-g-DOX prodrugs could self-assemble to form nano-sized micelles in aqueous solution, while DOX contents in the hydrophobic core were 9.9 and 12.5wt.%. DOX release rate from the prodrug micelles increased in acidic medium due to the acid-cleavable hydrazone linkage between the DOX and polycarbonates. MTT assays demonstrated that DOX prodrug micelles in this study showed effective cytotoxic effects to HeLa cells. Furthermore, confocal laser scanning microscopy (CLSM) observations also revealed that mPEG-b-P(ATMC-co-DTC)-g-DOX prodrugs could efficiently deliver and release DOX into the nuclei of HeLa cells.

Construction of a recombinant eukaryotic vector of Importin 8 and its expression in HeLa cells

Objective To clone the coding sequence of Importin 8 (IPO8) cDNA from HeLa cells and construct a eukaryotic expression vector for its green fluorescent protein (GFP)-IPO8 fusion protein expression in HeLa cells.Methods The total RNA was extracted from HeLa cells and the full length cDNA of IPO8 was amplified using reverse transcription-polymerase chain reaction (RT-PCR),and cloned into green fluorescence protein vector phosphorylated enhanced green fluorescent protein (pEGFP)-C1 to construct recombinant expression plasmid pEGFP-IPO8.After recombinant plasmids were transfected into HeLa cells,the expression of IPO8 was observed by fluorescent microscope.Results DNA sequence analysis showed that the amplified rat IPO8 gene was in concordance with that published on Gene Bank.The results of enzyme digestion and sequence analysis demonstrated that the recombinant plasmid pEGFP-IPO8 was constructed successfully.Fluorescence microscopy revealed the fusion protein GFP-IPO8 was mainly located in the nucleus of HeLa cells.Conclusion The IPO8 gene was cloned successfully and the recombinant plasmid were constructed successfully.The fusion protein GFP-IPO8 was demonstrated to be mainly located in the nucleus of HeLa cells,which provides a basis for biological functional study of IPO8 gene. Key words: Importin 8 gene; Green fluorescent protein; Fusion protein; Eukaryotic vector

Intracellular release of doxorubicin from core-crosslinked polypeptide micelles triggered by both pH and reduction conditions

Reduction and pH dual-sensitive reversibly core-crosslinked polypeptide micelles were developed from lipoic acid (LA) and cis-1,2-cyclohexanedicarboxylic acid (CCA) decorated poly(ethylene glycol)-b-poly(l-lysine) (PEG-P(LL-CCA/LA)) block copolymers for active loading and triggered intracellular release of doxorubicin (DOX). PEG-P(LL18-CCA4/LA14) and PEG-P(LL18-CCA8/LA10) (MnPEG = 5.0 kg/mol) formed nano-sized micelles that were readily crosslinked in the presence of a catalytic amount of dithiothreitol (DTT) in phosphate buffer (pH 7.4, 10 mm). PEG-P(LL18-CCA4/LA14) micelles displayed an elevated DOX loading over PEG-P(LL14-LA14) controls likely due to presence of ionic interactions between DOX and CCA. These core-crosslinked polypeptide micelles while exhibiting high stability against extensive dilution and high salt concentration were quickly dissociated into unimers in the presence of 10 mm DTT. The in vitro release studies showed that DOX release from PEG-P(LL18-CCA4/LA14) micelles at pH 7.4 and 37 °C was significantly inhibited by crosslinking (i.e. less than 20% release in 24 h). The release of DOX was, however, doubled under endosomal pH of 5.0, possibly triggered by cleavage of the acid-labile amide bonds of CCA. In particular, rapid DOX release was observed under a reductive condition containing 10 mm glutathione (GSH), in which 86.0% and 96.7% of DOX were released in 24 h at pH 7.4 and 5.0, respectively, under otherwise the same conditions. MTT assays demonstrated that these core-crosslinked polypeptide micelles were practically non-toxic up to a tested concentration of 1.0 mg/mL, while DOX-loaded micelles caused pronounced cytotoxic effects to HeLa and HepG2 tumor cells with IC50 (inhibitory concentration to produce 50% cell death) of ca. 12.5 μg DOX equiv/mL following 48 h incubation. Confocal microscopy observations revealed that DOX-loaded crosslinked PEG-P(LL18-CCA4/LA14) micelles more efficiently delivered and released DOX into the nuclei of HeLa cells than PEG-P(LL14-LA14) counterparts. These dual-bioresponsive core-crosslinked polypeptide micelles have appeared as an advanced platform for targeted cancer therapy.

