Kristine Mann

Listeriolysin O enhances cytoplasmic delivery by Her-2 targeting liposomes

To enhance cytoplasmic delivery of liposomal contents to breast cancer cells, the authors have attached the pore-forming protein, listeriolysin O (LLO), to thermosensitive liposomes. The antibody trastuzumab (Herceptin®) was also conjugated with the outer surface of the liposomes, resulting in highly specific binding and internalization into mammary epithelial cells that overexpress the human epidermal growth factor receptor 2 (Her-2). The liposomes were preloaded with a marker fluorescent dye, and the effect of LLO on the distribution of dye within the cells was monitored using fluorescence microscopy. Owing to the thermosensitive nature of the liposomes, hyperthermia at 42°C triggered the release of the encapsulated fluorescent calcein from the endocytosed liposomes into the interior of the endosomes. LLO, when conjugated to these liposomes, subsequently formed pores in the endosomal membrane, allowing calcein to flow out of the endosomal compartment into the cytoplasm. Her-2–targeted liposomes bearing LLO delivered a 22-fold greater concentration of calcein to mammary epithelial cells that overexpress Her-2 compared to cells with normal Her-2 expression. Thus, the addition of LLO to preformed liposomes offers a method for significantly enhancing delivery of liposomal contents to the cytoplasm of targeted cells.

Targeting Her-2+ Breast Cancer Cells with Bleomycin Immunoliposomes Linked to LLO

Bleomycin is a membrane impermeable chemotherapeutic agent that is relatively innocuous extracellularly but highly cytotoxic when delivered directly to the cytoplasm. We report on the development of a liposome delivery system that targets Her-2 overexpressing breast cancer cells and breaches the endosomal barrier, delivering bleomycin to the cytoplasm. The liposomes are conjugated to the antibody trastuzumab, which results in specific binding and internalization of liposomes into Her-2 overexpressing cells. In addition, the liposomes are disulfide bonded to a pore-forming protein listeriolysin O (LLO) which forms pores in the endosome and allows the liposomal cargo to pass into the cytoplasm. We demonstrate specific delivery to Her-2 positive MCF-7/Her18 cells relative to Her-2 negative MCF-7 cells using a fluorescent probe calcein within the immunoliposomes. When calcein is replaced by bleomycin, the liposomes effectively reduce viability of five different Her-2 overexpressing cell lines (BT-474, SKBR-3, MCF-7/Her18, HCC-1954 and MDA-453) while harming to a much lesser extent Her-2 negative breast cell lines (MCF-7, MCF-12a and MCF-10a). The liposomes also affect trastuzumab-resistant cells, reducing MDA-453 cell number by 97% compared to untreated cells. Importantly, the concentration of drug needed to reduce tumor cell growth and viability using this liposome therapy is approximately 57,000-fold less than the concentration needed if drug is delivered extracellularly, raising the possibility of increased therapeutic specificity with decreased side effects.

Glycosylation of simian virus 40 T antigen and localization of glycosylated T antigen in in the nuclear matrix

Evidence has been obtained for the glycosylation of simian virus 40 (SV40) T antigen in SV40-infected TC7 cells. Both [3H]mannose and [3H]glucosamine are incorporated into T antigen in cells grown and labeled in medium containing fructose instead of glucose. In addition, T antigen is visualized by a carbohydrate stain specific for mannose and/or glucose residues. Finally, lectin binding studies suggest that T antigen contains galactose and/or galactosamine, since T antigen is specifically eluted from soybean lectin by 0.2 M galactose. When gel-purified, [3H]glucosamine-labeled T antigen is subjected to tryptic peptide mapping, label is found in only one peptide, thought to correspond to the methionine-containing peptide extending from Asn-653 to Arg-691, near the carboxy-terminal end of T antigen. Insensitivity to tunicamycin and the localization of the glycosylation site in the carboxy-terminus of T antigen, and not at Asn-153, suggest that T antigen is not N-glycosylated. Cell fractionation studies show that [3H]glucosamine-labeled T antigen is preferentially associated with the nuclear matrix of SV40-infected TC7 cells.

Complement C3 mediated targeting of liposomes to granulocytic myeloid derived suppressor cells.

UNLABELLED In cancer patients, granulocytic myeloid derived suppressor cells (G-MDSCs) expand in number, infiltrating tumor and lymphatic tissues where they suppress an anti-tumor immune response. We report here the development of a liposomal drug delivery system that selectively targets G-MDSCs. The liposomes form a disulfide bond with activated complement C3 after intravenous injection and are taken up by G-MDSCs, which express the receptor for activated C3. In vitro experiments utilizing serum from a C3 knockout mouse demonstrate that G-MDSCs take up these liposomes in a C3-dependent manner. After systemic administration to tumor bearing mice, liposomes were incorporated by 22% of G-MDSCs in the blood and were also present in a percentage of G-MDSCs in the tumor (11%), spleen (22%), liver (35%) and lungs (26%). This liposomal system offers a versatile means of targeted drug delivery to G-MDSCs and could be an important tool for restoring anti-tumor immunity in cancer patients. FROM THE CLINICAL EDITOR It has been shown that the presence of granulocytic myeloid derived suppressor cells (G-MDSCs) in cancer patients suppress the tumor immune response of T cells. Many drugs can be used to reverse this process. In this article, the authors describe the development of a liposomal drug delivery system for targeted drug delivery to G- MDSCs. This system may prove to be useful adjunct in immunotherapy in the fight against cancers.

