Enhanced efficacy of radiation-induced gene therapy in mice bearing lung adenocarcinoma xenografts using hypoxia responsive elements

Abstract

The aim of the present study was to investigate whether the hypoxia responsive element (HRE) could be used to enhance suicide gene (HSV-tk) expression and tumoricidal activity in radiation-controlled gene therapy of human lung adenocarcinoma xenografts. A chimeric promoter, HRE-Egr, was generated by directly linking a 0.3-kb fragment of HRE to a 0.6-kb human Egr-1 promoter. Retroviral vectors containing luciferase or the HSV-tk gene driven by Egr-1 or HRE-Egr were constructed. A human adenocarcinoma cell line (A549) was stably transfected with the above vectors using the lipofectamine method. The sensitivity of transfected cells to prodrug ganciclovir (GCV) and cell survival rates were analyzed after exposure to a dose of 2 Gy radiation and hypoxia (1%). In vivo, tumor xenografts in BALB/c mice were transfected with the constructed retroviruses and irradiated to a total dose of 6 Gy, followed by GCV treatment (20 mg/kg for 14 days). When the HSV-tk gene controlled by the HRE-Egr promoter was introduced into A549 cells by a retroviral vector, the exposure to 1% O(2) and 2 Gy radiation induced significant enhancement of GCV cytotoxicity to the cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors infected with the hybrid promoter-containing virus gradually disappeared after GCV administration and radiation. These results indicate that HRE can enhance transgene expression and tumoricidal activity in HSV-tk gene therapy controlled by ionizing radiation in hypoxic human lung adenocarcinoma.

Figure 1

Retroviral constructs used in this study. A deletion in the U₃ region of the 3′ long terminal repeats (LTR) of the retroviral vector pBABA puro was introduced (dLTR) to generate the self‐inactivating (SIN) vector pBABE puro SIN. The inserts consisting of the HSV‐tk or Luc genes with the Egr‐1 promoter and hypoxia responsive element (HRE) enhancer are indicated.

Figure 2

Enhancement of radio‐inducible transcriptional activity from the Egr‐1 promoter by hypoxic response elements (HRE) after transient transfection exposure to hypoxia. A549 cells were infected with the luciferase reporter constructs SEL or HRE‐SEL and incubated in normoxia (N) or hypoxia (H) (1%) for 8 h with exposure to 2 Gy of radiation (R). Twelve hours later, these cells were harvested and assayed for luciferase activity. The luciferase activity were normalized to that in non‐irradiated cells under aerobic conditions. The error bars in all of the figures show the standard deviation (SD) of at least five independent samples. Versus SEL: **P < 0.01; versus N + R : #P < 0.05, ##P < 0.01.

Figure 3

HSV‐tk expression in parental and infected A549 cells. The HSV‐tk of the SET and HRE–SET groups and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)‐specific transcripts are shown. Lane 1, untreated infected cells at normoxia; lane 2, infected cells under hypoxia (1%); lanes 3–4, infected cells exposed to 2 Gy radiation with or without hypoxia (1%), respectively.

Figure 4

Concentration of ganciclovir (GCV) yielding 50% growth inhibition (IC₅₀) in vitro in parental A549 cells, and in SET‐ or HRE–SET‐infected cells exposed to radiation with normoxia (R + N) or hypoxia (1%) (R + H). Cell viability was assayed by using the trypan blue exclusion test. The IC₅₀ was calculated using curve‐fitting parameters. Versus SET: ##P < 0.01; versus R + N: **P < 0.01.

Figure 5

In vitro suicide gene therapy of A549 cells infected with either the SET or HRE–SET retrovirus. Cells infected with the recombinant retroviruses were incubated with 10 µM ganciclovir for 6 days, and exposed to 2 Gy radiation (R) and/or hypoxia (1%) (H) for 8 h previously every 2 days, followed by cell survival analysis. Data are representative of at least five separate experiments. Each bar represents the mean ± SD (n = 5–8).

Figure 6

CA IX staining photographs. (a) Peripheral view. (b) Central view. Both slices are shown at ×25 magnification. Scale bar = 40 µm. N, necrosis; P, CA IX positive staining; V, viable, well‐oxygenated tumor tissue. The arrow indicates a blood vessel.

Figure 7

In vivo suicide gene therapy in solid tumor xenografts consisting of A549 cells. The nude mice bearing the subcutaneous tumors were infected with either SET or HRE–SET retroviruses, followed by daily intraperitoneal injection of 20 mg/kg ganciclovir (GCV) for 2 weeks, with exposure to 2 Gy radiation on days 2, 4 and 6. Tumor size was measured every 2 days. Control, A549 cells injection only; H + G, HRE–SET transfection and GCV; H + R, HRE–SET transfection and radiation; H + R + G, HRE–SET transfection, radiation and GCV; R, A549 injection and radiation; S + G, SET transfection and GCV; S + R, SET transfection and radiation; S + R + G, SET transfection, radiation and GCV.