Discovery of a series of small molecules as potent histone deacetylase inhibitors

A series of small molecules were designed and synthesized based on our previous virtual screening approach, which was performed to discover potent histone deacetylase inhibitors (HDACIs) with novel structures. The derived compounds were tested by Hela cell nucleus extract for enzyme inhibition assay. Tumor cell growth inhibition assays were performed using a series of tumor cell lines. Molecule 4h has the best performance among these compounds with enzyme inhibition IC50 of 0.14 μM and tumor cell growth inhibition IC50 of 1.85 (U937), 2.02 (HL60), 2.67 (K562). Docking studies showed that multiple H-bonds and hydrophobic interactions make 4h binding to the active site of HDAC. 4h has the advantage of low molecular weight, so a variety of structural modifications can be performed in our further studies.

Degradation of initiator tRNA Met by Xrn1/2 via its accumulation in the nucleus of heat-treated HeLa cells

Stress response mechanisms that modulate the dynamics of tRNA degradation and accumulation from the cytoplasm to the nucleus have been studied in yeast, the rat hepatoma and human cells. In the current study, we investigated tRNA degradation and accumulation in HeLa cells under various forms of stress. We found that initiator tRNAMet (tRNA(iMet)) was specifically degraded under heat stress. Two exonucleases, Xrn1 and Xrn2, are involved in the degradation of tRNA(iMet) in the cytoplasm and the nucleus, respectively. In addition to degradation, we observed accumulation of tRNA(iMet) in the nucleus. We also found that the mammalian target of rapamycin (mTOR), which regulates tRNA trafficking in yeast, is partially phosphorylated at Ser2448 in the presence of rapamycin and/or during heat stress. Our results suggest phosphorylation of mTOR may correlate with accumulation of tRNA(iMet) in heat-treated HeLa cells.

A cell-penetrating peptide suppresses the hypoxia inducible factor-1 function by binding to the helix-loop-helix domain of the aryl hydrocarbon receptor nuclear translocator

The heterodimeric hypoxia inducible factor-1 (HIF-1) complex is composed of the hypoxia inducible factor-1 alpha (HIF-1α) and the aryl hydrocarbon receptor nuclear translocator (ARNT). Activation of the HIF-1 function is essential for tumor growth and metastasis. We previously showed that transfection of a plasmid containing an ARNT-interacting peptide (Ainp1) cDNA suppresses the HIF-1 signaling in Hep3B cells. Here we generated TAT fusion of the Ainp1 peptide (6His-TAT-Ainp1) to determine whether and how the Ainp1 peptide suppresses the HIF-1 function. The bacterially expressed 6His-TAT-Ainp1 was purified under denatured condition and then refolded by limited dialysis. The refolded 6His-TAT-Ainp1 interacts with the helix–loop–helix (HLH) domain of ARNT in a similar fashion as the native 6His-Ainp1. 6His-TAT-Ainp1 colocalizes with ARNT in the nucleus of HeLa and Hep3B cells after protein transduction. The transduced protein reaches the maximum intracellular levels within 2h while remains detectable up to 96h in HeLa cells. At 2μM concentration, 6His-TAT-Ainp1 is not cytotoxic in HeLa cells but suppresses the cobalt chloride-activated, hypoxia responsive enhancer-driven luciferase expression in a dose-dependent manner. In addition, it decreases the cobalt chloride-dependent induction of the HIF-1 target genes at both the message (vascular endothelial growth factor and aldolase C) and protein (carbonic anhydrase IX and glucose transporter 1) levels. The protein levels of HIF-1α and ARNT are not altered in the presence of 6His-TAT-Ainp1. In summary, we provided evidence to support that the Ainp1 peptide directly suppresses the HIF-1 function by interacting with the ARNT HLH domain, and in turn interfering with the heterodimerization of HIF-1α and ARNT.

