|
Drazen Raucher, Ph.D.
Telephone: (601) 984-1510
Graduated in 1995 from the Institute of Molecular Biophysics, Florida State University
Postdoctoral Studies (1995-1999), Dept. of Cell Biology, Duke University Medical Center
Assistant Research Professor (1999-2000), Dept. of Cell Biology, Duke University Medical Center
Assistant Research Professor (2001-2002), Dept. of Biomedical Engineering, Duke University
Assistant Professor (2002-Present), Dept. of Biochemistry, UMMC
RESEARCH INTERESTS
Membrane-cytoskeleton adhesion is of fundamental importance in the regulation of variety of cell functions such as formation of membrane processes, cell movement, signaling and endocytosis. Understanding the mechanism that regulates the membrane-cytoskeletal adhesion will enable us to gain new insights in molecular events that stimulate and modulate variety of cell functions. We have been studying the role of second messengers in the molecular mechanism of membrane-cytoskeleton adhesion using combination of video and fluorescence microscopy and molecular biology. This approach is used to examine the role of second messengers in the mediation and/or modulation of the cellular functions in normal and cancer cells. The plasma membrane of cells conforms to the shape of the cortical cytoskeleton, suggesting that significant adhesive interactions exist between the plasma membrane and the cytoskeleton. The regulation of this cytoskeleton-membrane adhesion energy is of fundamental importance not only for the overall cell shape but also in the regulation of a variety of cell functions. The overall interaction appears to be complex since many cytoskeletal proteins have been identified that bind to integral membrane proteins as well as to membrane phospholipids. Recent developments have made it possible to measure dynamic interaction between different cytoskeleton and plasma membrane components by separating the plasma membrane from the underlying cytoskeleton using optical tweezers. Tethers are formed by pulling on membrane-attached beads with laser tweezers and the displacement of the beads in the laser tweezers gives a rapid readout of the force on the beads, which reflects membrane-cytoskeleton adhesion.
The addition of amphiphilic organic molecules to cells often produces dramatic changes in cell functions. Some changes are due to specific effects of those molecules as enzyme inhibitors or substrates but there are other changes that occur with many amphiphilic compounds with very different molecular structures. Those changes may be due to the more general effects of the amphiphilic compounds on hydrophobic-hydrophilic interfaces in the cell such as at membrane surfaces. A class of compounds that has been characterized includes the local anesthetics, but the mechanism is poorly understood. We have also examined what are the requirements for local anesthetics that decrease tension and enhance endocytosis. We have found that membrane-impermeable local anesthetics which intercalate mainly into the lipid in the exterior half of the bilayer and expand that layer relative to the cytoplasmic half, have little or no effect on cytoskeleton-membrane adhesion and consequently on the endocytosis rate. However, thei r membrane-permeable analogs, which expand the cytoplasmic half caused a dramatic decrease in cytoskeleton-membrane adhesion, as well as an increase in the endocytosis rate and detachment of the plasma membrane from the underlying cytoskeleton. These findings indicate that enzymes that regulate PIP2 concentration and act at the membrane-cytoplasm interface are particularly sensitive to the effects of amphiphilic compounds. These properties of local anesthetics will give us not only insight in phophatydilinositol signalling mechanism and cell function but they are also applicable in new therapies for cancer. One of the major characteristics of cancer cells is rapid growth and frequent cell division. Since cell division is accompanied by a dramatic inhibition of endocytosis, there is a possibility that local anesthetics which reduce membrane-cytoskeleton adhesion may increase susceptibility of dividing cells to antitumor drugs by increasing their rate of endocytosis. This hypothesis is confirmed in my preliminary experiments where I have shown that a combination of anti-tumor drugs taxol or navelbine with various membrane-cytoskeleton adhesion reducing reagents increased drug uptake and decreased cell proliferation in various cancer cell lines. If these compounds and antineoplastic combinations do not cause an increase of the toxicity to normal cell populations, then standard doses of antineoplastic agents could be delivered with membrane-cytoskeleton adhesion reducing reagents, resulting in marked potentiation of cytotoxicity to the tumor with no increase in side effects. Alternatively, a reduced dose of antineoplastic agents could be delivered in combination with membrane--cytoskeleton adhesion reducing reagents to produce an equivalent tumor cell kill as a full dose, but with fewer systemic side effects. Further studies are required to elucidate whether therapeutic administration of biologically compatible membrane-cytoskeleton adhesion reducing reagents in combination with antitumor drugs at lower concentrations may effectively inhibit tumor growth.
