Adenoviruses synergize with nuclear localization signals to enhance nuclear delivery and photodynamic action of internalizable conjugates containing chlorin e6

Abstract

Photosensitizers, molecules that produce active oxygen species upon activation by visible light, are currently being used in photodynamic therapy (PDT) to treat cancer and other conditions, where limitations include normal cells and tissue damage and associated side effects, and the fact that cytotoxic effects are largely restricted to the plasma and other peripheral membranes. In this study, we used insulincontaining conjugates to which variants of the simian-virus-SV40 large-tumor antigen (T-ag) nuclear localization signal (NLS) were linked in order to target the photosensitizer chlorin e 6 to the nucleus. NLSs were included either as peptides coupled co-valently to the carrier bovine serum albumin, or within the coding sequence of ␤-galactosidase fusion proteins. The most potent photosensitizing conjugate was the NLS-containing T-ag ␤-galactosidase fusion protein (P10)-(chlorin e 6 )-insulin, exhibiting an EC 50 more than 2400-fold lower than the value for free chlorin e 6 , and more than 15-fold lower than that of an NLS-deficient ␤-galactosidase-(chlorin e 6 )-insulin construct, thus demonstrating that NLSs can increase the photosensitizing activity of chlorin e 6 . Attenuated adenoviruses were used to increase the nuclear delivery of conjugates through its endosomal-membrane-disrupting activity. In the case of the NLS-containing P10-conjugate, co-incubation with adenovirus increased the proportion of cells whose nuclear photosensitizing activity was higher than that in the cytoplasm by 2.5-fold. This use of adenoviruses in conjunction with photosensitizers has clear implications for achieving efficient cell-type-specific PDT. Int. J. Cancer 81:734-740, 1999.1999 Wiley-Liss, Inc.

Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery

“…35,36 Specific recognition sequences for the DNAbinding protein can be included within the plasmid to be expressed in the target cell, to enable it to be recognized by the protein and thus be cotransported into the target cells. GAL4 is particularly interesting in this respect since its DNA binding domain, which recognizes a specific 17 bp sequence, 26 appears to include or overlap with an efficient NLS.…”

Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency

“…8,9 Conjugates were constructed using either BSA, β-galactosidase or the T-ag-β-galactosidase fusion protein P10 as carriers to which chlorin e6, insulin and, in the case of the BSA-containing conjugates, NLS-containing peptides were successively linked. The various constructs are listed schematically, together with the sequences of the derivatives of T-ag NLS used, in Table 1.…”

“…We first compared the photodynamic activity of constructs carrying or lacking the minimal T-ag NLS (amino acids 126-132), 8,9 with or without the amino-terminal flanking sequence of T-ag (amino acids 111-125, see Table 1), which has been shown to enhance T-ag nuclear transport (see Jans and Huebner, 91 Jans et al 114,118 ). The CK2 phosphorylation site (Ser 112 ) within this flanking region increases the rate of T-ag nuclear import by approximately 50-fold, whereas phosphorylation by the cyclin-dependent kinase cdc2 at another site (Thr 124 ) reduces the maximal level of transport.…”

“…[3][4][5] That uneven intracellular distribution of PS can lead to differences in toxicity has been shown using laser microbeam irradiation. 6 In contrast to cell membranes and other cytoplasmic organelles, the cell nucleus [7][8][9] is known to be a very sensitive target for reactive oxygen species. In order to reduce the dose of PS administered to patients and hence minimize the harmful side-effects of PDT, new approaches have been devised to increase the effectiveness of tumour-cell killing through targeted delivery of PS to hypersensitive subcellular sites.…”

“…69 2′,7′-Dichlorofluorescin diacetate penetrates into living cells and is deacetylated by intracellular esterases; because reactive oxygen species react with 2′,7′-dichlorofluorescin to yield fluorescent 2′,7′-dichlorofluorescein, this can be used to visualize the sites of intracellular production of reactive oxygen species and sites of PS subcellular location, as well as being an indicator of intracellular photodynamic activity in situ. 8,9,69,86 The insulin-BSA-chlorin e6 conjugate at low concentrations suppresses the propagation of hepatoma cells (half-maximal effect at about 1 nmol/L). Other ligands that have been used in internalizable PS conjugates include transferrin, 87 epidermal growth factor 88 and maleylated BSA, a ligand for 'scavenger' receptors.…”