Factors influencing the nuclear targeting ability of nuclear localization signals.

A nuclear localization signal (NLS) is a short amino acid sequence derived from eukaryotic nuclear proteins and viral proteins. Many NLS peptides can efficiently mediate the intranucleus transport of cargo molecules, so they have been widely used for non-viral gene transfer and shown potential ability to improve nuclear delivery of DNA. In order to maximally utilize NLS peptides to enhance gene transfer, several factors such as methods of incorporating NLS peptide, type and property of NLS peptide, number of NLS peptide, and spacer between NLS peptide and DNA should be considered. This review article summarizes how these factors influence the ability of NLS peptides in enhancing non-viral gene delivery and aids in defining the requirements for successful NLS-enhanced transfection.

Antitumor effects and normal-tissue toxicity of 111In-nuclear localization sequence-trastuzumab in athymic mice bearing HER-positive human breast cancer xenografts.

UNLABELLED: (111)In-nuclear localization sequence-trastuzumab is a radioimmunotherapeutic agent consisting of trastuzumab modified with NLS peptides (CGYGPKKKRKVGG) and labeled with the Auger electron emitter (111)In. Our objectives were to evaluate the tumor growth-inhibitory properties and normal-tissue toxicity of (111)In-NLS-trastuzumab in mice after intraperitoneal administration. METHODS: The pharmacokinetics of (111)In-NLS-trastuzumab after intravenous (tail vein) or intraperitoneal injection in BALB/c mice were compared. Normal-tissue toxicity was determined in BALB/c mice at 2 wk after intraperitoneal injection of 3.7-18.5 MBq (4 mg/kg) of (111)In-NLS-trastuzumab by monitoring body weight, histopathologic examination of tissues, and hematology (white blood cell, platelet, red blood cell, and hemoglobin) or clinical biochemistry (alanine transaminase and creatinine) parameters. A no-observable-adverse-effect-level (NOAEL) dose was defined. Athymic mice bearing subcutaneous MDA-MB-361 or MDA-MB-231 human breast cancer xenografts (5.0 x 10(5) or 0.5 x 10(5) HER2/cell, respectively) were treated with a single NOAEL dose or 2 doses administered intraperitoneally and separated by 2 wk. Control groups were administered (111)In-trastuzumab, trastuzumab, nonspecific (111)In-NLS-human IgG (hIgG), or normal saline. RESULTS: The bioavailability of (111)In-NLS-trastuzumab after intraperitoneal injection was 0.7. The NOAEL dose was 9.25 MBq (4 mg/kg); doses greater than or equal to 18.5 MBq decreased white blood cell or platelet counts, and doses of 27.7 MBq decreased red blood cell counts. There was no increase in alanine transaminase or creatinine at any doses tested. There were no morphologic changes to the liver, kidneys, heart, or spleen or loss of body weight. A single dose of (111)In-NLS-trastuzumab (9.25 MBq)-compared with mice receiving (111)In-trastuzumab, trastuzumab, (111)In-NLS-hIgG, or normal saline-significantly slowed the rate of growth of MDA-MB-361 tumors over 60 d (0.014 d(-1) vs. 0.033 d(-1), 0.046 d(-1), 0.030 d(-1), and 0.061 d(-1), respectively; P < 0.05). (111)In-NLS-trastuzumab had no effect on the growth of MDA-MB-231 tumors. Two doses of (111)In-NLS-trastuzumab (9.25 MBq; 4 mg/kg) separated by 2 wk increased the survival of mice with MDA-MB-361 tumors, compared with mice treated with trastuzumab or normal saline (>140 d vs. 96 and 84 d, respectively; P < 0.001 or 0.027, respectively). CONCLUSION: (111)In-NLS-trastuzumab is a promising radioimmunotherapeutic agent that could be effective for treatment of HER2-overexpressing breast cancer in humans.

Lack of mutagenicity and clastogenicity of PNAEmu-NLS targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma.

Peptide nucleic acids (PNAs) are synthetic homolog of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. They bind complementary polynucleotide sequences with higher affinity and specificity than their natural counterparts. PNAs linked to the appropriate nuclear localization signal (NLS) peptide have been used to selectively down-regulate the expression of several genes in viable cells. For example in Burkitt's lymphoma (BL) cells the c-myc oncogene is translocated in proximity to the Emu enhancer of the Ig gene locus and upregulated. PNAs complementary to the second exon of c-myc or to the Emu enhancer sequence (PNAEmu-NLS), selectively and specifically block the expression of the c-myc oncogene and inhibit cell growth in vitro and in vivo. PNAEmu-NLS administration to mice did not exhibit toxic effects even at the highest concentration allowed by the experimental conditions. Because of the accumulating data confirming PNAEmu-NLS potential therapeutic value, PNAEmu-NLS was evaluated for the inability to induce mutations in tester strains of Salmonella typhimurium, Escherichia coli, and at the hprt locus in Chinese hamster ovary cells (CHO). Moreover, the induction of chromosomal aberrations in CHO cells and of micronuclei in human lymphocytes were investigated. We may conclude that PNAEmu-NLS neither induces mutations nor has clastogenic effects as detectable by treatment under the standard test conditions.

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity.

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.