Improved retroviral suicide gene transfer in colon cancer cell lines after cell synchronization with methotrexate

Abstract

Background: Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk) in two colon cancer cell lines, DHDK12 and HT29.

Methods: Synchronization was induced by methotrexate (MTX), aracytin (ara-C) or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV) towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry.

Results: DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV.

Conclusions: Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy.

Figure 1

Distribution in cell cycle-phase after MTX treatment. Cell cycle phases of DHDK12 cells (A) and HT29 cells (B) were obtained by uniparametric flow cytometry analysis of DNA content (propidium iodide red-fluorescence intensity in fluorescence units) at various time after MTX removal. On the ordinate is shown the number of cells corresponding to the fluorescence units.

Figure 2

Infection efficiency of the β-gal retroviral vector. DHDK12 cells (A) and HT29 cells (B) were treated for 24 hr with (filled circle) or whithout (open circle) MTX. Cells were transduced with TG 5391 at the indicated times after MTX removal. The level of β-galactosidase activity was obtained 48 hr after the transduction by flow cytometry analysis using FDG, a fluorescent substrate of β-galactosidase. The percentage of cells in S phase (open triangle) at various time after MTX removal was determined by flow cytometry analysis of DNA content. Data are expressed as the mean ± SE from at least three separate experiments.

Figure 3

Detection of HSV-TK protein. DHDK12 cells (A) and DHDK12 cells transduced with the HSV-tk retroviral vector (B) were immunostained for HSV-TK. Cells seeded on chamber were transduced with TG 9344. After 48 hr, cells were fixed with 4% paraformaldehyde and stained with a mouse monoclonal 4C8 antibody against HSV-TK protein.

Figure 4

Infection efficiency of the HSV-tk retroviral vector. DHDK12 cells (A) and HT29 cells (B) were treated for 24 hr with (filled square) or without (open square) MTX. Cells were transduced with TG 9344 at the indicated times after MTX washout. The HSV-TK expression level was determined 48 hr after transduction by flow cytometry using a mouse monoclonal 4C8 antibody against HSV-TK protein. Data are expressed as the mean ± SE from at least three separate experiments. *P <.05 vs. untreated cells, # P <.05 vs. MTX-treated cells at 12 and 16 hr after MTX withdrawal.

Figure 5

Internucleosomal DNA fragmentation induced by GCV. Lane 1 and lane 4 show DHDK12 cells and HT29 cells transduced with TG 9344 and treated for 96 hr with 20 μM GCV, respectively. Lane 3 and 5 show DHDK12 cells and HT29 cells transduced with TG 9344 after a 24 hr pretreatment with MTX and treated for 96 hr with 20 μM GCV, respectively. Lane 6 and 7 show DHDK12 cells and HT29 cells treated for 24 h with MTX, respectively. Lane 2 shows pBR 322 base pair size markers. Qualitative detection of DNA was achieved by ethidium bromide staining.

Figure 6

Induction of apoptosis. Untransduced DHDK12 cells (A) were treated with MTX, GCV or the combination of MTX plus GCV for 24 h. Transduced DHDK12 cells (B) and transduced HT29 cells (C) were treated for 24 hr with (filled square) or without (open square) MTX. Cells were transduced with TG 9344 at the indicated times after MTX washout and 48 hr after transduction were treated with 20 μM GCV for 72 hr. Quantitative detection of apoptosis was determined by biparametric flow cytometry analysis of fluorescein labeled-annexin V cells and PI. Apoptotic cells were annexin V positive, PI negative. Data are expressed as the mean ± SE from at least three separate experiments. * P <.05 vs untreated cells