Surgical resection, followed by chemo- and/or radiotherapy are the most common therapeutic modalities indicated for localized tumors. Since, these modalities fail to control local disease in a large number of cases, it is necessary to consider alternative novel therapies. Antisense oligodeoxynucleotides (ODN), targeted against several oncogene mRNA, have been shown to inhibit cell proliferation in many malignant cells, making them attractive candidates for cancer therapy. However, the limitations of currently available ODN therapies, particularly for the treatment of diseases localized in a specific organ or tissue, have motivated the consideration of alternative methods of ODN delivery to increase their specificity and decrease toxicity. Motivated by the limitations of current therapeutic approaches for localized tumors, the objective of the proposed research is to develop a targeted approach using thermally responsive polypeptides to specifically deliver antisense ODNs to a tumor site and induce apoptosis of cancer cells. The thermally responsive polypeptide derived from the elastin repeat is currently synthesized using standard molecular biology techniques . These elastin-like polypeptides (ELPs), composed of a VPGXG repeat (the “guest residue”, X, any amino acid), undergo a reversible phase transition in aqueous solution. They are hydrophilic and soluble below their transition temperature (Tt), but become hydrophobic and aggregate when the temperature is raised above their Tt. The underlying hypothesis is that intravenously delivered ELP polymers carrying ODN (ELP-ODN) are likely to be rapidly cleared under physiological conditions (T< Tt) and aggregate of ELP-ODN at sites where T>Tt, which should allow preferential accumulation of ELP-ODN only at the targeted diseased sites. In our preliminary study we demonstrated that cellular uptake of a thermally responsive ELP is significantly enhanced by the thermally triggered phase transition of the polypeptide. Therefore, greater amounts of ODN will enter the cell, which will improve ability to inhibit gene expression of targeted protein.
Recent Publications
Bidwell, G. L., 3rd and Raucher, D. Application of thermally responsive polypeptides directed against c-Myc transcriptional function for cancer therapy. Mol. Cancer Ther., 4: 1076-1085, 2005.
Massodi, I., Bidwell, G. L., 3rd, and Raucher, D. Evaluation of cell penetrating peptides fused to elastin-like polypeptide for drug delivery. J. Control Release, Sep 9; [Epub ahead of print] 2005.
Bidwell, G. L., 3rd and Raucher, D. Enhancing the Antiproliferative Effect of Topoisomerase II Inhibitors Using a Polypeptide Inhibitor of c-Myc. Biochem. Pharm., in press, 2005.
Raucher D., and A. Chilkoti, Enhanced uptake of thermally responsive polypeptide drug carrier by tumor cells in response to its hyperthermia mediated phase transition. Cancer Res. 61: 7163-7177, 2001. [link to PDF]
Raucher D., and M. P. Sheetz Local anesthetics cause decrease in membrane-cytoskeleton adhesion and PIP2 levels. J. Cell Sci. 114:3759-3766, 2001. [link to PDF]
Raucher D., Stauffer, T, Chen, W., Shen, K., Guo S.,York, J. D.,Sheetz, M. P., and Meyer, T. Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell 100:221-228, 2000. [link to PDF]
Raucher D., and M. P. Sheetz Important role of membrane tension in lamellipodial extension. J. Cell. Biol. 148(1):127-136, 2000. [link to PDF]
Raucher D., and M. P. Sheetz Membrane tension regulates endocytosis in mitosis. J.Cell Biol. 144(3):497-506, 1999. [link to PDF]
Raucher D., and M. P. Sheetz. Characteristics of a membrane reservoir studied by membrane tethers extracted by a laser tweezers trap. Biophys. J. 77(4):1992-2002, 1999. [link to PDF]
Hochmuth R.M, Raucher, D., Ting-Beall, H. P.and M.P. Sheetz Membrane-cytoskeleton adhesion and detachment from cells with “excess” area. Proceedings of the First Joint BMES/EMBS Conference Vol 1: 47, 1999.
