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

Abstract

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...

Regression of established subcutaneous B16‐F10 murine melanoma tumors after gef gene therapy associated with the mitochondrial apoptotic pathway

“…Although activation of this protein induces arrest of respiration and death in bacterial cells the mechanism of action in tumoral cells is unclear. We have previously demonstrated that gef protein is able to induce changes in proliferation rate and differentiation degree of tumoral cells without having to use prodrugs (14,15). However, its possible in vivo application could not be demonstrated yet.…”

Gef gene therapy enhances the therapeutic efficacy of doxorubicin to combat growth of MCF-7 breast cancer cells

“…Activation of this protein induces arrest of cellular respiration and cell death [15]. In human tumour cells, gef gene induces cell cycle arrest and apoptosis [16,17], which may be a complementary strategy for classic cancer treatments, since it has been demonstrated that apoptosis deficiency is a critical factor for treatment failure in advanced cancer [18]. In this context, gef gene may improve current chemotherapy options in cancer, including MBC, and could be of use as an adjuvant therapy to increase the effectiveness of conventional tumour treatment.…”

Tumour malignancy loss and cell differentiation are associated with induction ofgefgene in human melanoma cells

“…One possibility is modulation of the degree of differentiation in melanoma cells, which has been related to the proliferation capacity and aggressiveness of the tumour 23 . Our group recently developed a novel therapeutic system using the gef killer gene, which modulates the cell cycle in melanoma cells (MS‐36) 19 . The present study explored the ability of the gef gene to increase the degree of maturation in melanoma cells and thereby induce a differentiation process.…”

“…RNA, DNA and protein were extracted from MS‐36 and MS‐36TG to confirm correct transfection and transcription of the gef gene as described by Boulaiz et al. 19 …”