Functional Poly(ε-caprolactone)s via Copolymerization of ε-Caprolactone and Pyridyl Disulfide-Containing Cyclic Carbonate: Controlled Synthesis and Facile Access to Reduction-Sensitive Biodegradable Graft Copolymer Micelles

Pyridyl disulfide-functionalized cyclic carbonate (PDSC) monomer was obtained in four straightforward steps from 3-methyl-3-oxetanemethanol and exploited for facile preparation of functional poly(e-caprolactone) (PCL) containing pendant pyridyl disulfide (PDS) groups via ring-opening copolymerization with e-caprolactone. The results showed that PDS-functionalized PCL polymers were prepared with controlled molecular weights and functionalities. The exchange reaction between PDS-functionalized PCL and thiolated poly(ethylene glycol) (PEG-SH) at a PEG-SH/PDS molar ratio of 2/1 afforded PCL-g-SS-PEG graft copolymers in high yields. The dynamic light scattering (DLS) analyses showed that PCL-g-SS-PEG copolymer self-assembled into micelles with a diameter of 110–120 nm and a low polydispersity (PDI) in phosphate buffer (pH 7.4, 10 mM). PCL-g-SS-PEG micelles while sufficiently stable under physiological conditions were prone to rapid shell shedding and aggregation under a reductive condition. Doxorubicin (DOX) was loaded into PCL-g-SS-PEG micelles with a decent drug loading content of 10.1 wt %. Notably, in vitro release studies revealed that ca. 82.1% DOX was released in 12 h under a reductive environment analogous to that of the intracellular compartments such as cytosol and the cell nucleus whereas only ca. 17.5% DOX was released in 24 h under nonreductive conditions. Confocal microscopy observation indicated that DOX was delivered into the nuclei of HeLa cells following 8 h incubation with DOX-loaded PCL-g-SS-PEG micelles. MTT assays in HeLa cells demonstrated that DOX-loaded PCL-g-SS-PEG micelles retained high antitumor activity with low IC50 (half-maximal inhibitory concentration) of 0.82–0.95 μg DOX equiv/mL while blank PCL-g-SS-PEG micelles were nontoxic up to a tested concentration of 1.0 mg/mL. This study presents a versatile and controlled synthesis of PDS-functionalized biodegradable polymers and reduction-sensitive biodegradable graft copolymer micelles that are of particular interest for active intracellular drug release

Human Eb Peptide: Not just a By-product of Pre-pro-IGF1b Processing?

Several physiological activities have been assigned to E-peptides derived from pre-pro-insulin-like growth factor (IGF1) processing; however, the whole range of the E-peptides' functions is still unknown. The objective of this study was to investigate human Eb peptide (hEb) in terms of its bioactivity, cellular localization, and intracellular trafficking using human cancer cells. Human Eb fused with red fluorescence protein (RFP) or green fluorescence protein (GFP) localizes strongly to nucleoli and to a lesser extent to nuclei of HeLa and U2-OS cells. Mutagenesis of hEb nucleolus localization sequence (NoLS) leads to its partial delocalization from nuclei and nucleoli to cytoplasm of transfected cells. Thus, NoLS is not sufficient for the hEb to be localized in nucleoli of the cells and a different mechanism may be involved in hEb targeting. A BrdU ELISA showed that the proliferation index of cells expressing hEb hybrid proteins increased up to 28%. For comparison, the same assay was performed using HeLa cells treated extracellularly with synthetic hEb. A significant increase in the proliferation index was observed (41-58% for concentrations ranging from 10-100 nM, respectively). Additionally, a cell migration assay was performed using stable U2-OS cell lines expressing hEb fused with RFP or RFP alone as a negative control. The migration index of hEb expressing cells was 38.3% greater. The increase in cell proliferation index and in motile properties of hEb expressing cells demonstrate that hEb is more than a pre-pro-IGF1b processing product, and has intrinsic activity of biological significance.