|
Membrane-cytoskeleton adhesion is of fundamental importance in the regulation of variety of cell functions such as formation of membrane processes, cell movement, signaling and endocytosis. Understanding the mechanism that regulates the membrane-cytoskeletal adhesion will enable us to gain new insights in molecular events that stimulate and modulate variety of cell functions. We have been studying the role of second messengers in the molecular mechanism of membrane-cytoskeleton adhesion using combination of video and fluorescence microscopy and molecular biology. This approach is used to examine the role of second messengers in the mediation and/or modulation of the cellular functions in normal and cancer cells. The plasma membrane of cells conforms to the shape of the cortical cytoskeleton, suggesting that significant adhesive interactions exist between the plasma membrane and the cytoskeleton. The regulation of this cytoskeleton-membrane adhesion energy is of fundamental importance not only for the overall cell shape but also in the regulation of a variety of cell functions. The overall interaction appears to be complex since many cytoskeletal proteins have been identified that bind to integral membrane proteins as well as to membrane phospholipids. Recent developments have made it possible to measure dynamic interaction between different cytoskeleton and plasma membrane components by separating the plasma membrane from the underlying cytoskeleton using optical tweezers. Tethers are formed by pulling on membrane-attached beads with laser tweezers and the displacement of the beads in the laser tweezers gives a rapid readout of the force on the beads, which reflects membrane-cytoskeleton adhesion.
The addition of amphiphilic organic molecules to cells often produces dramatic changes in cell functions. Some changes are due to specific effects of those molecules as enzyme inhibitors or substrates but there are other changes that occur with many amphiphilic compounds with very different molecular structures. Those changes may be due to the more general effects of the amphiphilic compounds on hydrophobic-hydrophilic interfaces in the cell such as at membrane surfaces. A class of compounds that has been characterized includes the local anesthetics, but the mechanism is poorly understood. We have also examined what are the requirements for local anesthetics that decrease tension and enhance endocytosis. We have found that membrane-impermeable local anesthetics which intercalate mainly into the lipid in the exterior half of the bilayer and expand that layer relative to the cytoplasmic half, have little or no effect on cytoskeleton-membrane adhesion and consequently on the endocytosis rate. However, thei r membrane-permeable analogs, which expand the cytoplasmic half caused a dramatic decrease in cytoskeleton-membrane adhesion, as well as an increase in the endocytosis rate and detachment of the plasma membrane from the underlying cytoskeleton. These findings indicate that enzymes that regulate PIP2 concentration and act at the membrane-cytoplasm interface are particularly sensitive to the effects of amphiphilic compounds. These properties of local anesthetics will give us not only insight in phophatydilinositol signalling mechanism and cell function but they are also applicable in new therapies for cancer. One of the major characteristics of cancer cells is rapid growth and frequent cell division. Since cell division is accompanied by a dramatic inhibition of endocytosis, there is a possibility that local anesthetics which reduce membrane-cytoskeleton adhesion may increase susceptibility of dividing cells to antitumor drugs by increasing their rate of endocytosis. This hypothesis is confirmed in my preliminary experiments where I have shown that a combination of anti-tumor drugs taxol or navelbine with various membrane-cytoskeleton adhesion reducing reagents increased drug uptake and decreased cell proliferation in various cancer cell lines. If these compounds and antineoplastic combinations do not cause an increase of the toxicity to normal cell populations, then standard doses of antineoplastic agents could be delivered with membrane-cytoskeleton adhesion reducing reagents, resulting in marked potentiation of cytotoxicity to the tumor with no increase in side effects. Alternatively, a reduced dose of antineoplastic agents could be delivered in combination with membrane--cytoskeleton adhesion reducing reagents to produce an equivalent tumor cell kill as a full dose, but with fewer systemic side effects. Further studies are required to elucidate whether therapeutic administration of biologically compatible membrane-cytoskeleton adhesion reducing reagents in combination with antitumor drugs at lower concentrations may effectively inhibit tumor growth.
