Multidrug Resistance Phenotype in the RMS‐GR Human Rhabdomyosarcoma Cell Line Obtained after Polychemotherapy

Prados, José; Melguizo, Consolación; Marchal, Juan Antonio; Vélez, Celia; Álvarez, Luis; Aránega, Amelia

doi:10.1111/j.1349-7006.1999.tb00816.x

Cited by 1 publication

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References 28 publications

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“…The integrity of RNA was assessed by amplifying β-actin mRNA using the 5′-primer nt 1854 to 1873 and 3′-primer nt 2151 to 2170. 16) Immunoblotting was done with a polyclonal antibody against the Gef protein, which was produced by immunizing rabbits with a 25-amino-acid synthetic peptide (carboxyl terminus) having the sequence predicted from the DNA sequence of the gef gene. 12) Immunization was done at the Technical Services of the University of Seville (Seville, Spain).…”

Section: Methodsmentioning

confidence: 99%

Transfection of MS‐36 melanoma cells with gef gene inhibits proliferation and induces modulation of the cell cycle

et al. 2003

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The gef gene, found in Escherichia coli DNA, encodes a small (50 amino acids) protein which is related to cell-killing functions. We echnologies for gene transfer to tumor cells are providing new possibilities for cancer therapy. One potential approach involves genetic modification of tumor cells 1, 2) based on the transfer of suicide genes. Most strategies that are being developed involve killer-suicide genes, which are able to convert a nontoxic prodrug into a toxic metabolite. The herpes simplex virus thymidine kinase encoding gene (HSVtk) and Escherichia coli (E. coli) cytosine deaminase (CD) are two examples of genes utilized for these strategies.3, 4) HSVtk converts the antiviral drug gancyclovir to a monophosphorylated molecule that is then metabolized to the toxic triphosphate form by cellular kinases, inhibiting DNA synthesis.5-7) The enzyme encoded by the CD gene converts 5-fluorocytosine into the toxic anabolyte 5-fluorouracil (5-FU), which is subsequently processed either to 5-fluorouracil triphosphate or to 5-fluoro-2′-deoxyuridine 5′-monophosphate, which inhibits RNA and DNA synthesis. 7)However, the release of toxic metabolites and their bioavailability are two important limitations to the use of these systems. 8) Similar strategies using E. coli β-galactosidase have similar limitations. 9) Thus, the transfer of genes which are not dependent on the use of a prodrug is receiving more attention.10)The gef gene from E. coli, identified as a member of a gene family which encodes homologous cell-killing functions, 11) may be a new candidate for cancer gene therapy. This gene codes a membrane protein of about 50 amino acids which is anchored in the cytoplasmic membrane at the N-terminal portion. The Cterminal part is located in the periplasm. Mutagenesis studies with the Gef protein have shown that its periplasmic portion encodes the toxic domain and that its dimerization is not essential for the toxic effect.12) Studies of suicide cassettes consisting of members of the gef gene family in combination with inducible promoters have documented the toxic effect, 13) which is currently being studied to determine its relation to the ability to control bacterial population death. 14)We constructed a mammalian expression vector containing the gef gene under the control of the mouse mammary tumor virus (MMTV) promoter, which is inducible by dexamethasone (Dex), to evaluate the effect of the expression of this gene in MS-36 cells in vitro. Our data show that expression of the gef gene modifies the cell cycle, induces morphologic changes and significantly decreases cell growth. These results suggest that the gef gene may merit testing as a new approach to cancer therapy. Materials and MethodsCell line. The human melanoma MS-36 cell line was obtained from the Immunology Service, Virgen de las Nieves Hospital (Granada, Spain). MS-36 cells were grown at 37°C in an atmosphere containing 5% CO 2 , with Iscove's modified Dulbecco's medium (IMDM) (Gibco, Grand Island, NY), 10% heat-inactivated fetal bovine serum (FBS) (G...

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Section: Methodsmentioning

confidence: 99%