Redox and pH-responsive degradable micelles for dually activated intracellular anticancer drug release

Redox and pH dual-responsive biodegradable micelles were developed based on poly(ethylene glycol)-SS-poly(2,4,6-trimethoxybenzylidene-pentaerythritol carbonate) (PEG-SS-PTMBPEC) copolymer and investigated for intracellular doxorubicin (DOX) release. PEG-SS-PTMBPEC copolymer with an Mn of 5.0–4.1kg/mol formed micellar particles with an average diameter of 140nm and a low polydispersity of 0.12. DOX was loaded into PEG-SS-PTMBPEC micelles with a decent drug loading content of 11.3wt.%. The in vitro release studies showed that under physiological conditions only ca. 24.5% DOX was released from DOX-loaded micelles in 21h. The release of DOX was significantly accelerated at pH5.0 or in the presence of 10mM glutathione (GSH) at pH7.4, in which 62.8% and 74.3% of DOX was released, respectively, in 21h. The drug release was further boosted under 10mM GSH and pH 5.0 conditions, with 94.2% of DOX released in 10h. Notably, DOX release was also facilitated by 2 or 4h incubation at pH 5.0 and then at pH 7.4 with 10mM GSH, which mimics the intracellular pathways of endocytosed micellar drugs. Confocal microscopy observation indicated that DOX was delivered and released into the nuclei of HeLa cells following 8h incubation with DOX-loaded PEG-SS-PTMBPEC micelles, while DOX was mainly located in the cytoplasm for reduction-insensitive PEG-PTMBPEC controls. MTT assays revealed that DOX-loaded PEG-SS-PTMBPEC micelles had higher anti-tumor activity than reduction-insensitive controls, with low IC50 of 0.75 and 0.60μg/mL for HeLa and RAW 264.7 cells, respectively, following 48h incubation. PEG-SS-PTMBPEC micelles displayed low cytotoxicity up to a concentration of 1.0mg/mL. These redox and pH dual-bioresponsive degradable micelles have appeared as a promising platform for targeted intracellular anticancer drug release.

Димерные бисбензимидазолы: цитотоксичность и влияние на метилирование ДНК в нормальных и раковых клетках человека

Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors cause re-activation of these genes that allows considering DNA methyltransferases as targets for anticancer therapy. As it was previously shown by us, dimeric bisbenzimidazoles, DB(n), differing in length of the oligomethylene linker between the two bisbenzimidazole fragments (n--number of methylene groups in linker) effectively inhibit the methylation of DNA duplexes by murine methyltransferase Dnmt3a. Here, the cytotoxicity of some of these compounds, their penetration into cells and influence on the methylation of genomic DNA in fetal lung fibroblasts line F-977 and cervical cancer cells HeLa have been studied. In the 0-60 microM concentration range, only the DB(11) displayed a significant toxic effect on the normal cells, whereas the effect of DB(n) investigated on the cancer cells was not significant. Interestingly, the DB(1) and DB(3) to a small extent stimulate the proliferation of HeLa and F-977 cells, respectively. DB(1) and DB(3) display ability to penetrate into the nucleus of HeLa and F-977 cells and accumulate in various parts of the nuclei. DB(11) is not able to penetrate into the nuclei of these cells. The incubation of F-977 cells with 26 microM of DB(1) or DB(3) led to a decrease of the methylation of 18S rRNA gene, which is located in the region of DB(1) and DB(3) accumulation. A similar effect produces the same concentration of DB (3) in the F-977 cells. However, the overall level of genomic DNA methylation was not changed. These data suggest that DB(n) can be directed to act on specific genes demethylation and in the future may selectively inhibit the proliferation of cancer cells.

Blue two-photon fluorescence metal cluster probe precisely marking cell nuclei of two cell lines

A bifunctional peptide was designed to in situ reduce Cu ions and anchor a Cu cluster. The peptide-Cu cluster probe, mainly composed of Cu14, emitted blue two-photon fluorescence under femtosecond laser excitation. Most important, the probe can specifically mark the nuclei of HeLa and A549 cells, respectively.