Targeted delivery of antisense oligonucleotides to tumors by genetically engineered thermally responsive polymers
/A> Surgical resection, followed by chemo- and/or radiotherapy are the most common therapeutic modalities indicated for localized tumors. Since, these modalities fail to control local disease in a large number of cases, it is necessary to consider alternative novel therapies. Antisense oligodeoxynucleotides (ODN), targeted against several oncogene mRNA, have been shown to inhibit cell proliferation in many malignant cells, making them attractive candidates for cancer therapy. However, the limitations of currently available ODN therapies, particularly for the treatment of diseases localized in a specific organ or tissue, have motivated the consideration of alternative methods of ODN delivery to increase their specificity and decrease toxicity. Motivated by the limitations of current therapeutic approaches for localized tumors, the objective of the proposed research is to develop a targeted approach using thermally responsive polypeptides to specifically deliver antisense ODNs to a tumor site and induce apoptosis of cancer cells. The thermally responsive polypeptide derived from the elastin repeat is currently synthesized using standard molecular biology techniques . These elastin-like polypeptides (ELPs), composed of a VPGXG repeat (the “guest residue”, X, any amino acid), undergo a reversible phase transition in aqueous solution. They are hydrophilic and soluble below their transition temperature (Tt), but become hydrophobic and aggregate when the temperature is raised above their Tt. The underlying hypothesis is that intravenously delivered ELP polymers carrying ODN (ELP-ODN) are likely to be rapidly cleared under physiological conditions (T< Tt) and aggregate of ELP-ODN at sites where T>Tt, which should allow preferential accumulation of ELP-ODN only at the targeted diseased sites. In our preliminary study we demonstrated that cellular uptake of a thermally responsive ELP is significantly enhanced by the thermally triggered phase transition of the polypeptide. Therefore, greater amounts of ODN will enter the cell, which will improve ability to inhibit gene expression of targeted protein.
Recent Publications
Bidwell, G. L., 3rd and Raucher, D. Application of thermally responsive polypeptides directed against c-Myc transcriptional function for cancer therapy. Mol. Cancer Ther., 4: 1076-1085, 2005.
Massodi, I., Bidwell, G. L., 3rd, and Raucher, D. Evaluation of cell penetrating peptides fused to elastin-like polypeptide for drug delivery. J. Control Release, Sep 9; [Epub ahead of print] 2005.
Bidwell, G. L., 3rd and Raucher, D. Enhancing the Antiproliferative Effect of Topoisomerase II Inhibitors Using a Polypeptide Inhibitor of c-Myc. Biochem. Pharm., in press, 2005.
Raucher D., and A. Chilkoti, Enhanced uptake of thermally responsive polypeptide drug carrier by tumor cells in response to its hyperthermia mediated phase transition. Cancer Res. 61: 7163-7177, 2001. [link to PDF]
Raucher D., and M. P. Sheetz Local anesthetics cause decrease in membrane-cytoskeleton adhesion and PIP2 levels. J. Cell Sci. 114:3759-3766, 2001. [link to PDF]
Raucher D., Stauffer, T, Chen, W., Shen, K., Guo S.,York, J. D.,Sheetz, M. P., and Meyer, T. Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell 100:221-228, 2000. [link to PDF]
Raucher D., and M. P. Sheetz Important role of membrane tension in lamellipodial extension. J. Cell. Biol. 148(1):127-136, 2000. [link to PDF]
Raucher D., and M. P. Sheetz Membrane tension regulates endocytosis in mitosis. J.Cell Biol. 144(3):497-506, 1999. [link to PDF]
Raucher D., and M. P. Sheetz. Characteristics of a membrane reservoir studied by membrane tethers extracted by a laser tweezers trap. Biophys. J. 77(4):1992-2002, 1999. [link to PDF]
Hochmuth R.M, Raucher, D., Ting-Beall, H. P.and M.P. Sheetz Membrane-cytoskeleton adhesion and detachment from cells with “excess” area. Proceedings of the First Joint BMES/EMBS Conference Vol 1: 47, 1999.
|