Association of topoisomerases I and II with the chromatin in SV40-infected monkey cells.

The subcellular localization of topoisomerase I and topoisomerase II has been compared in Simian virus (SV40)-infected and uninfected TC7 monkey cells. In SV40-infected cells, both of these enzymes are preferentially associated with the chromatin. Some topoisomerase I is associated with the nuclear matrix, whereas topoisomerase II shows no such association. In uninfected TC7 cells, topoisomerase I is present in both the chromatin and nuclear matrix fractions. Topoisomerase II, on the other hand, is not detected in any of the subcellular fractions of uninfected cells. After SV40 infection, there is a marked increase in the level of chromatin-associated topoisomerase II.

Phosphorylation of SV40 large T antigen in SV40 nucleoprotein complexes

Abstract The SV40 large T antigen associated with SV40 nucleoprotein complexes containing SV40 (I) DNA is phosphorylated. Tryptic peptide analysis reveals two major phosphopeptides in nucleoprotein complex large T antigen similar to those found in unbound SV40 large T antigen, one peptide containing mainly phosphoserine and the other containing mainly phosphothreonine. In addition to the two major phosphopeptides, unbound SV40 large T antigen contains a number of minor phosphopeptides. There is a minor phosphopeptide present in nucleoprotein complex large T antigen which is not apparent in the unbound large T antigen.

Complement C3-dependent uptake of targeted liposomes into human macrophages, B cells, dendritic cells, neutrophils, and MDSCs

Antitumor immunity in cancer patients is heavily modulated by cells of the innate immune system. Antigen-presenting cells, including dendritic cells, macrophages, and B cells, initiate immune recognition of tumor antigen by displaying antigen to effector cells. Countering this immune stimulation are immunosuppressive cells which include M2 macrophages, N2 neutrophils, and myeloid-derived suppressor cells (MDSCs). To create effective cancer immunotherapies, it is critical that we can target these important cell types of the immune system with immunostimulatory compounds. A commonality of these cell types is the complement receptor, which recognizes pathogens that are bound to activated complement C3 in human blood. To target the complement receptor, we have created a liposome that has a small molecule, orthopyridyl disulfide (OPSS), conjugated to its surface. OPSS forms a disulfide bond with activated complement C3, which then targets liposomes for uptake by dendritic cells, macrophages, B cells, MDSCs, and neutrophils in human blood. Internalization is efficient and specific to cells that display the complement receptor. Liposomes are a versatile drug delivery device. Possible applications for this system include delivery of toll-receptor agonists or tumor antigen to antigen-presenting cells and delivery of immunostimulatory drugs to M2, N2, and MDSC immunosuppressive cells.

Characterization of SV40 T antigen associated with the nuclear matrix.

Simian virus 40 (SV40) T antigen associated with the nuclear matrix of SV40-infected TC7 cells has been characterized. Pulse-chase studies on the turnover of T antigen in the different subcellular fractions show that T antigen turns over most rapidly in its association with the purified SV40 nucleoprotein complexes (NPCs) and undergoes a slower rate of turnover in its association with the nuclear matrix. In contrast, turnover of SV40 T antigen in its association with the other subcellular fractions is not detected during the same period of time. Tryptic peptide maps establish that NPC-associated T antigen and nuclear matrix-associated T antigen are chemically related, in that they have two additional methionine-containing peptides that are not found in the majority of T antigen molecules. The association of T antigen with the nuclear matrix is independent of SV40 DNA replication since T antigen is still present in the nuclear matrix after a 1-hr shift-up of tsA58-infected cells to the nonpermissive temperature. In addition, T antigen is associated with the nuclear matrices of both C6 and Cos7 transformed cells, indicating that the association of T antigen with the nuclear matrix is independent of its ability to initiate and support SV40 DNA replication.

A comparison of methods for the extraction of nucleoprotein complexes from SV40-infected cells

A comparison was made between two alternative methods for the nuclear extraction of Simian virus 40 (SV40) virions and nucleoprotein complexes (NPCs) from SV40-infected TC7 cells. The low-salt hypotonic method of Su and DePamphilis (1976) was compared with the detergent method of Garber et al. (1978), since other methods had been shown to result in virion breakdown. There was no disruption of mature SV40 virions with either of these extraction procedures. There was, however, considerably more effective extraction of SV40 NPCs, known to contain large tumor (T) antigen, using the low-salt hypotonic method as opposed to the detergent method. Thus, the low-salt hypotonic method for extraction should be the method of choice when studying SV40 DNA replication or the function of SV40 T antigen in SV40 nucleoprotein complexes.

Methods for the Study of Redox-Mediated Changes in p53 Structure and Function

It is now generally accepted that both the structure and function of a number of specific transcriptional factors, including p53, are subject to redox regulation in cells in which these factors are expressed. The present chapter describes methods for the analysis of redox changes in the structure of p53 and the effect of redox modulation on binding of p53 to a DNA consensus sequence. In addition, methods are described for studying the effect of redox perturbations of cells on the functioning of p53 in the cell cycle and in apoptosis. By studying the effect ofredox agents on p53, we have concluded that p53 is subject to structural redox modulation and that this modulation affects the functional ability of the protein to bind to DNA, to cause cell cycle arrest, and to trigger apoptosis.