Irregular Folding of Human Genome DNA in the Cells

How is 2-m-long genomic DNA organized into a mitotic chromosome or nucleus? The nucleosome fiber has long been assumed to be folded into a 30-nm chromatin fiber, and further helically folded larger fiber. However, when we observed frozen hydrated human mitotic cells using cryoelectron microscopy (cryo-EM), no higher-order structures including 30-nm chromatin fibers were found. To further investigate the bulk structure of mitotic chromosomes, we performed small-angle X-ray scattering (SAXS) at SPring-8. No structural feature larger than 11 nm was detected, even on a chromosome-diameter scale (~1 μm). We also found a similar scattering pattern in interphase nuclei of HeLa cells in the range up to ~275 nm. Our findings suggest a common structural feature in interphase and mitotic chromatins: compact and irregular folding of nucleosome fibers occurs without a 30-nm chromatin structure.

Identification of novel CDK9 and Cyclin T1-associated protein complexes (CCAPs) whose siRNA depletion enhances HIV-1 Tat function

BackgroundHIV-1 Tat activates RNA Polymerase II (RNAP II) elongation of the integrated provirus by recruiting a protein kinase known as P-TEFb to TAR RNA at the 5′ end of nascent viral transcripts. The catalytic core of P-TEFb contains CDK9 and Cyclin T1 (CCNT1). A human endogenous complexome has recently been described – the set of multi-protein complexes in HeLa cell nuclei. We mined this complexome data set and identified 12 distinct multi-protein complexes that contain both CDK9 and CCNT1. We have termed these complexes CCAPs for CDK9/CCNT1-associated protein complexes. Nine CCAPs are novel, while three were previously identified as Core P-TEFb, the 7SK snRNP, and the Super-Elongation Complex. We have investigated the role of five newly identified CCAPs in Tat function and viral gene expression.ResultsWe examined five CCAPs that contain: 1) PPP1R10/TOX3/WDR82; 2) TTF2; 3) TPR; 4) WRNIP1; 5) FBXO11/CUL1/SKP1. SiRNA depletions of protein subunits of the five CCAPs enhanced Tat activation of an integrated HIV-1 LTR-Luciferase reporter in TZM-bl cells. Using plasmid transfection assays in HeLa cells, we also found that siRNA depletions of TTF2, FBXO11, PPP1R10, WDR82, and TOX3 enhanced Tat activation of an HIV-1 LTR-luciferase reporter, but the depletions did not enhance expression of an NF-κB reporter plasmid with the exception of PPP1R10. We found no evidence that depletion of CCAPs perturbed the level of CDK9/CCNT1 in the 7SK snRNP. We also found that the combination of siRNA depletions of both TTF2 and FBXO11 sensitized a latent provirus in Jurkat cells to reactivation by sub-optimal amounts of αCD3/CD28 antibodies.ConclusionsOur results identified five novel CDK9/CCNT1 complexes that are capable of negative regulation of HIV-1 Tat function and viral gene expression. Because siRNA depletions of CCAPs enhance Tat function, it is possible that these complexes reduce the level of CDK9 and CCNT1 available for Tat, similar to the negative regulation of Tat by the 7SK snRNP. Our results highlight the complexity in the biological functions of CDK9 and CCNT1.

Labeling of Polyethylenimine with Fluorescent Dye to Image Nucleus, Nucleolus, and Chromosomes in Digitonin-Permeabilized HeLa Cells

The visualization of nuclear architecture, which changes dynamically depending on the physiological and the pathological situation, remains an important challenge. Here we report that exposure of fluoresceinisothiocyanate-labeled polyethylenimine (FITC-PEI) to digitonin-permeabilized cervical cancer HeLa cells enable rapid detection of the morphology of the nuclear rim, the nucleolus, and mitotic chromosomes. This simple detection strategy can aid in scientific investigation for both basic research and diagnostic purposes.

