16 research outputs found
Assessment of viral and non-viral gene transfer into adult rat brains using HSV-1, calcium phosphate and PEI-based methods
CNS gene transfer could provide new approaches to the modelling of neurodegenerative
diseases and devising potential therapies. One such disorder is Parkinson’s
disease (PD), in which dysfunction of several different metabolic processes
has been implicated. Here we review the literature on gene transfer systems
based on herpes simplex virus type 1 (HSV-1) and non-viral
polyethyleneimine (PEI) and calcium phosphate nanoparticle methods. We also
assess the usefulness of various CNS gene delivery methods and present some
of our own data to exemplify such usefulness. Our data result from vectors
stereotaxically introduced to the substantia nigra (SN) of adult rats and evaluated
1 week and/or 1 month post injection using histochemical methods to assess
recombinant ß-galactosidase enzyme activity. Gene transfer using PEI or calcium
phosphate-mediated transfections was observed for both methods and PEI was
comparable to that of HSV-1 amplicon. Our data show that the amplicon delivery
was markedly increased when packaged with a helper virus and was similar
to the expression profile achieved with a full-size replication-defective HSV-1
recombinant (8117/43). We also examine whether PEI or HSV-1 amplicon-mediated
gene transfer could facilitate assessment of the biological effects induced
by a dominant negative FGF receptor-1 mutant to model the reduced FGF signalling
thought to occur in Parkinson’s disease
Factors Controlling Fibroblast Growth Factor Receptor-1's Cytoplasmic Trafficking and Its Regulation as Revealed by FRAP Analysis
Biochemical and microscopic studies have indicated that FGFR1 is a transmembrane and soluble protein present in the cytosol and nucleus. How FGFR1 enters the cytosol and subsequently the nucleus to control cell development and associated gene activities has become a compelling question. Analyses of protein synthesis, cytoplasmic subcompartmental distribution and movement of FGFR1-EGFP and FGFR1 mutants showed that FGFR1 exists as three separate populations (a) a newly synthesized, highly mobile, nonglycosylated, cytosolic receptor that is depleted by brefeldin A and resides outside the ER-Golgi lumen, (b) a slowly diffusing membrane receptor population, and (c) an immobile membrane pool increased by brefeldin A. RSK1 increases the highly mobile cytosolic FGFR1 population and its overall diffusion rate leading to increased FGFR1 nuclear accumulation, which coaccumulates with RSK1. A model is proposed in which newly synthesized FGFR1 can enter the (a) “nuclear pathway,” where the nonglycosylated receptor is extruded from the pre-Golgi producing highly mobile cytosolic receptor molecules that rapidly accumulate in the nucleus or (b) “membrane pathway,” in which FGFR1 is processed through the Golgi, where its movement is spatially restricted to trans-Golgi membranes with limited lateral mobility. Entrance into the nuclear pathway is favored by FGFR1's interaction with kinase active RSK1
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Regulation of targeted chemotherapy with cytotoxic lutenizing hormone-releasing hormone analogue by epidermal growth factor.
Targeting chemotherapy selectively to cancers can reduce the toxic
side effects. AN-152, a conjugate of doxorubicin and[
d-Lys6]-luteinizing hormone-releasing
hormone (LH-RH), is more potent against LH-RH receptor-bearing cancers
and produces less peripheral toxicity than doxorubicin. Many cancers,
e.g., 50% of breast cancers, but few normal tissues
express these receptors, providing a selective target for this
cytotoxic conjugate. In this study, the effectiveness of AN-152 was
heightened by receptor up-regulation. The cytotoxic effect of AN-152
can be regulated by the number of active LH-RH receptors on cancer
cells. LH-RH receptor-positive (MCF-7) and -negative (UCI-107) cancer
cells were treated with epidermal growth factor (EGF) or the
somatostatin analogue, RC-160. EGF and RC-160 have been shown
previously to regulate LH-RH receptors through phosphorylation. The
effect of receptor regulation, by hormone exposure, on the cytotoxicity
of AN-152 and doxorubicin and on the cellular uptake of AN-152,[
d-Lys6]LH-RH, or doxorubicin was assessed by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
and by two-photon laser scanning microscopy. The results demonstrated
that the cellular entry of the conjugate was: (a)
specific for cancers with LH-RH receptors; (b)
up-regulated by EGF; (c) down-regulated by RC-160; and
(d) the cytotoxicity of the AN-152 paralleled the
efficiency of entry. This study illustrates the potential use of
receptor regulation for increasing the efficacy of chemotherapeutic
approaches that are directed to cell surface receptors
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Studies on the mechanism of action of a targeted chemotherapeutic drug in living cancer cells by two photon laser scanning microspectrofluorometry
In this study, we present a spectroscopic study of the entry pattern of a chemotherapeutic drug (AN-152) and its carrier hormone
into living cancer cells, with the help of our two-photon probes and a home-built localized microspectrofluorometer coupled with two photon laser scanning microscope (TPLSM). Due to the inherent localization ability of TPLSM, we were able to identify the drug and carrier location in different compartments of the cancer cells
. The apparent doxorubicin-assisted nucleic accumulation of AN-152 suggests a possible nuclear action of the drug on cell proliferation.
A chemically labeled cytotoxic agent: Two-photon fluorophore for optical tracking of cellular pathway in chemotherapy
Chemotherapy is commonly used in the treatment of cancers. However, the mechanism of action of many of these agents is not well understood. We present the synthesis of a two-photon fluorophore (C625) and its biological application when chemically linked to a chemotherapeutic agent (AN-152). By using two-photon laser-scanning microscopy, the drug:fluorophore conjugate can be observed directly as it interacts with receptor-positive cell lines. The results of this project visually show the receptor-mediated entry of AN-152 into the cell cytoplasm and subsequently into the nucleus. These observations will allow for better understanding of the drug’s therapeutic mechanism, which is a subject of ongoing research aimed at improving present methods for cancer therapy