Reduction-Responsive Disassemblable Core-Cross-Linked Micelles Based on Poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)–Lipoic Acid Conjugates for Triggered Intracellular Anticancer Drug Release

Reduction-sensitive reversibly core-cross-linked micelles were developed based on poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)-lipoic acid (PEG-b-PHPMA-LA) conjugates and investigated for triggered doxorubicin (DOX) release. Water-soluble PEG-b-PHPMA block copolymers were obtained with M(n,PEG) of 5.0 kg/mol and M(n,HPMA) varying from 1.7 and 4.1 to 7.0 kg/mol by reversible addition-fragmentation chain transfer (RAFT) polymerization. The esterification of the hydroxyl groups in the PEG-b-PHPMA copolymers with lipoic acid (LA) gave amphiphilic PEG-b-PHPMA-LA conjugates with degrees of substitution (DS) of 71-86%, which formed monodispersed micelles with average sizes ranging from 85.3 to 142.5 nm, depending on PHPMA molecular weights, in phosphate buffer (PB, 10 mM, pH 7.4). These micelles were readily cross-linked with a catalytic amount of dithiothreitol (DTT). Notably, PEG-b-PHPMA(7.0k)-LA micelles displayed superior DOX loading content (21.3 wt %) and loading efficiency (90%). The in vitro release studies showed that only about 23.0% of DOX was released in 12 h from cross-linked micelles at 37 °C at a low micelle concentration of 40 μg/mL, whereas about 87.0% of DOX was released in the presence of 10 mM DTT under otherwise the same conditions. MTT assays showed that DOX-loaded core-cross-linked PEG-b-PHPMA-LA micelles exhibited high antitumor activity in HeLa and HepG2 cells with low IC(50) (half inhibitory concentration) of 6.7 and 12.8 μg DOX equiv/mL, respectively, following 48 h incubation, while blank micelles were practically nontoxic up to a tested concentration of 1.0 mg/mL. Confocal laser scanning microscope (CLSM) studies showed that DOX-loaded core-cross-linked micelles released DOX into the cell nuclei of HeLa cells in 12 h. These reduction-sensitive disassemblable core-cross-linked micelles with excellent biocompatibility, superior drug loading, high extracellular stability, and triggered intracellular drug release are promising for tumor-targeted anticancer drug delivery.

PH-sensitive degradable chimaeric polymersomes for the intracellular release of doxorubicin hydrochloride

pH-sensitive degradable chimaeric polymersomes were developed based on asymmetric poly(ethylene glycol)-b-poly(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate)-b-poly(acrylic acid) (PEG-PTTMA-PAA) triblock copolymers for active loading as well as triggered intracellular release of hydrophilic doxorubicin hydrochloride (DOX·HCl). PEG-PTTMA-PAA copolymers were readily prepared with Mn PAA ranging from 1.5, 2.1 to 2.7 kg/mol by sequential reversible addition-fragmentation chain transfer (RAFT) copolymerization of 2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate (TTMA) and acrylic acid (AA) using PEG-CPADN (Mn PEG = 5.0 kg/mol; CPADN: 4-cyanopentanoic acid dithionaphthalenoate) as a macro-RAFT agent. PEG-PTTMA-PAA copolymers formed mono-disperse polymersomes with average sizes of 63.9–112.1 nm, which decreased with increasing Mn PAA. The polymersomal structure was confirmed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Notably, the acetals in polymersomes while sufficiently stable at pH 7.4 were prone to rapid hydrolysis at mildly acidic pHs of 4.0 and 5.0, which resulted in swelling and eventually disassembly of polymersomes. These chimaeric polymersomes could actively load DOX·HCl resulting in remarkably high drug loading contents (up to 15.9 wt.%) and loading efficiencies (up to 88.8%). The in vitro release studies showed that DOX·HCl was released from chimaeric polymersomes in a controlled and pH-dependent manner. CLSM observations revealed that these chimaeric polymersomes could efficiently deliver and release DOX·HCl into the nuclei of HeLa cells. MTT assays in HeLa cells demonstrated that DOX·HCl-loaded PEG-PTTMA-PAA polymersomes exhibited high anti-tumor activity with IC50 (inhibitory concentration to produce 50% cell death) of 1.48–1.67 μg/mL, close to that of free DOX·HCl, while blank polymersomes were practically non-toxic up to a tested concentration of 2.0 mg/mL. These pH-sensitive degradable chimaeric polymersomes have appeared to be a promising alternative to liposomes for tumor-targeted delivery of DOX·HCl.

Aminomethylene Peptide Nucleic Acid (am-PNA): Synthesis, Regio-/Stereospecific DNA Binding, And Differential Cell Uptake of (α/γ,R/S)am-PNA Analogues

Inherently chiral, cationic am-PNAs having pendant aminomethylene groups at α(R/S) or γ(S) sites on PNA backbone have been synthesized. The modified PNAs are shown to stabilize duplexes with complementary cDNA in a regio- and stereo-preferred manner with γ(S)-am PNA superior to α(R/S)-am PNAs and α(R)-am PNA better than the α(S) isomer. The enhanced stabilization of am-PNA:DNA duplexes is accompanied by a greater discrimination of mismatched bases. This seems to be a combined result of both electrostatic interactions and conformational preorganization of backbone favoring the cDNA binding. The am-PNAs are demonstrated to effectively traverse the cell membrane, localize in the nucleus of HeLa cells, and exhibit low toxicity to cells.

HIV-2 viral protein X (Vpx) ubiquitination is dispensable for ubiquitin ligase interaction and effects on macrophage infection

HIV-2 Vpx, a virus-associated accessory protein, is critical for infection of non-dividing myeloid cells. To understand the function of Vpx ubiquitination, interaction with an E3 ubiquitin ligase complex, and ability to overcome an inhibition of reverse transcription, we analyzed Vpx lysine mutants for their function and replication capability in macrophages. Both Wt Vpx and Vpx TA (lysine-less Vpx) localized to the cytoplasm and nucleus in HeLa cells. All HIV-2 Vpx lysine mutants were functional in virion packaging. However, ubiquitination was absent with Vpx TA and Vpx K84A mutants, indicating a lack of ubiquitin on positions K68 and K77. Mutants Vpx K68A and K77A were unable to infect macrophages due to impaired reverse transcription from loss of interaction with the ubiquitin substrate receptor, DCAF1. Even though Vpx K84A lacked ubiquitination, it bound DCAF1, and infected macrophages comparable to Wt Vpx.

Fabrication and characterization of nuclear localization signal-conjugated glycol chitosan micelles for improving the nuclear delivery of doxorubicin

BackgroundSupramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS)-modified polymeric micelles were studied with the aim of improving nuclear drug delivery.MethodsIn this research, cholesterol-modified glycol chitosan (CHGC) was synthesized. NLS-conjugated CHGC (NCHGC) was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa and HepG2 cells.ResultsThe degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC) micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater cellular uptake and higher amounts of DOX in the nuclei of HeLa cells than free DOX and DOX-loaded CHGC (DCHGC) micelles. The half maximal inhibitory concentration (IC50) values of free DOX, DCHGC, and DNCHGC micelles against HepG2 cells were 4.063, 0.591, and 0.171 μg/mL, respectively. Moreover, the IC50 values of free DOX (3.210 μg/mL) and the DCHGC micelles (1.413 μg/mL) against HeLa cells were nearly 6.96- and 3.07-fold (P < 0.01), respectively, higher than the IC50 value of the DNCHGC micelles (0.461 μg/mL).ConclusionThe results of this study suggest that novel NCHGC micelles could be a potential carrier for nucleus-targeting delivery.

Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion

There is little quantitative information regarding how much splicing occurs co-transcriptionally in higher eukaryotes, and it remains unclear where precisely splicing occurs in the nucleus. Here we determine the global extent of co- and post-transcriptional splicing in mammalian cells, and their respective subnuclear locations, using antibodies that specifically recognize phosphorylated SF3b155 (P-SF3b155) found only in catalytically activated/active spliceosomes. Quantification of chromatin- and nucleoplasm-associated P-SF3b155 after fractionation of HeLa cell nuclei, reveals that ~80% of pre-mRNA splicing occurs co-transcriptionally. Active spliceosomes localize in situ to regions of decompacted chromatin, at the periphery of or within nuclear speckles. Immunofluorescence microscopy with anti-P-SF3b155 antibodies, coupled with transcription inhibition and a block in splicing after SF3b155 phosphorylation, indicates that post-transcriptional splicing occurs in nuclear speckles and that release of post-transcriptionally spliced mRNA from speckles is coupled to the nuclear mRNA export pathway. Our data provide new insights into when and where splicing occurs in cells.